Methods for treating non-muscle invasive bladder cancer (NMIBC) using antibody-drug conjugates (ADCs) that bind to 191P4D12 protein

Abstract

Provided herein are methods for intravesical treatment of bladder cancer and methods for treating non-muscle invasive bladder cancer with antibody drug conjugates (ADCs) that bind to the 191P4D12 protein (Nectin-4).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Application No. 63 / 233,048, filed August 13, 2021, U.S. Application No. 63 / 242,380, filed September 9, 2021, and U.S. Application No. 63 / 328,441, filed April 7, 2022, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Reference to an electronically submitted sequence listing This application contains a computer readable Sequence Listing which has been submitted herewith in XML file format, the entire contents of which are incorporated herein by reference. The Sequence Listing XML file submitted herewith is entitled "14369-281-228_SEQ_LISTING.xml", was created on August 2, 2022, and is 34,743 bytes in size.

[0003] 1. Field Provided herein are methods for treating non-muscle invasive bladder cancer (NMIBC) using antibody drug conjugates (ADCs) that bind to the 191P4D12 protein (Nectin-4). [Background technology]

[0004] 2.Background Bladder cancer, the most common form of urothelial carcinoma (UC) and the sixth most common cancer in the United States (US), is estimated to cause approximately 200,000 patient deaths annually worldwide, including over 65,000 in Europe and nearly 18,000 in the US (Bray 2018; Ferlay 2018; Siegel 2019). The annual number of new cases of bladder cancer diagnosed worldwide was estimated to be over 549,000 in 2018. In 2020, there were an estimated 81,400 new cases of bladder cancer in the US, a figure that is expected to increase to over 83,000 in 2021 (American Cancer Society (ACS) 2021; National Cancer Institute (NCI) 2021). Bladder cancer incidence and mortality rates increase strongly with age, making it an increasingly problematic disease as the elderly population grows.

[0005] Approximately 70%–80% of bladder cancer diagnoses are non-muscle-invasive disease (Chang 2016; Woldu 2017; Kates 2020; Li 2020). Non-muscle-invasive bladder cancer (NMIBC) represents a heterogeneous group of cancers, including those that are essentially papillary and confined to the mucosa (Ta), those that are high-grade and flat and confined to the epithelium (Tis or carcinoma in situ [CIS]), and those that invade the submucosa or lamina propria (T1) (Pasin 2008). Among NMIBC patients, papillary disease is the most common, affecting approximately 70% of patients, while T1 disease and CIS affect approximately 20% and 10% of patients, respectively (Kirkali 2005; Anastasiadis 2012).

[0006] The standard means of treating NMIBC involves surgical removal of the bladder tumor via transurethral resection of the bladder tumor (TURBT) and the intravesical administration of therapeutic agents for additional antitumor activity (Kawai 2013; Chang 2016; Woldu 2017; Jamil 2019; Kates 2020). Intravesical Bacillus Calmette-Guérin (BCG) is considered the treatment of choice for NMIBC patients, particularly those with high-risk disease features, as it induces a local immune response that correlates with antitumor activity (Kassouf 2015; Chang 2016).

[0007] BCG-nonresponsive disease represents a subgroup of NMIBC patients who do not respond to adequate treatment with BCG and remain at high risk for disease recurrence and progression to subsequent stages of UC. For these patients, further treatment with BCG is not an option, and radical cystectomy remains the best option. Although intravesical chemotherapy agents such as gemcitabine, mitomycin, and barbicanine have shown some efficacy, current guidelines state that treatments other than radical cystectomy are inferior for the treatment of BCG-nonresponsive disease (Navai 2016; Taylor 2020) (EAU. Guidelines for Non-muscleinvasive Bladder Cancer. 2020. https: / / uroweb.org / guideline / non-muscle-invasive-bladdercancer / Accessed Feb 16, 2021.). Recently, systemic pembrolizumab was approved for the treatment of patients with BCG-nonresponsive carcinoma in situ (CIS); however, more than half of patients treated with pembrolizumab do not have a complete response to treatment, and safe and effective intravesical treatments remain needed in this patient population.

[0008] 191P4D12 (also known as nectin-4) is a 66 kDa type I transmembrane protein that belongs to the nectin family of adhesion molecules. 191P4D12 is composed of an extracellular domain (ECD) containing three immunoglobulin (Ig)-like subdomains, a transmembrane helix, and an intracellular region (Takai et al., Annu Rev Cell Dev Biol (2008); 24:309-42). Nectins mediate Ca transport through both homophilic and heterophilic trans-interactions at adherens junctions, which can recruit cadherins and regulate cytoskeletal rearrangements. 2+It is thought to mediate cell-cell adhesion independent of the nuclei (Rikitake et al., Cell Mol Life Sci (2008); 65(2):253-63). The sequence identity of nectin-4 to other nectin family members is low, ranging from 25% to 30% in the ECD (Reymond et al., Biol Chem (2001); 276(46):43205-15).

[0009] Nectin-4 has been shown to be expressed in several cancers, particularly urothelial, breast, lung, pancreatic, and ovarian cancers. Higher expression levels are associated with disease progression and / or poor prognosis (Fabre-Lafay et al., BMC Cancer (2007); 7:73).

[0010] For patients with BCG-unresponsive NMIBC who are ineligible or unsuitable for radical cystectomy, or who make an informed decision not to undergo radical cystectomy, there is an unmet need for a safe and effective intravesical treatment. Summary of the Invention

[0011] 3. Overview Provided herein are methods for treating bladder cancer in a human subject using intravesical administration of an antibody drug conjugate (ADC) that binds to 191P4D12.

[0012] Embodiment 1. A method of treating bladder cancer in a human subject, comprising intravesically administering to the subject an effective amount of an antibody drug conjugate (ADC), wherein the ADC: An antibody or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethyl auristatin E (MMAE) The method comprising:

[0013] Embodiment 2. The method of embodiment 1, wherein the bladder cancer is non-muscle-invasive bladder cancer (NMIBC).

[0014] Embodiment 3. The method of embodiment 2, wherein the NMIBC is histologically confirmed and is carcinoma in situ (CIS).

[0015] Embodiment 4. The method of embodiment 3, wherein the subject has a papillary disease.

[0016] Embodiment 5. The method of embodiment 3, wherein the subject does not have papillary disease.

[0017] Embodiment 6. The method of any of embodiments 2-5, wherein the NMIBC is histologically confirmed and the predominant histological component (>50%) is urothelial (transitional cell) carcinoma.

[0018] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the subject has high-risk Bacillus Calmette-Guerin (BCG)-non-responsive disease.

[0019] Embodiment 8 The method of any one of embodiments 1 to 7, wherein the subject is ineligible for or refuses to undergo radical cystectomy.

[0020] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the subject has had all visible papillary Ta / T1 tumors completely resected within 60 days prior to the treatment.

[0021] Embodiment 10. The method of embodiment 9, wherein the subject has residual pure CIS.

[0022] Embodiment 11. The method of embodiment 9, wherein the subject does not have residual pure CIS.

[0023] Embodiment 12. The method of any one of embodiments 1 to 11, wherein the subject has an Eastern Cooperative Oncology Group (ECOG) performance status score of 0.

[0024] Embodiment 13. The method of any one of embodiments 1 to 11, wherein the subject has an Eastern Cooperative Oncology Group (ECOG) performance status score of 1.

[0025] Embodiment 14. The method of any one of embodiments 1 to 11, wherein the subject has an Eastern Cooperative Oncology Group (ECOG) performance status score of 2.

[0026] Embodiment 15. The method of embodiment 14, wherein the subject has a glomerular filtration rate (GFR) of 50 mL / min or greater, and the subject does not have New York Heart Association (NYHA) Class III heart failure.

[0027] Embodiment 16. The subject is a. Absolute neutrophil count (ANC) ≥ 1500 / μL; b. Hemoglobin (Hgb) is 10 g / dL or more; c. Platelet count is 100,000 / μL or more. d. Serum bilirubin less than 1.5 × upper limit of normal (ULN) or less than 3 × ULN for patients with Gilbert's disease; e. Calculated creatinine clearance (CrCl) ≥ 30 mL / min (GFR can be used instead of creatinine or CrCl), CrCl should be calculated using the Cockcroft-Gault method or the Modified Diet in Renal Disease (MDRD) formula, and subjects with ECOG activity status 2 must have a GFR ≥ 50 mL / min. f. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels less than 3 × ULN, or g. International normalized ratio (INR), or prothrombin time (PT), activated partial thromboplastin time (aPTT), or partial thromboplastin time (PTT) of 1.5 ULN or less, unless the subject is undergoing anticoagulant therapy, as long as the PT or aPTT is within the therapeutic range for which the anticoagulant is intended. 16. The method of any one of embodiments 1 to 15, wherein the patient has one or more of the conditions selected from the group consisting of:

[0028] Embodiment 17. The method of embodiment 16, wherein the subject has all of conditions (a) through (g) of embodiment 16.

[0029] Embodiment 18. The method of any one of embodiments 1 to 17, wherein the subject has an estimated life expectancy of greater than 2 years.

[0030] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO: 22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 23.

[0031] Embodiment 20. The antibody or antigen-binding fragment thereof comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, CDR-H2 comprising the amino acid sequence of SEQ ID NO:10, CDR-H3 comprising the amino acid sequence of SEQ ID NO:11, CDR-L1 comprising the amino acid sequence of SEQ ID NO:12, CDR-L2 comprising the amino acid sequence of SEQ ID NO:13, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:14; or The antibody or antigen-binding fragment thereof comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21. 20. The method according to any one of embodiments 1 to 19.

[0032] Embodiment 21. the antibody or antigen-binding fragment thereof comprises CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 9, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 10, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 11, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 12, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 13, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 14; or The method according to any one of embodiments 1 to 19, wherein the antibody or antigen-binding fragment thereof comprises CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 16, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 17, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 18, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 19, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 20, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 21.

[0033] Embodiment 22. The method of any one of embodiments 1 to 21, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23.

[0034] Embodiment 23. The method of any one of embodiments 1 to 22, wherein the antibody comprises a heavy chain comprising an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7, and a light chain comprising an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8.

[0035] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the antigen-binding fragment is a Fab, F(ab')2, Fv, or scFv.

[0036] Embodiment 25. The method of any one of embodiments 1 to 24, wherein the antibody is a fully human antibody.

[0037] Embodiment 26. The method of any one of embodiments 1 to 25, wherein the antibody is an IgG1 and the light chain is a kappa light chain.

[0038] Embodiment 27. The method of any one of embodiments 1 to 26, wherein the antibody or antigen-binding fragment thereof is recombinantly produced.

[0039] Embodiment 28. The method of any one of embodiments 1 to 27, wherein the antibody or antigen-binding fragment is conjugated to each unit of MMAE via a linker.

[0040] Embodiment 29. The method of embodiment 28, wherein the linker is an enzyme-cleavable linker and forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof.

[0041] Embodiment 30. The method of embodiment 28 or 29, wherein the linker has the formula -Aa-Ww-Yy-, where -A- is an extender unit, a is 0 or 1, -W- is an amino acid unit, w is an integer ranging from 0 to 12, -Y- is a spacer unit, and y is 0, 1, or 2.

[0042] Embodiment 31. The method of embodiment 30, wherein the extender unit has the structure of formula (1): TIFF2024534012000002.tif75165.

[0043] Embodiment 32. The method of embodiment 30 or 31, wherein the extender unit forms a bond with a sulfur atom of an antibody or antigen-binding fragment thereof, and the spacer unit is linked to MMAE via a carbamate group.

[0044] Embodiment 33. The method of any one of embodiments 1 to 32, wherein the ADC comprises 1 to 20 units of MMAE per antibody or antigen-binding fragment thereof.

[0045] Embodiment 34. The method of any one of embodiments 1 to 33, wherein the ADC comprises 1 to 10 units of MMAE per antibody or antigen-binding fragment thereof.

[0046] Embodiment 35. The method of any one of embodiments 1 to 34, wherein the ADC comprises 2 to 8 units of MMAE per antibody or antigen-binding fragment thereof.

[0047] Embodiment 36. The method of any one of embodiments 1 to 35, wherein the ADC comprises 3 to 5 units of MMAE per antibody or antigen-binding fragment thereof.

[0048] Embodiment 37. The ADC has the structure: TIFF2024534012000003.tif42165, wherein L- represents an antibody or an antigen-binding fragment thereof, and p is 1 to 10.

[0049] Embodiment 38. The method of embodiment 37, wherein p is 2 to 8.

[0050] Embodiment 39. The method of embodiment 37 or 38, wherein p is 3 to 5.

[0051] Embodiment 40. The method of any one of embodiments 37 to 39, wherein p is 3 to 4.

[0052] Embodiment 41. The method of any one of embodiments 37 to 40, wherein p is about 4.

[0053] Embodiment 42. The method of any one of embodiments 37 to 40, wherein the effective dose of the antibody-drug conjugate has a mean p-value of about 3.8.

[0054] Embodiment 43. The method of any one of embodiments 1 to 42, wherein the ADC is formulated in a pharmaceutical composition comprising L-histidine, polysorbate-20 (TWEEN-20), and trehalose anhydrous.

[0055] Embodiment 44. The method of any one of embodiments 1 to 43, wherein the ADC is formulated in a pharmaceutical composition comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and hydrochloride, and the pH of the pharmaceutical composition is about 6.0 at 25°C.

[0056] Embodiment 45. The method of any one of embodiments 1 to 43, wherein the ADC is formulated in a pharmaceutical composition comprising about 9 mM histidine, about 11 mM histidine hydrochloride monohydrate, about 0.02% (w / v) TWEEN-20, and about 5.5% (w / v) trehalose dihydrate, wherein the pH of the pharmaceutical composition is about 6.0 at 25°C.

[0057] Embodiment 46. The method of any one of embodiments 1 to 45, wherein the effective amount of the ADC is a dose of about 100 mg to about 1000 mg, about 125 mg to about 950 mg, about 125 mg to about 900 mg, about 125 mg to about 850 mg, about 125 mg to about 800 mg, or about 125 mg to about 750 mg in an infusion volume of about 10 mL to about 100 mL.

[0058] Embodiment 47. The method of any one of embodiments 1 to 46, wherein the effective amount of the ADC is a dose of about 125 mg to about 750 mg in an infusion volume of about 25 mL.

[0059] Embodiment 48. The method of any one of embodiments 1 to 47, wherein the effective amount of the ADC is a dose of about 125 mg in an infusion volume of about 25 mL.

[0060] Embodiment 49. The method of any one of embodiments 1 to 47, wherein the effective amount of the ADC is a dose of about 250 mg in an infusion volume of about 25 mL.

[0061] Embodiment 50. The method of any one of embodiments 1-47, wherein the effective amount of the ADC is a dose of about 500 mg in an infusion volume of about 25 mL.

[0062] Embodiment 51. The method of any one of embodiments 1 to 47, wherein the effective amount of the ADC is a dose of about 750 mg in an infusion volume of about 25 mL.

[0063] Embodiment 52. The method of any one of embodiments 1 to 51, wherein the maximum residence time of each intravesical administration is about 90 minutes.

[0064] Embodiment 53. The method of any one of embodiments 1 to 51, wherein the maximum residence time of each intravesical administration is about 120 minutes.

[0065] Embodiment 54. The method of any one of embodiments 1 to 51, wherein the residence time of each intravesical administration is about 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, or 120 minutes.

[0066] Embodiment 55. The method of any one of embodiments 1 to 54, wherein the ADC is administered intravesically during two phases: an induction phase and a maintenance phase.

[0067] Embodiment 56. The method of embodiment 55, wherein the maintenance phase begins 6 to 10 weeks, 6 to 9 weeks, or 6 to 8 weeks after the induction phase.

[0068] Embodiment 57. The method of embodiment 55 or 56, wherein the ADC is administered intravesically once a week for 6 weeks during the induction phase.

[0069] Embodiment 58. The method of any one of embodiments 55-57, wherein the ADC is administered intravesically once a month for 9 months during the maintenance phase.

[0070] Embodiment 59. The ADC has the structure: TIFF2024534012000004.tif44165, wherein L- represents an antibody or antigen-binding fragment thereof, and p is about 3 to about 4, and the antibody comprises a heavy chain comprising an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO: 7, and a light chain comprising an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO: 8, wherein the ADC is administered intravesically at a dose of about 125 mg, in an instillation volume of about 25 mL, and with a maximum dwell time of 90 minutes, and the dose is administered intravesically once weekly for 6 weeks during the induction phase and once monthly for 9 months during the maintenance phase, wherein the maintenance phase begins 6 to 10 weeks after the induction phase.

[0071] Embodiment 60. The ADC has the structure: TIFF2024534012000005.tif42165, wherein L- represents an antibody or antigen-binding fragment thereof, and p is about 3 to about 4, and the antibody comprises a heavy chain comprising an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO: 7, and a light chain comprising an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO: 8, wherein the ADC is administered intravesically at a dose of about 250 mg, in an instillation volume of about 25 mL, and with a maximum dwell time of 90 minutes, and the dose is administered intravesically once weekly for 6 weeks during the induction phase and once monthly for 9 months during the maintenance phase, wherein the maintenance phase begins 6 to 10 weeks after the induction phase.

[0072] Embodiment 61. The ADC has the structure: TIFF2024534012000006.tif38165, wherein L- represents an antibody or antigen-binding fragment thereof, and p is about 3 to about 4, and the antibody comprises a heavy chain comprising an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO: 7, and a light chain comprising an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO: 8, wherein the ADC is administered intravesically at a dose of about 500 mg, in an instillation volume of about 25 mL, and with a maximum dwell time of 90 minutes, and the dose is administered intravesically once weekly for 6 weeks during the induction phase and once monthly for 9 months during the maintenance phase, wherein the maintenance phase begins 6 to 10 weeks after the induction phase.

[0073] Embodiment 62. The ADC has the structure: TIFF2024534012000007.tif37165, wherein L- represents an antibody or antigen-binding fragment thereof, and p is about 3 to about 4, and the antibody comprises a heavy chain comprising an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO: 7, and a light chain comprising an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO: 8, wherein the ADC is administered intravesically at a dose of about 750 mg, in an instillation volume of about 25 mL, and with a maximum dwell time of 90 minutes, and the dose is administered intravesically once weekly for 6 weeks during the induction phase and once monthly for 9 months during the maintenance phase, wherein the maintenance phase begins 6 to 10 weeks after the induction phase. [Brief explanation of the drawings]

[0074] 4. Brief description of the drawings [Figure 1A-1] 1 shows the nucleotide and amino acid sequences of the Nectin-4 protein. [Figure 1A-2] See legend to Figure 1A-1. [Figure 1B] The nucleotide and amino acid sequences of the heavy chain of Ha22-2(2.4)6.1 are shown. [Figure 1C] The nucleotide and amino acid sequences of the light chain of Ha22-2(2.4)6.1 are shown. [Figure 1D] The amino acid sequence of the heavy chain of Ha22-2(2.4)6.1 is shown. [Figure 1E] The amino acid sequence of the light chain of Ha22-2(2.4)6.1 is shown. [Figure 2] We demonstrate the cytotoxic activity of enfortumab vedotin (EV) in vitro in bladder cancer cells overexpressing Nectin-4 (i.e., UM-UC-3-hNectin-4+) using conditions mimicking intravesical administration. [Figure 3A] This figure shows the efficacy of intravesical administration of enfortumab vedotin (EV) in a Nectin-4+ bladder orthotopic xenograft mouse model. The generation of SCID mice orthotopically implanted with UM-UC-3-hNectin4+-Luc+ cells after chemical abrasion is shown, along with the administration schedule for intravesical EV administration to mice and histological analysis of bladder tissue. [Figure 3B-1] Efficacy of intravesical administration of enfortumab vedotin (EV) in a Nectin-4+ bladder orthotopic xenograft mouse model. Bioluminescence imaging results confirm tumor engraftment and EV activity. SWFI, sterile water for injection. [Figure 3B-2] See legend to Figure 3B-1. [Figure 3C] Figure 3C shows the efficacy of intravesical administration of enfortumab vedotin (EV) in a Nectin-4 bladder orthotopic xenograft mouse model. Anti-Nectin-4 immunohistochemistry results confirm EV activity. The bladder tissues in the five right panels of Figure 3C were derived from five mice treated with intravesical administration of EV, as shown in Figure 3B. [Figure 3D] Figure 3B shows the efficacy of intravesical administration of enfortumab vedotin (EV) in a Nectin-4 bladder orthotopic xenograft mouse model. Quantitative analysis of bioluminescence imaging results is shown in Figure 3B. [Figure 3E]This figure shows the efficacy of intravesical administration of enfortumab vedotin (EV) in a Nectin-4+ bladder orthotopic xenograft mouse model. Immunohistochemical (IHC) staining of Nectin-4 and MMAE in bladder tumor tissue demonstrates colocalization of Nectin-4 and MMAE. [Figure 4] Panels A-B show free MMAE in bladder tissue from Sprague-Dawley rats treated with a single intravesical administration of EV at various concentrations and doses. [Figure 5] Shown is intravesical EV systemic exposure. [Figure 6] Figure 1 shows free MMAE in bladder tissue from Sprague-Dawley rats treated with a single intravesical administration of EVs with different residence times. [Figure 7] A schema for the clinical trial described in Section 6.1 is shown, which is a Phase 1, open-label, multicenter, dose-escalation and dose-expansion study designed to evaluate the safety, tolerability, PK, and antitumor activity of intravesical enfortumab vedotin in adults with NMIBC. (BCG = bacillus Calmette-Guerin; CIS = carcinoma in situ; EV = enfortumab vedotin; mTPI = modified toxicity probability interval; q3 = every 3; q6 = every 6; TURBT = transurethral resection of bladder tumor; wkly = weekly. Investigation of safety at a given dose or at dose levels below or intermediate to the planned dose levels is contemplated.) DETAILED DESCRIPTION OF THE INVENTION

[0075] 5. Detailed Description Before the present disclosure is further described, it is to be understood that the present disclosure is not limited to particular embodiments described herein and that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0076] In the efficient use of intravesical therapy, various factors must be considered, including at least the infusion volume, residence time, dose concentration, total dose, infusion pH, urine pH, urine output reduction before and during treatment, etc., to achieve an appropriate balance between efficacy and safety. The present disclosure has surprisingly determined that dose concentration and total dose are the most important factors for the efficient and safe delivery of ADCs, such as the MMAE ADCs disclosed herein. The present disclosure has used this knowledge to aid in the development of various methods using intravesical administration of ADCs to treat bladder cancer (e.g., non-muscle-invasive bladder cancer (NMIBC)).

[0077] 5.1 Definition The techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed by those of skill in the art using conventional methodology, such as, for example, the widely utilized methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2nd ed. 2010).

[0078] Unless otherwise defined herein, the technical and scientific terms used in this description have the meanings that are commonly understood by those skilled in the art. For the purposes of interpreting this specification, the following explanations of terms shall apply, and where appropriate, terms used in the singular shall also include the plural, and vice versa. In the event that the explanations of terms set forth conflict with documents incorporated herein by reference, the explanations of terms set forth below shall prevail.

[0079] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense, specifically encompassing, for example, monoclonal antibodies (including agonist, antagonist, neutralizing, full-length, or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or univalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, and fragments thereof, as described below. Antibodies may be human, humanized, chimeric, and / or affinity-matured antibodies, as well as antibodies from other species, e.g., mouse and rabbit. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides that can bind to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2nd ed. 1995); and Kuby, Immunology (3rd ed. 1997). In specific embodiments, specific molecular antigens can be bound by the antibodies provided herein, including polypeptides or epitopes. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, where a functional fragment refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab') fragments, F(ab') fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules containing an antigen-binding domain or site that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. The antibodies can be agonist or antagonist antibodies.

[0080] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which may include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0081] An "antigen" is a structure to which an antibody can selectively bind. Target antigens can be polypeptides, carbohydrates, nucleic acids, lipids, haptens, or other natural or synthetic compounds. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, e.g., present on or within a cell, e.g., on or within a cancer cell.

[0082] An "intact" antibody is one that comprises an antigen-binding site and a CL and at least a heavy chain constant region, CH1, CH2, and CH3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0083] The terms "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," and similar terms refer to a portion of an antibody (e.g., a CDR) that contains amino acid residues that interact with an antigen and confer specificity and affinity for the antigen to the binding agent. As used herein, "antigen-binding fragment" includes an "antibody fragment" that contains a portion of an intact antibody, e.g., the antigen-binding region or variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), and Fv fragments; diabodies and di-diabodies (e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. 90:6444-48; Lu et al., 2005, J. Biol. Chem. 280:19665-72; Hudson et al., 2005, J. Biol. Chem. 280:19665-72). al., 2003, Nat. Med. 9:129-34; WO 93 / 11161; and U.S. Pat. Nos. 5,837,242 and 6,492,123); single-chain antibody molecules (see, e.g., U.S. Pat. Nos. 4,946,778; 5,260,203; 5,482,858; and 5,476,786); dual variable domain antibodies (see, e.g., U.S. Pat. No. 7,612,181); single variable domain antibodies (sdAbs) (see, e.g., Woolven et al., 1999, Immunogenetics 50:98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA. 101:12444-49); and multispecific antibodies formed from antibody fragments.

[0084] The terms "bind" or "binding" refer to interactions between molecules, including, for example, forming a complex. The interaction can be a non-covalent interaction, including, for example, hydrogen bonding interactions, ionic bonding interactions, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of all non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The dissociation rate (k off ) and association rate (k on ) ratio (k off / k on ) is the dissociation constant K D which is inversely proportional to the affinity. D The lower the value, the higher the affinity of the antibody. D The value of k varies for different antibody-antigen complexes. on and k off The dissociation constant K of the antibodies provided herein depends on both D The affinity of an antibody can be determined using any method provided herein or any other method known to those skilled in the art. The affinity at one binding site does not necessarily reflect the true strength of the interaction between an antibody and an antigen. When a complex antigen containing multiple repeat antigenic determinants, such as a multivalent antigen, contacts an antibody containing multiple binding sites, the interaction between the antibody and the antigen at one site increases the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity.

[0085] In connection with the antibodies or antigen-binding fragments thereof described herein, terms such as "binds to," "specifically binds to," and similar terms are also used interchangeably herein and refer to binding molecules of an antigen-binding domain, such as polypeptides, that specifically bind to an antigen. Antibodies or antigen-binding fragments that bind to or specifically bind to an antigen may be cross-reactive with related antigens. In certain embodiments, antibodies or antigen-binding fragments that bind to or specifically bind to an antigen do not cross-react with other antigens. Antibodies or antigen-binding fragments that bind to or specifically bind to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques known to those skilled in the art. In some embodiments, an antibody or antigen-binding fragment binds to or specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigens as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response is at least twice the background signal or noise, and may be greater than 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In certain embodiments, the extent of binding of an antibody or antigen-binding fragment to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its specific target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. Terms such as "specific binding," "specifically binds to," or "specific for" refer to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target.Antibodies or antigen-binding fragments that bind to an antigen include those that can bind to the antigen with sufficient affinity so that the binding molecule is useful, for example, as a diagnostic agent in targeting the antigen. In certain embodiments, antibodies or antigen-binding fragments that bind to an antigen have an affinity of less than 1000 nM, less than 800 nM, less than 500 nM, less than 250 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM a dissociation constant (K) of less than 1000 nM, 800 nM, 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. D In certain embodiments, the antibody or antigen-binding fragment binds to an epitope of an antigen that is conserved among antigens from different species (e.g., between the human species and the cynomolgus monkey species).

[0086] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure. Specific exemplary embodiments include the following. In one embodiment, "K D " or "K DThe "K value" can be measured by assays known in the art, for example, binding assays. D K may be measured, for example, in an RIA performed using the Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81). D or K D The value may also be measured by using biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays, for example by Octet® using an Octet® QK384 system, or by Biacore® using, for example, a Biacore® TM-2000 or Biacore® TM-3000. "On-rate" or "rate of association" or "association rate" or "k" may also be determined using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, for example, using an Octet® QK384, Biacore® TM-2000, or Biacore® TM-3000 system.

[0087] In certain embodiments, antibodies or antigen-binding fragments can include "chimeric" sequences in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55).

[0088] In certain embodiments, an antibody or antigen-binding fragment can comprise a portion of a "humanized" form of a non-human (e.g., murine) antibody, which is a chimeric antibody comprising a human immunoglobulin (e.g., recipient antibody) in which native CDR residues are replaced by residues from a corresponding CDR (e.g., donor antibody) of a non-human species, such as mouse, rat, rabbit, or non-human primate, containing the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. The heavy or light chain of a humanized antibody can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-29; Presta, 1992, Curr. Op. Struct. Biol. 2:593-96; Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; U.S. Patent Nos. 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

[0089] In certain embodiments, an antibody or antigen-binding fragment can comprise a portion of a "fully human antibody" or "human antibody," which terms are used interchangeably herein and refer to an antibody comprising a human variable region and, for example, a human constant region. In specific embodiments, these terms refer to an antibody comprising variable and constant regions of human origin. A "fully human" antibody, in certain embodiments, can also encompass antibodies that bind to a polypeptide and are encoded by nucleic acid sequences that are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequences. The term "fully human antibody" includes antibodies comprising variable and constant regions that correspond to human germline immunoglobulin sequences described by Kabat et al. (See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or has been produced using any of the techniques for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581) and yeast display libraries (Chao et al., 2006, Nature Protocols 1:755-68). Methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77(1985); Boerner et al., 1991, J. Immunol. 147(1):86-95; and van Dijk and van de Winkel, 2001, Curr. Opin. Pharmacol. 5:368-74 can also be used to prepare human monoclonal antibodies.Human antibodies can be prepared by administering antigen to transgenic animals, e.g., mice, that have been engineered to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been disabled (see, e.g., Jakobovits, 1995, Curr. Opin. Biotechnol. 6(5):561-66; Bruggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; and U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li et al., 2006, Proc. Natl. Acad. Sci. USA 103:3557-62 regarding human antibodies generated via human B-cell hybridoma technology.

[0090] In certain embodiments, an antibody or antigen-binding fragment can comprise a portion of a "recombinant human antibody," which phrase includes human antibodies that are prepared, expressed, generated, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or a cow) that is transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo.

[0091] In certain embodiments, an antibody or antigen-binding fragment can comprise a portion of a "monoclonal antibody," a term used herein to refer to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations, and each monoclonal antibody typically recognizes a single epitope on an antigen. In specific embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to a particular method for producing the antibody. For example, monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma method first described by Kohler et al., 1975, Nature 256:495, or may be produced using recombinant DNA methods in bacterial or eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., 1991, Nature 352:624-28 and Marks et al., 1991, J. Mol. Biol. 222:581-97. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0092] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. The pairing of a VH and a VL forms a single antigen-binding site. The structure and properties of various classes of antibodies are described, for example, in Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds., 5th ed. 1995). th ed. 2001).

[0093] The term "Fab" or "Fab region" refers to the antibody region that binds to an antigen. Conventional IgGs usually contain two Fab regions, each present in one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region from each of the heavy and light chains. More specifically, the heavy chain variable and constant regions in the Fab region are the VH and CH1 regions, and the light chain variable and constant regions in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL regions within the Fab region can be arranged in various ways to confer antigen-binding capability according to the present disclosure. For example, as with the Fab region of a conventional IgG, the VH and CH1 regions can be on one polypeptide, while the VL and CL regions can be on separate polypeptides. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in different orders, as described in more detail in the following sections.

[0094] The terms "variable region," "variable domain," "V region," or "V domain" refer to the portion of an antibody light or heavy chain, generally located at the amino terminus of the light or heavy chain, approximately 120 to 130 amino acids in length for heavy chains and approximately 100 to 110 amino acids in length for light chains, that is used to determine the binding and specificity of each particular antibody for its specific antigen. The variable region of a heavy chain may be referred to as "VH." The variable region of a light chain may be referred to as "VL." The term "variable" refers to the fact that certain segments of the variable region vary significantly in sequence among antibodies. The V region mediates antigen binding and determines the specificity of a particular antibody for a particular antigen. However, variability is not uniformly distributed across the 110-amino acid span of the variable region. Instead, the V region consists of less variable (e.g., relatively invariant) stretches of approximately 15 to 30 amino acids called framework regions (FRs) separated by shorter regions of greater variability (e.g., extreme variability) called "hypervariable regions," each approximately 9 to 12 amino acids in length. The heavy and light chain variable regions each contain four FRs, primarily adopting a β-sheet configuration, connected by three hypervariable regions that form loops that connect and, in some cases, form part of the β-sheet structure. The hypervariable regions of each chain are held in close proximity together by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The sequences of the variable regions vary significantly among different antibodies. In a specific embodiment, the variable regions are human variable regions.

[0095] The terms "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of, or insertions into, FRs or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat numbered sequence at the regions of homology. The Kabat numbering system is commonly used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is commonly used when referring to residues within the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems are described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0096] The term "heavy chain," when used in reference to an antibody, refers to a polypeptide chain of approximately 50 to 70 kDa, the amino-terminal portion of which contains a variable region of approximately 120 to 130 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The constant region can be one of five different types (e.g., isotypes), designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the heavy chain constant region. Different heavy chains vary in size, with α, δ, and γ containing approximately 450 amino acids, and μ and ε containing approximately 550 amino acids. When combined with light chains, these different types of heavy chains give rise to five well-known classes (e.g., isotypes) of antibodies: IgA, IgD, IgE, IgG, and IgM, each of which contains four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4.

[0097] The term "light chain," when used in reference to an antibody, refers to a polypeptide chain of about 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion of which contains a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain.

[0098] As used herein, the terms "hypervariable region," "HVR," "complementarity-determining region," and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of an antibody VL β-sheet framework. Thus, CDRs are variable region sequences interspersed within framework region sequences.

[0099] CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra). Instead, Chothia refers to the position of the structural loop (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). When numbered using Kabat's numbering rules, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dubel eds., 2nd ed. 2010)). The "contact" hypervariable regions are based on the analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). IMGT is an integrated information system specialized for immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. Herein, CDRs are referred to both with respect to amino acid sequence and location within the light or heavy chain. Because the "locations" of CDRs within the structure of immunoglobulin variable domains are conserved across species and reside within structures called loops, CDR and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features.This information can be used to graft and replace CDR residues from one species' immunoglobulin onto an acceptor framework, typically from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, 2001, J. Mol. Biol. 309:657-70. For example, the correspondence between numbering systems, including the Kabat numbering and the IMGT-specific numbering system, is well known to those of skill in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are shown in Table 1 below.

[0100] (Table 1) TIFF2024534012000008.tif74161

[0101] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof, e.g., a variable region, and individual CDRs of an antibody or region thereof (e.g., "CDR-H1, CDR-H2"), should be understood to encompass complementarity determining regions defined by any of the known schemes described hereinabove. In some cases, a scheme for identifying a particular CDR or CDRs is specified, such as CDRs defined by the Kabat, Chothia, or Contact methods. In other cases, the specific amino acid sequences of the CDRs are given.

[0102] The hypervariable regions may include "extended hypervariable regions" such as: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0103] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that are not directly involved in binding an antibody to an antigen but exhibit various effector functions, such as interaction with Fc receptors. This term refers to the portion of an immunoglobulin molecule that contains a more conserved amino acid sequence compared to other portions of the immunoglobulin, the variable region, which contains the antigen-binding site. The constant region may include the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0104] The term "framework" or "FR" refers to variable region residues that flank the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region or CDR residues.

[0105] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is often defined to extend from the amino acid residue at position Cys226 or the amino acid residue at position Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations containing a mixture of antibodies with and without the K447 residue. A "functional Fc region" possesses the "effector functions" of a native-sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and the like. Such effector functions generally require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays known to those skilled in the art. A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution compared to the native-sequence Fc region or the Fc region of a parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, or from about 1 to about 5 amino acid substitutions in the native-sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein may have at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, for example, at least about 95% homology thereto.

[0106] As used herein, "epitope" is a term of art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody) can specifically bind. An epitope can be a linear epitope, a conformational epitope, a non-linear epitope, or a discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be contiguous amino acids of the polypeptide (a "linear" epitope), or it can include amino acids from two or more discontinuous regions of the polypeptide (a "conformational," "non-linear," or "discontinuous" epitope). In general, those skilled in the art will understand that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids regardless of whether they are folded into a native three-dimensional protein structure. In other embodiments, the binding molecule requires the amino acid residues that make up the epitope to adopt a particular conformation (eg, a bend, twist, turn, or fold) in order to recognize and bind to the epitope.

[0107] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified, naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are polypeptides containing one or more analogs of an amino acid, including, but not limited to, unnatural amino acids, as well as other modifications known in the art. Because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, it is understood that in certain embodiments, a "polypeptide" can exist as a single chain or as two or more associated chains.

[0108] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, and more particularly in humans.

[0109] "Excipient" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives such as absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, humectants, lubricants, fragrances, preservatives, propellants, releasing agents, sterilizing agents, sweeteners, solubilizing agents, wetting agents, and mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle.

[0110] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other components of the pharmaceutical formulation and suitable for use in contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0111] The abbreviation "MMAE" refers to monomethyl auristatin E.

[0112] Unless the context dictates otherwise, a hyphen (-) designates the point of attachment to the pendant molecule.

[0113] The term "chemotherapeutic agent" refers to any chemical compound that is effective in inhibiting tumor growth. Non-limiting examples of chemotherapeutic agents include alkylating agents, such as nitrogen mustard, ethyleneimine compounds, and alkylsulfonates; antimetabolites, such as folic acid, purine, or pyrimidine antagonists; mitotic inhibitors, such as antitubulin agents, such as derivatives of vinca alkaloids, auristatins, and podophyllotoxins; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, such as DNA minor groove binders; and growth factor receptor antagonists. In addition, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biological molecules, and small molecules.

[0114] As used herein, the term "conservative substitution" is known to those skilled in the art and generally refers to an amino acid substitution that may be made without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin / Cummings Pub. Co., p. 224 (4th Edition 1987)). Such exemplary substitutions are preferably made in accordance with the substitutions shown in Tables 2 and 3. For example, such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; substituting glutamic acid (E) with aspartic acid (D) and vice versa; substituting asparagine (N) with glutamine (Q) and vice versa; and substituting threonine (T) with serine (S) and vice versa. Other substitutions can also be considered conservative, depending on the particular amino acid's environment and its role in the protein's three-dimensional structure. For example, glycine (G) and alanine (A) can often be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can often be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are often interchangeable in positions where the important feature of an amino acid residue is its charge, and the different pKs of these two amino acid residues are not important. Still other changes may be considered "conservative" in particular circumstances (see, e.g., Table 3 herein; pages 13-15 of "Biochemistry," 2nd ed., Lubert Stryer ed. (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19;270(20):11882-11886). Other substitutions are permissible and may be determined empirically or in accordance with known conservative substitutions.

[0115] Table 2: Amino acid abbreviations TIFF2024534012000009.tif140157

[0116] Table 3: Amino acid substitution or similarity matrix Adapted from GCG software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely the substitution is found in related native proteins. TIFF2024534012000010.tif129135

[0117] The term "homology" or "homologous" is intended to refer to the sequence similarity between two polynucleotides or two polypeptides. Similarity can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a given position in two polypeptide sequences is not identical, the similarity or conservation of that position can be determined by evaluating the amino acid similarity at that position, for example, according to Table 3. The degree of similarity between sequences is a function of the number of matching or homologous positions shared by the sequences. Alignment of two sequences to determine percent sequence similarity can be performed using software programs known in the art, such as those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). Preferably, default parameters are used for alignment, an example of which is provided below. One alignment program known in the art that can be used is BLAST set to default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort order = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. More information about these programs can be found at the National Center for Biotechnology Information.

[0118] The term "homolog" of a given amino acid or nucleic acid sequence is intended to indicate a corresponding sequence of the "homolog" having substantial identity or homology to the given amino acid or nucleic acid sequence.

[0119] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed, for example, using the NBLAST nucleotide program parameters set to score=100 and word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using, for example, XBLAST program parameters set to score 50 and word length = 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17.Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0120] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0121] The term "cytotoxic agent" refers to a substance that inhibits or prevents the development, activity, function, and / or causes destruction of cells. This term is intended to include radioisotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants. Examples of cytotoxic agents include auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureomycin, maytansinoids, ricin, ricin A chain, combrestatins, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α-sarcin, gelonin, mitogenin, restrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, soapwort (Sapaonaria officinalis) inhibitors, and glucocorticoids and other chemotherapeutic agents, as well as At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 or Bi213 , P 32 Radioactive isotopes such as Lu 177 Examples of suitable anti-cancer prodrug-activating enzymes include, but are not limited to, radioactive isotopes of Lu, including:

[0014] The antibodies may also be conjugated to anti-cancer prodrug-activating enzymes capable of converting a prodrug to its active form.

[0122] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a binding molecule (e.g., an antibody) or pharmaceutical composition provided herein sufficient to effect a desired result.

[0123] The terms "subject" and "patient" may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, who has been diagnosed with a condition or disorder. In another embodiment, a subject is a mammal, e.g., a human, who is at risk of developing a condition or disorder.

[0124] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body to a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0125] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of a disease or condition resulting from the administration of one or more therapies. Treatment may be determined by assessing whether there has been a reduction, alleviation, and / or relief of one or more symptoms associated with the underlying disease, such that an improvement is observed for the patient, even though the patient may still be suffering from the underlying disease. The term "treating" includes both management and amelioration of disease. The terms "manage," "managing," and "management" refer to the beneficial effects a subject derives from a therapy that does not necessarily result in a cure of the disease.

[0126] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of the occurrence (or recurrence) of a disease, disorder, condition, or one or more associated symptoms (e.g., cancer).

[0127] The terms "cancer" or "cancer cells" are used herein to refer to tissue or cells found in neoplasms that have characteristics that distinguish them from normal tissue or tissue cells. Such characteristics include, but are not limited to, the degree of anaplasia, irregular shape, unclear cell outlines, nuclear size, changes in nuclear or cytoplasmic structure, other phenotypic changes, the presence of cellular proteins indicative of cancer or precancerous conditions, an increased number of mitoses, and the ability to metastasize. Words related to "cancer" include carcinoma, sarcoma, tumor, epithelioma, leukemia, lymphoma, polyp, and scirrhous carcinoma, transformation, neoplasia, etc.

[0128] As used herein, "locally advanced" cancer refers to cancer that has spread from where it began to nearby tissues or lymph nodes.

[0129] As used herein, "metastatic" cancer refers to cancer that has spread from where it began to another part of the body.

[0130] The term "intravesical administration" refers to the infusion of a therapeutic agent directly into the bladder via insertion of a urethral catheter.

[0131] The term "residence time" refers to the length of time that a therapeutic substance is retained in a particular part or organ (eg, the bladder) of a treated subject.

[0132] The terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0133] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0134] Whenever an embodiment is described herein using the term "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. Also, whenever an embodiment is described herein using the phrase "consisting essentially of," it is understood that other similar embodiments described in terms of "consisting of" are also provided.

[0135] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0136] The term "variant" refers to a molecule that exhibits variation from a described type or reference, such as a protein with one or more different amino acid residues at the corresponding position(s) of a specifically described protein (e.g., the 191P4D12 protein shown in Figure 1A). Analogs are an example of a variant protein. Splice isoforms and single nucleotide polymorphisms (SNPs) are further examples of variants.

[0137] "191P4D12 proteins" and / or "191P4D12-related proteins" of the present disclosure include those specifically identified herein (see FIG. 1A), as well as allelic variants, conservatively substituted variants, analogs, and homologs, which can be isolated / produced and characterized without undue experimentation according to the methods outlined herein or readily available in the art. Also included are fusion proteins that combine portions or fragments of different 191P4D12 proteins, and fusion proteins of a 191P4D12 protein with a heterologous polypeptide. Such 191P4D12 proteins are collectively referred to as 191P4D12-related proteins, proteins of the present disclosure, or 191P4D12. The term "191P4D12-related protein" refers to a 191P4D12-related protein that is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids, or at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least The term "191P4D12" refers to a polypeptide fragment or 191P4D12 protein sequence of at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 330, at least 335, at least 339 or more amino acids. The term "191P4D12" is used interchangeably with Nectin-4.

[0138] 5.2 Treatment of Non-Muscle-Invasive Bladder Cancer (NMIBC) in Selected Patients Provided herein are methods for treating bladder cancer in a human subject via intravesical administration of an antibody drug conjugate (ADC) that binds to 191P4D12.

[0139]

[0013] In one aspect, provided herein is a method of treating bladder cancer in a human subject, comprising intravesically administering to the subject an effective amount of an antibody drug conjugate (ADC), wherein the ADC: An antibody or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethyl auristatin E (MMAE) The present invention provides a method comprising:

[0140] In some embodiments, the bladder cancer is non-muscle invasive bladder cancer (NMIBC). In some embodiments, the NMIBC is histologically confirmed. In some embodiments, the NMIBC is carcinoma in situ (CIS). In some embodiments, the NMIBC is histologically confirmed as carcinoma in situ (CIS). In certain embodiments, the subject has papillary disease. In certain embodiments, the subject does not have papillary disease. In certain embodiments, the NMIBC is histologically confirmed, with the predominant histological component (>50%) being urothelial (transitional cell) carcinoma.

[0141] In some embodiments, the human subject treated with the methods provided herein has high-risk Bacillus Calmette-Guérin (BCG)-unresponsive disease. In certain embodiments, high-risk BCG-unresponsive disease is defined as persistent or recurrent CIS alone or recurrent Ta / T1 (non-invasive papillary disease / tumor invading the subepithelial connective tissue) disease within 12 months of completing adequate BCG therapy. In certain embodiments, adequate BCG therapy is defined as five of six initial induction courses plus at least two of three maintenance courses. In certain embodiments, adequate BCG therapy is defined as five of six initial induction courses plus at least two of six second induction courses.

[0142] In some embodiments, the human subject treated with the methods provided herein is ineligible for radical cystectomy, hi some embodiments, the human subject treated with the methods provided herein refuses to undergo radical cystectomy.

[0143] In some embodiments, the subject has had all visible papillary Ta / T1 tumors completely resected within 60 days prior to said treatment. In some embodiments, the subject has residual pure CIS. In some embodiments, the subject does not have residual pure CIS.

[0144] In some embodiments, a human subject treated with the methods provided herein has satisfactory bladder function and the ability to sustain an infusion of an ADC provided herein for at least 1 hour, even with premedication. In some embodiments, the human subject is at least 18 years old. In some embodiments, the human subject has an estimated life expectancy of greater than 2 years.

[0145] In some embodiments, a human subject treated with the methods provided herein has an Eastern Cooperative Oncology Group (ECOG) performance status score of 0. In some embodiments, a human subject treated with the methods provided herein has an Eastern Cooperative Oncology Group (ECOG) performance status score of 1. In some embodiments, a human subject treated with the methods provided herein has an Eastern Cooperative Oncology Group (ECOG) performance status score of 2. In some embodiments, a human subject treated with the methods provided herein has an Eastern Cooperative Oncology Group (ECOG) performance status score of 2, the subject's glomerular filtration rate (GFR) is 50 mL / min or greater, and the subject does not have New York Heart Association (NYHA) Class III heart failure.

[0146] In further embodiments of the methods provided herein, including the method of the preceding paragraph, human subjects for whom the methods provided herein can be used are human subjects with a variety of other conditions. In some embodiments, a human subject treated with the methods provided herein has an absolute neutrophil count (ANC) of 1500 / μL or greater. In some embodiments, a human subject treated with the methods provided herein has a hemoglobin (Hgb) of 10 g / dL or greater. In some embodiments, a human subject treated with the methods provided herein has a platelet count of 100,000 / μL or greater. In some embodiments, a human subject treated with the methods provided herein has a serum bilirubin of 1.5×ULN or less of normal (ULN), or 3×ULN or less in the case of subjects with Gilbert's disease. In some embodiments, a human subject treated with the methods provided herein has a calculated creatinine clearance (CrCl) of 30 mL / min or greater. In some embodiments, CrCl is calculated using the Cockcroft-Gault method or the Modified Diet in Renal Disease (MDRD) formula. In some aspects, a human subject treated with the methods provided herein has a GFR of 30 mL / min or greater. In some embodiments, a human subject treated with the methods provided herein has an ECOG activity status of 2 and a GFR of 50 mL / min or greater. In some embodiments, a human subject treated with the methods provided herein has an alanine aminotransferase (ALT) and aspartate aminotransferase (AST) status of 3×ULN or less. In some embodiments, a human subject treated with the methods provided herein has an international normalized ratio (INR), or a prothrombin time (PT), activated partial thromboplastin time (aPTT), or partial thromboplastin time (PTT) of 1.5 ULN or less, except in the case of a human subject receiving anticoagulant therapy, so long as the PT or aPTT is within the therapeutic range for the intended use of the anticoagulant. In some embodiments, a human subject treated with the methods provided herein has more than one of the conditions described in this paragraph.In some embodiments, the human subject treated with the methods provided herein has all of the conditions described in this paragraph.

[0147] In further embodiments of the methods provided herein, including the method of the preceding paragraph, the human subject in whom the methods provided herein can be used is a human subject without a specific condition. In some embodiments, the human subject treated with the methods provided herein does not have a current or previous history of muscle-invasive urothelial carcinoma (i.e., T2, T3, or T4 disease) or metastatic disease. In some embodiments, the human subject treated with the methods provided herein does not have nodal or metastatic disease as shown by computed tomography (CT) or magnetic resonance imaging (MRI) performed within three months prior to treatment with an ADC. In some embodiments, the human subject treated with the methods provided herein does not have concomitant upper tract urothelial carcinoma as shown by a CT or MRI urogram with abdominal / pelvic imaging performed within three months prior to treatment with an ADC. In some embodiments, the human subject treated with the methods provided herein does not have a history of or concomitant urothelial carcinoma of the prostatic urethra within six months prior to treatment with an ADC. In some embodiments, a human subject treated with the methods provided herein does not have tumor-associated hydronephrosis prior to administration of the ADC. In some embodiments, a human subject treated with the methods provided herein has not received systemic anti-cancer therapy (e.g., chemotherapy, biologic therapy, immunotherapy, targeted therapy, endocrine therapy, investigational drug) within 4 weeks of the first dose of treatment with the methods provided herein, or any intravesical therapy for the treatment of NMIBC within 6 weeks prior to the initiation of treatment with the methods provided herein. In some embodiments, a human subject treated with the methods provided herein receives a single infusion of a cytotoxic agent (e.g., mitomycin C, doxorubicin, and gemcitabine) immediately after the TURBT procedure within 14 to 60 days prior to the initiation of treatment with the methods provided herein. In some embodiments, a human subject treated with the methods provided herein does not have symptoms (grade 2 or higher) secondary to an adverse event (AE) associated with a prior treatment for NMIBC. In some embodiments, a human subject treated with the methods provided herein has not previously received bladder radiation for the treatment of urothelial carcinoma.In some embodiments, a human subject treated with the methods provided herein does not have an active infection, and the subject is treated with a systemic (e.g., oral or intravenous) antibiotic within 14 days prior to initiating treatment with the ADC. In some embodiments, a human subject treated with the methods provided herein is resistant to intravesical administration or intravesical surgical manipulation. In some embodiments, a human subject treated with the methods provided herein does not have a history of malignancy or any evidence of residual disease from a previously diagnosed malignancy within three years prior to treatment with the methods provided herein. In some embodiments, a human subject treated with the methods provided herein has a negligible risk of metastasis or death (e.g., a 5-year overall survival rate [OS] of ≥ 90%), such as adequately treated cervical CIS, non-melanoma skin cancer, breast ductal CIS, or stage I uterine cancer. In some embodiments, a human subject treated with the methods provided herein has a history of prostate cancer (T2N0M0 or lower, Gleason score ≦7) that was treated with definite intent (surgery or radiation therapy) at least one year prior to treatment with the methods provided herein, provided that the subject is not considered to have prostate cancer, and the following criteria are met: (1) for subjects who have undergone radical prostatectomy, the prostate-specific antigen (PSA) is undetectable for more than one year prior to administration of the ADC; and (2) for subjects who have been irradiated, the PSA doubling time is greater than one year (based on at least three values ​​measured more than one month apart) and the total PSA value does not meet the Phoenix criteria for biochemical recurrence (i.e., <2.0 ng / mL from the nadir). In some embodiments, a human subject treated with the methods provided herein has never been exposed to a Nectin-4 targeted therapy or a monomethyl auristatin E (MMAE)-containing agent. In some embodiments, a human subject treated with the methods provided herein does not have an autoimmune or inflammatory skin disorder. In some embodiments, the human subject treated with the methods provided herein does not have psoriasis or atopic dermatitis. In some embodiments, the human subject treated with the methods provided herein does not have ongoing sensory or motor neuropathy Grade 2 or higher.In some embodiments, a human subject treated with the methods provided herein does not have positive hepatitis B surface antigen and / or anti-hepatitis B core antibodies. In some embodiments, a human subject treated with the methods provided herein has a negative polymerase chain reaction (PCR) assay for hepatitis B and is on appropriate antiviral prophylaxis. In some embodiments, a human subject treated with the methods provided herein does not have active hepatitis C infection or known human immunodeficiency virus (HIV) infection. In some embodiments, a human subject treated with the methods provided herein does not have active tuberculosis. In some embodiments, a human subject treated with the methods provided herein does not have uncontrolled diabetes. In some embodiments, uncontrolled diabetes is defined as a subject with a hemoglobin A1c (HbA1c) of ≥ 8%, or an HbA1c of < 7%-8%, accompanied by associated diabetic symptoms (polyuria or polydipsia). In some embodiments, a human subject treated with the methods provided herein does not have a cerebrovascular event (e.g., stroke or transient ischemic attack), unstable angina, myocardial infarction, or cardiac condition consistent with NYHA class III-IV within 6 months prior to the first administration of the ADC. In some embodiments, a human subject treated with the methods provided herein does not have severe (≧Grade 3) hypersensitivity to enfortumab vedotin or any excipients contained in the drug formulation of enfortumab vedotin (e.g., histidine, trehalose dihydrate, and / or polysorbate 20). In some embodiments, a human subject treated with the methods provided herein does not have active keratitis or corneal ulcers. In some embodiments, a human subject treated with the methods provided herein has superficial punctate keratitis.

[0148] In certain embodiments, the methods provided herein are used to treat a subject with non-muscle-invasive bladder cancer (NMIBC) that expresses 191P4D12 RNA, expresses 191P4D12 protein, or expresses both 191P4D12 RNA and 191P4D12 protein.

[0149] In some embodiments, 191P4D12 RNA expression in cancer is determined by polynucleotide hybridization, sequencing (to assess relative abundance of sequences), and / or PCR (including RT-PCR). In some embodiments, 191P4D12 protein expression in cancer is determined by IHC, analysis in fluorescence-activated cell sorting (FACS), and / or Western blotting. In some embodiments, 191P4D12 protein expression in cancer is determined by more than one method. In some embodiments, 191P4D12 protein expression in cancer is determined by two methods: IHC.

[0150] In some embodiments, non-muscle-invasive bladder cancer (NMIBC) is confirmed histologically, cytologically, or both histologically and cytologically.

[0151] In another aspect, provided herein is a method of treating NMIBC in a human subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate: An antibody or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethyl auristatin E (MMAE) wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO: 22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 23, wherein the subject has any suitable characteristic provided in Section 6.

[0152] In another aspect, provided herein is a method of preventing or treating cancer in a human subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate: An antibody or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethyl auristatin E (MMAE) wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO: 22 and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 23, wherein the cancer has any suitable marker and / or characteristic provided in Section 6.

[0153] In yet another aspect, provided herein is a method of preventing or treating cancer in a human subject, comprising administering to the subject an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate: An antibody or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethyl auristatin E (MMAE) wherein the subject has any suitable characteristic provided in Section 6. In a further aspect, provided herein is a method of preventing or treating cancer in a human subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate: An antibody or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethyl auristatin E (MMAE) wherein the cancer has any suitable marker and / or characteristic provided in Section 6.

[0154] In all of the methods provided herein, particularly those described in the preceding paragraphs, ADCs that may be used are described in Sections 3, 5.2, 5.3, 5.4, 5.5, 5.6, and 6; patient selection for treatment is described herein and exemplified in this section (Section 5.2) and Sections 3 and 6; dosing regimens and pharmaceutical compositions for administering therapeutic agents are described in Sections 5.4, 5.6, and 6, below, and can be used to identify therapeutic agents, select patients, determine the outcome of these methods, and / or serve as criteria for these methods. Biomarkers are described herein and exemplified in this section and Section 6, biomarkers are determined in Section 5.7 or as known in the art, treatment outcomes for the methods provided herein are described in this section (Section 5.2) and Sections 3 and 6, additional treatment outcomes for the methods provided herein can be an improvement in a biomarker described herein, e.g., those described and exemplified in this section (Section 5.2) and Sections 3 and 6, and combination therapies including ADCs and other therapeutic agents are described in this section and Section 5.5. Accordingly, one of skill in the art will understand that the methods provided herein include all permutations and combinations of the patients, therapeutic agents, dosing regimens, biomarkers, and treatment outcomes described above and below.

[0155] 5.3 Antibody Drug Conjugates for the Method In various embodiments of the methods provided herein, including those provided in Section 5.2, the ADC used in the method comprises or is an anti-191P4D12 ADC described herein and / or in U.S. Patent No. 8,637,642, which is incorporated by reference in its entirety. In some embodiments, the anti-191P4D12 antibody-drug conjugates provided for the methods herein comprise an antibody or antigen-binding fragment thereof that binds to 191P4D12, as provided herein, including those in Sections 3, 5.3.1, and 6, conjugated to one or more units (drug units, or D) of a cytotoxic agent, as provided herein, including those in this section (Section 5.3), including the further disclosure in Sections 3 and 6 and 5.3.2 and 5.3.4. In certain embodiments, the cytotoxic agent (Drug Unit, or D) may be covalently attached directly or via a Linker Unit (LU) as provided herein, including those in this section (Section 5.3), including the further disclosure in Sections 3 and 6 and Section 5.3.3.

[0156] In some embodiments, the antibody drug conjugate compound has the following formula: L-(LU-D) p (I) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody unit, e.g., an anti-Nectin-4 antibody or antigen-binding fragment thereof, e.g., as provided in Sections 3, 5.3.1, and 6; (LU-D) is the Linker Unit-Drug Unit moiety, wherein: LU- is a linker unit as provided in this section (Section 5.3), e.g., including the further disclosure in Sections 3 and 6 and Section 5.3.3; D is a Drug Unit having cytostatic or cytotoxic activity against a target cell, e.g., as provided in this Section (Section 5.3), including the further disclosure in Sections 3 and 6 and Sections 5.3.2 and 5.3.4; p is an integer from 1 to 20, including further examples provided in Sections 3 and 6 and this section (Section 5.3).

[0157] In some embodiments, p is in the range of 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is in the range of 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p is in the range of 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, or 3 to 4. In some embodiments, p is about 1. In some embodiments, p is about 2. In some embodiments, p is about 3. In some embodiments, p is about 4. In some embodiments, p is about 3.8. In some embodiments, p is about 5. In some embodiments, p is about 6. In some embodiments, p is about 7. In some embodiments, p is about 8. In some embodiments, p is about 9. In some embodiments, p is about 10. In some embodiments, p is about 11. In some embodiments, p is about 12. In some embodiments, p is about 13. In some embodiments, p is about 14. In some embodiments, p is about 15. In some embodiments, p is about 16. In some embodiments, p is about 17. In some embodiments, p is about 18. In some embodiments, p is about 19. In some embodiments, p is about 20.

[0158] In some embodiments, the antibody drug conjugate compound has the following formula: L-(A a -W w -Y y -D) p (II) or a pharmaceutically acceptable salt or solvate thereof, wherein L is an antibody unit, e.g., an anti-Nectin-4 antibody or antigen-binding fragment thereof, e.g., as provided in Sections 3, 5.3.1, and 6; -A a -W w -Y y - is a linker unit (LU), wherein -A- is an extender unit, a is 0 or 1, each -W- is independently an amino acid unit; w is an integer ranging from 0 to 12, -Y- is a self-immolative spacer unit; y is 0, 1, or 2; each as provided in this section (Section 5.3), including, for example, Sections 3 and 6 and the further disclosure in Section 5.3.3; D is a Drug Unit having cytostatic or cytotoxic activity against a target cell, e.g., as provided in this Section (Section 5.3), including the further disclosure in Sections 3 and 6 and Sections 5.3.2 and 5.3.4; p is an integer from 1 to 20, including further examples provided in Sections 3 and 6 and this section (Section 5.3).

[0159] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1, or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, p is in the range of 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is in the range of 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p is in the range of 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, or 3 to 4. In some embodiments, p is about 1. In some embodiments, p is about 2. In some embodiments, p is about 3. In some embodiments, p is about 4. In some embodiments, p is about 3.8. In some embodiments, p is about 5. In some embodiments, p is about 6. In some embodiments, p is about 7. In some embodiments, p is about 8. In some embodiments, p is about 9. In some embodiments, p is about 10. In some embodiments, p is about 11. In some embodiments, p is about 12. In some embodiments, p is about 13. In some embodiments, p is about 14. In some embodiments, p is about 15. In some embodiments, p is about 16. In some embodiments, p is about 17. In some embodiments, p is about 18. In some embodiments, p is about 19. In some embodiments, p is about 20. In some embodiments, when w is not 0, y is 1 or 2. In some embodiments, when w is 1-12, y is 1 or 2. In some embodiments, w is 2-12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

[0160] In some specific embodiments of the methods provided herein, including those provided in Section 5.2, the cytotoxic agent as part of any of the ADCs provided herein for the methods comprises, consists of, or is MMAE.

[0161] For compositions comprising multiple antibodies or antigen-binding fragments thereof, drug loading is represented by p, the average number of drug molecules per antibody unit. Drug loading can range from 1 to 20 drugs (D) per antibody. The average number of drugs per antibody in a conjugation reaction preparation can be characterized by conventional means, such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of antibody-drug conjugates with respect to p can also be determined. In some cases, separation, purification, and characterization of homogeneous antibody-drug conjugates with a constant p from antibody-drug conjugates with other drug loads can be performed by means such as reverse-phase HPLC or electrophoresis. In certain exemplary embodiments, p is 2 to 8.

[0162] Additional embodiments of ADCs for the methods provided herein are described in U.S. Pat. No. 8,637,642 and International Application No. PCT / US2019 / 056214 (Publication No. WO2020 / 117373), both of which are incorporated by reference herein in their entireties.

[0163] In some embodiments of the methods provided herein, including Sections 3, 5.2, and 6 and this section (Section 5.3), the ADC is enfortumab vedotin. In certain embodiments of the methods provided herein, including Sections 3, 5.2, and 6 and this section (Section 5.3), the ADC is a biosimilar of enfortumab vedotin.

[0164] 5.3.1 Anti-191P4D12 Antibodies or Antigen-Binding Fragments In one embodiment, the antibody or antigen-binding fragment that binds to a Nectin-4-related protein is an antibody or antigen-binding fragment that specifically binds to a Nectin-4 protein comprising the amino acid sequence of SEQ ID NO: 2 (see Figure 1A). The corresponding cDNA encoding the 191P4D12 protein has the sequence of SEQ ID NO: 1 (see Figure 1A).

[0165] Antibodies that specifically bind to a Nectin-4 protein comprising the amino acid sequence of SEQ ID NO: 2 include antibodies that can bind to other Nectin-4-related proteins. For example, an antibody that binds to a Nectin-4 protein comprising the amino acid sequence of SEQ ID NO: 2 can bind to Nectin-4-related proteins such as Nectin-4 variants and their homologs or analogs.

[0166] In some embodiments, the anti-Nectin-4 antibodies provided herein are monoclonal antibodies.

[0167] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:4 (cDNA sequence of SEQ ID NO:3) and / or a light chain comprising the amino acid sequence of SEQ ID NO:6 (cDNA sequence of SEQ ID NO:5), as shown in Figures 1B and 1C.

[0168] In some embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR having the amino acid sequence of the complementarity-determining region (CDR) of the heavy chain variable region set forth in SEQ ID NO: 22 (which is the amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 136th amino acid (serine) of SEQ ID NO: 7), and a light chain variable region comprising a CDR having the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 23 (which is the amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) of SEQ ID NO: 8). In certain embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity-determining region 1 (CDR-H1), CDR-H2, and CDR-H3, which contain the corresponding amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region sequence shown in SEQ ID NO: 22 (which is the amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 136th amino acid (serine) of SEQ ID NO: 7), and a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3, which contain the corresponding amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region sequence shown in SEQ ID NO: 23 (which is the amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) of SEQ ID NO: 8). In some embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR consisting of the amino acid sequence of the complementarity-determining region (CDR) of the heavy chain variable region set forth in SEQ ID NO: 22 (which is the amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 136th amino acid (serine) of SEQ ID NO: 7), and a light chain variable region comprising a CDR consisting of the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 23 (which is the amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) of SEQ ID NO: 8).In certain embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region including complementarity-determining region 1 (CDR-H1), CDR-H2, and CDR-H3 consisting of the corresponding amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 in the heavy chain variable region sequence shown in SEQ ID NO: 22 (which is the amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 136 (serine) of SEQ ID NO: 7), and a light chain variable region including CDR-L1, CDR-L2, and CDR-L3 consisting of the corresponding amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 in the light chain variable region sequence shown in SEQ ID NO: 23 (which is the amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 130 (arginine) of SEQ ID NO: 8). SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 7, and SEQ ID NO: 8 are shown in Figures 1D and 1E and are listed below. SEQ ID NO: 22 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSS SEQ ID NO: 23 DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIKR SEQ ID NO:7 MELGLCWVFLVAILEGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCA RAYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKR VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:8 MDMRVPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPP TFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0169] CDR sequences can be determined according to well-known numbering systems. As mentioned above, CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (see, for example, Kabat et al., supra). Instead, Chothia refers to the position of the structural loop (see, for example, Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). When numbered using Kabat's numbering rules, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dubel eds., 2nd ed. 2010)). The "contact" hypervariable regions are based on the analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). IMGT is an integrated information system specialized for immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. Herein, CDRs are referred to both with respect to amino acid sequence and location within the light or heavy chain. Because the "locations" of CDRs within the structure of immunoglobulin variable domains are conserved across species and reside within structures called loops, CDR and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features.This information can be used to graft and replace CDR residues from one species' immunoglobulin onto an acceptor framework, typically from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, 2001, J. Mol. Biol. 309:657-70. For example, the correspondence between numbering systems, including the Kabat numbering and the IMGT-specific numbering system, is well known to those of skill in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are set forth in Table 1 above.

[0170] In some embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO: 22 according to the Kabat numbering, and a light chain variable region including CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to the Kabat numbering.

[0171] In some embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO: 22 according to AbM numbering, and a light chain variable region including CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to AbM numbering.

[0172] In other embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO: 22 according to Chothia numbering, and a light chain variable region including CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to Chothia numbering.

[0173] In other embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO: 22 by Contact numbering, and a light chain variable region including CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 by Contact numbering.

[0174] In still other embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO: 22 according to the IMGT numbering system, and a light chain variable region including CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to the IMGT numbering system.

[0175] In some embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) consisting of the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO: 22 according to the Kabat numbering, and a light chain variable region including CDRs consisting of the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to the Kabat numbering.

[0176] In some embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) consisting of the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO: 22 according to AbM numbering, and a light chain variable region including CDRs consisting of the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to AbM numbering.

[0177] In another embodiment, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) consisting of the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO: 22 according to Chothia numbering, and a light chain variable region including CDRs consisting of the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to Chothia numbering.

[0178] In other embodiments, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) consisting of the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO: 22 by Contact numbering, and a light chain variable region including CDRs consisting of the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 by Contact numbering.

[0179] In still another embodiment, the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) consisting of the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO: 22 according to the IMGT numbering system, and a light chain variable region including CDRs consisting of the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to the IMGT numbering system.

[0180] As noted above, CDR sequences according to different numbering systems can be readily determined using online tools such as those provided by the Antigen Receptor Numbering and Receptor Classification (ANARCI). For example, the heavy chain CDR sequences in SEQ ID NO: 22 and the light chain CDR sequences in SEQ ID NO: 23 according to the Kabat numbering as determined by ANARCI are listed in Table 4 below.

[0181] (Table 4) TIFF2024534012000011.tif26159

[0182] As another example, the heavy chain CDR sequences in SEQ ID NO: 22 and the light chain CDR sequences in SEQ ID NO: 23 according to the IMGT numbering as determined by ANARCI are listed in Table 5 below.

[0183] (Table 5) TIFF2024534012000012.tif26159

[0184] In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

[0185] In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21.

[0186] In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 9, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 10, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 11, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 12, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 13, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 14.

[0187] In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 16, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 17, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 18, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 19, CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 20, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 21.

[0188] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:23.

[0189] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO:22 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:23.

[0190] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7, and a light chain comprising the amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8.

[0191] In some embodiments, the antibody comprises a heavy chain consisting of the amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7, and a light chain consisting of the amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8.

[0192] In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody, including, but not limited to, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Thus, in addition to the antibodies described herein, it is contemplated that antibody variants can be prepared. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesis of the desired antibody or polypeptide. Those skilled in the art will recognize that amino acid changes can alter post-translational processing of the antibody, for example, by changing the number or position of glycosylation sites or altering membrane anchoring properties.

[0193] In some embodiments, the antibodies provided herein are chemically modified, for example, by covalent attachment of any type of molecule to the antibody. Antibody derivatives can include antibodies chemically modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody can contain one or more non-classical amino acids.

[0194] Mutations can be substitutions, deletions, or insertions of one or more codons encoding a single domain antibody or polypeptide, resulting in a change in the amino acid sequence compared to the original antibody or polypeptide. Amino acid substitutions can be, for example, conservative amino acid substitutions, resulting from the replacement of one amino acid with another amino acid containing similar structural and / or chemical properties, such as the replacement of a leucine with a serine. Standard techniques known to those of skill in the art can be used to introduce mutations into the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Insertions or deletions can range from about 1 to 5 amino acids. In certain embodiments, substitutions, deletions, or insertions comprise fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the original molecule. In specific embodiments, substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Acceptable mutations can be determined by systematically inserting, deleting, or substituting amino acids in a sequence and testing the resulting variants for activity exhibited by the parent antibody.

[0195] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue.

[0196] Antibodies generated by conservative amino acid substitutions are included in the present disclosure. In conservative amino acid substitutions, an amino acid residue is replaced with an amino acid residue containing a side chain with a similar charge. As described above, families of amino acid residues containing side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed, the activity of the protein can be determined, and conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) can be made to maintain or not significantly change the properties.

[0197] Amino acids can be grouped according to similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0198] For example, any cysteine ​​residue not involved in maintaining the proper conformation of the antibody may be substituted with another amino acid, e.g., alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

[0199] Modifications can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, 1986, Biochem J. 237:1-7; and Zoller et al., 1982, Nucl. Acids Res. 10:6487-500), cassette mutagenesis (see, e.g., Wells et al., 1985, Gene 34:315-23), or other known techniques can be performed on cloned DNA to produce anti-anti-MSLN antibody variant DNA.

[0200] Covalent modifications of antibodies are included within the scope of the present disclosure. Covalent modifications include reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or the N- or C-terminal residues of the antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0201] Other types of covalent modifications of antibodies within the scope of the present disclosure include altering the native glycosylation pattern of the antibody or polypeptide (see, e.g., Beck et al., 2008, Curr. Pharm. Biotechnol. 9:482-501; and Walsh, 2010, Drug Discov. Today 15:773-80), and linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, e.g., by methods described in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.

[0202] In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain that has a particular homology or identity to the heavy chain set forth in SEQ ID NO:7 and a light chain that has a particular homology or identity to the light chain set forth in SEQ ID NO:8. Embodiments of heavy / light chains with such homology or identity are further set forth below. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain that has greater than 70% homology or identity to the heavy chain set forth in SEQ ID NO:7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain that has greater than 75% homology or identity to the heavy chain set forth in SEQ ID NO:7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain that has greater than 80% homology or identity to the heavy chain set forth in SEQ ID NO:7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain that has greater than 85% homology or identity to the heavy chain set forth in SEQ ID NO:7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 90% homology or identity to the heavy chain set forth in SEQ ID NO: 7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 95% homology or identity to the heavy chain set forth in SEQ ID NO: 7. In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having any of the indicated homology or identity to the heavy chain set forth in SEQ ID NO: 7, and its CDRs (CDR-H1, CDR-H2, and CDR-H3) are identical to the CDRs in the heavy chain set forth in SEQ ID NO: 7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 70% homology or identity to the light chain set forth in SEQ ID NO: 8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 75% homology or identity to the light chain set forth in SEQ ID NO: 8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 80% homology or identity to the light chain set forth in SEQ ID NO:8.In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 85% homology or identity to the light chain set forth in SEQ ID NO: 8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 90% homology or identity to the light chain set forth in SEQ ID NO: 8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 95% homology or identity to the light chain set forth in SEQ ID NO: 8. In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having any of the indicated homology or identity to the light chain set forth in SEQ ID NO: 8, and whose CDRs (CDR-L1, CDR-L2, and CDR-L3) are identical to the CDRs in the light chain set forth in SEQ ID NO: 8. In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise any homologous light chain and any homologous heavy chain provided in this paragraph, in any combination or permutation.

[0203] In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having particular homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22 and a light chain variable region having particular homology or identity to the light chain variable region set forth in SEQ ID NO: 23. Embodiments of heavy chain variable regions and light chain variable regions having such homology or identity are further set forth below. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having greater than 70% homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having greater than 75% homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having greater than 80% homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having greater than 85% homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having greater than 90% homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having greater than 95% homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22. In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having any of the indicated homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22, and its CDRs (CDR-H1, CDR-H2, and CDR-H3) are identical to the CDRs in the heavy chain variable region set forth in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain variable region having greater than 70% homology or identity to the light chain variable region set forth in SEQ ID NO: 23. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain variable region having greater than 75% homology or identity to the light chain variable region set forth in SEQ ID NO:23.In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain variable region having greater than 80% homology or identity to the light chain variable region set forth in SEQ ID NO: 23. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain variable region having greater than 85% homology or identity to the light chain variable region set forth in SEQ ID NO: 23. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain variable region having greater than 90% homology or identity to the light chain variable region set forth in SEQ ID NO: 23. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain variable region having greater than 95% homology or identity to the light chain variable region set forth in SEQ ID NO: 23. In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain variable region having any of the indicated homology or identity to the light chain variable region set forth in SEQ ID NO: 23, and the CDRs (CDR-L1, CDR-L2, and CDR-L3) are identical to the CDRs in the light chain variable region set forth in SEQ ID NO: 23. In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise any of the homologous light chain variable regions and any of the homologous heavy chain variable regions provided in this paragraph, in any combination or permutation.

[0204] In some embodiments, the anti-Nectin-4 antibodies provided herein comprise the heavy and light chain CDR regions of the antibody designated Ha22-2(2,4)6.1 produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, or heavy and light chain CDR regions comprising amino acid sequences homologous to the amino acid sequences of the heavy and light chain CDR regions of Ha22-2(2,4)6.1, and the antibodies retain desirable functional properties of the anti-Nectin-4 antibody designated Ha22-2(2,4)6.1 produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0205] In some embodiments, the anti-Nectin-4 antibody provided herein comprises the heavy and light chain CDR regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of the antibody designated Ha22-2(2,4)6.1 produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, or heavy and light chain CDR regions consisting of amino acid sequences homologous to the amino acid sequences of the heavy and light chain CDR regions of Ha22-2(2,4)6.1, and the antibody retains desirable functional properties of the anti-Nectin-4 antibody designated Ha22-2(2,4)6.1 produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0206] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a humanized heavy chain variable region and a humanized light chain variable region, in which case: (a) the heavy chain variable region comprises CDRs (CDR-H1, CDR-H2, and CDR-H3) comprising the amino acid sequences of the heavy chain variable region CDRs shown in the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267; (b) The light chain variable region comprises CDRs (CDR-L1, CDR-L2, and CDR-L3) comprising the amino acid sequences of the light chain variable region CDRs shown in the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0207] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a humanized heavy chain variable region and a humanized light chain variable region, in which case: (a) the heavy chain variable region comprises CDRs (CDR-H1, CDR-H2, and CDR-H3) consisting of the amino acid sequences of the heavy chain variable region CDRs shown in the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267; (b) The light chain variable region contains CDRs (CDR-L1, CDR-L2, and CDR-L3) consisting of the amino acid sequences of the light chain variable region CDRs shown in the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0208] In some embodiments, the anti-Nectin-4 antibodies provided herein comprise the heavy and light chain variable regions of the antibody designated Ha22-2(2,4)6.1 produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, or heavy and light chain variable regions comprising amino acid sequences homologous to the amino acid sequences of the heavy and light chain variable regions of Ha22-2(2,4)6.1, and the antibodies retain the desirable functional properties of the anti-Nectin-4 antibodies provided herein. In some embodiments, the anti-Nectin-4 antibodies provided herein comprise the heavy and light chain variable regions of the antibody designated Ha22-2(2,4)6.1, which is produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, or heavy and light chain variable regions consisting of amino acid sequences homologous to the amino acid sequences of the heavy and light chain variable regions of Ha22-2(2,4)6.1, and the antibodies retain the desired functional properties of the anti-Nectin-4 antibodies provided herein. The constant region of the antibody of the present disclosure can be selected from any subclass. In one embodiment, a human IgG1 constant region can be used as the heavy chain constant region, and a human Ig kappa constant region can be used as the light chain constant region.

[0209] In some embodiments, the anti-Nectin-4 antibodies provided herein comprise the heavy and light chains of the antibody designated Ha22-2(2,4)6.1 produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, or heavy and light chain variable regions comprising amino acid sequences homologous to the amino acid sequences of the heavy and light chain variable regions of Ha22-2(2,4)6.1, and the antibodies retain desirable functional properties of the anti-Nectin-4 antibodies provided herein. In some embodiments, the anti-Nectin-4 antibodies provided herein comprise the heavy and light chains of the antibody designated Ha22-2(2,4)6.1 produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, or heavy and light chain variable regions consisting of amino acid sequences homologous to the amino acid sequences of the heavy and light chain variable regions of Ha22-2(2,4)6.1, and the antibodies retain the desirable functional properties of the anti-Nectin-4 antibodies provided herein.

[0210] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous or identical to the heavy chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267; and (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous or identical to the light chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0211] In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having a certain degree of homology or identity to the heavy chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, and a light chain variable region having a certain degree of homology or identity to the light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. Embodiments of heavy chain and light chain variable regions having such homology or identity are further set forth below. In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 85% homologous or identical to the heavy chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 90% homologous or identical to the heavy chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In yet other embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 95% homologous or identical to the heavy chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the heavy chain variable region may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the heavy chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.In some embodiments, the light chain variable region comprises an amino acid sequence that is at least 85% homologous or identical to the light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the light chain variable region comprises an amino acid sequence that is at least 90% homologous or identical to the light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In yet other embodiments, the light chain variable region comprises an amino acid sequence that is at least 95% homologous or identical to the light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the light chain variable region can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the light chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA- 11267. In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise any homologous light chain variable region and any homologous heavy chain variable region provided in this paragraph, in any combination or permutation.

[0212] In other embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain and a light chain, in which case: (a) the heavy chain comprises an amino acid sequence that is at least 80% homologous or identical to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267; and (b) the light chain comprises an amino acid sequence that is at least 80% homologous or identical to the light chain amino acid sequence of an antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0213] In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having a certain degree of homology or identity to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, and a light chain having a certain degree of homology or identity to the light chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. Embodiments of heavy and light chains having such homology or identity are further set forth below. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 85% homologous or identical to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the heavy chain comprises an amino acid sequence that is at least 90% homologous or identical to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In yet other embodiments, the heavy chain comprises an amino acid sequence that is at least 95% homologous or identical to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the heavy chain may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.In some embodiments, the light chain comprises an amino acid sequence that is at least 85% homologous or identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the light chain comprises an amino acid sequence that is at least 90% homologous or identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In yet other embodiments, the light chain comprises an amino acid sequence that is at least 95% homologous or identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the light chain can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to the light chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA- 11267. In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise any homologous light chain and any homologous heavy chain provided in this paragraph, in any combination or permutation.

[0214] In some embodiments, the antibodies or antigen-binding fragments provided herein bind to a specific epitope of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to the VC1 domain of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to the VC1 domain but not the C1C2 domain of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 1 to 147 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to an epitope located at amino acid residues 1 to 147 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 1 to 10 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 11 to 20 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 21 to 30 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 31 to 40 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 41 to 50 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 51 to 60 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 61 to 70 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 71 to 80 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 81 to 90 of 191P4D12.In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 91 to 100 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 101 to 110 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 111 to 120 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 121 to 130 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 131 to 140 of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to amino acid residues 141 to 147 of 191P4D12. The binding epitopes of certain embodiments of the antibodies or antigen-binding fragments thereof provided herein have been determined and are described in WO2012 / 047724, which is incorporated herein by reference in its entirety.

[0215] In some embodiments, the antibodies or antigen-binding fragments provided herein bind to an epitope of 191P4D12 that is shared among 191P4D12 variants observed in humans. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to an epitope of 191P4D12 that is shared among 191P4D12 polymorphisms observed in humans. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to an epitope of 191P4D12 that is shared among 191P4D12 polymorphisms observed in human cancer. In some embodiments, the antibodies or antigen-binding fragments provided herein bind to an epitope of 191P4D12 that will bind, internalize, disrupt, or modulate the biological function of 191P4D12 or a 191P4D12 variant. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to an epitope of 191P4D12 that will disrupt interactions between 191P4D12 and ligands, substrates, and binding partners.

[0216] The engineered antibodies provided herein include those in which modifications have been made to framework residues within the VH and / or VL (e.g., to improve the properties of the antibody). Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived. To return framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence (e.g., "backmutating" leucine to methionine), for example, by site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the present disclosure.

[0217] Another type of framework modification involves mutating one or more residues within the framework regions, or even within one or more CDR regions, to remove T-cell epitopes and thereby reduce the potential immunogenicity of the antibody. This approach, also referred to as "deimmunization," is described in further detail in U.S. Patent Application Publication No. 2003 / 0153043 by Carr et al.

[0218] In addition to, or instead of, modifications made within the framework or CDR regions, antibodies of the disclosure can be engineered to contain modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Furthermore, the anti-191P4D12 antibodies provided herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter its glycosylation, also to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

[0219] In one embodiment, the hinge region of CH1 is modified such that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the anti-191P4D12 antibody.

[0220] In another embodiment, the Fc-hinge region of the antibody is mutated to decrease the biological half-life of the anti-191P4D12 antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment so that the antibody has reduced Staphylococcus protein A (SpA) binding compared to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0221] In another embodiment, an anti-191P4D12 antibody is modified to increase its biological half-life. Various approaches are possible. For example, mutations can be introduced as described in U.S. Patent No. 6,277,375 to Ward. Alternatively, to increase biological half-life, the antibody can be altered in the CH1 or CL region to include salvage receptor binding epitopes obtained from two loops in the CH2 domain of the IgG Fc region, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al.

[0222] In yet other embodiments, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from specific amino acid residues can be replaced with a different amino acid residue such that the antibody has altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0223] The reactivity of anti-191P4D12 antibodies with 191P4D12-related proteins can be established by a number of well-known means, including Western blot, immunoprecipitation, ELISA, and FACS analysis, using 191P4D12-related proteins, 191P4D12-expressing cells, or extracts thereof, as appropriate. The 191P4D12 antibody or a fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymes. Furthermore, bispecific antibodies specific for two or more 191P4D12 epitopes can be generated using methods commonly known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53:2560-2565).

[0224] In yet another specific embodiment, the anti-191P4D12 antibody provided herein is an antibody comprising the heavy and light chains of the antibody designated Ha22-2(2,4)6.1, wherein the heavy chain of Ha22-2(2,4)6.1 consists of the amino acid sequence ranging from E residue 20 to K residue 466 of SEQ ID NO:7, and the light chain of Ha22-2(2,4)6.1 consists of the amino acid sequence ranging from D residue 23 to C residue 236 of SEQ ID NO:8.

[0225] The hybridoma producing the antibody designated Ha22-2(2,4)6.1 was sent (via Federal Express) to the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108 on August 18, 2010, and was assigned accession number PTA-11267.

[0226] Additional embodiments of anti-Nectin-4 antibodies are described in U.S. Pat. No. 8,637,642 and International Application No. PCT / US2019 / 056214 (Publication No. WO2020 / 117373), both of which are incorporated by reference herein in their entireties.

[0227] 5.3.2 Cytotoxic Agents (Drug Units) Where the ADCs used in the methods provided herein comprise an antibody or antigen-binding fragment thereof conjugated to a cytotoxic agent, the disclosure further provides various embodiments of the cytotoxic agent as part of the ADC for use in the methods. In various embodiments of the methods provided herein, including those provided in Section 5.2, the cytotoxic agent as part of any of the ADCs provided herein for the methods comprises, consists of, or is a tubulin disrupting agent. In one embodiment, the cytotoxic agent is a tubulin disrupting agent. In some embodiments, the tubulin disrupting agent is selected from the group consisting of dolastatin, auristatin, hemiasterlin, vinca alkaloid, maytansinoid, eribulin, colchicine, procabulin, fomopsin, epothilone, cryptophycin, and taxane. In a specific embodiment, the tubulin disrupting agent is an auristatin. In a further specific embodiment, the auristatin is monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), AFP, or auristatin T. In yet another specific embodiment, the auristatin is monomethylauristatin E (MMAE).

[0228] In various embodiments of the methods provided herein, including those provided in Section 5.2, the cytotoxic agent as part of any of the ADCs provided herein for the methods comprises, consists of, or is any agent selected from the cytotoxic agents described in U.S. Pat. No. 8,637,642 and International Application No. PCT / US2019 / 056214 (Publication No. WO2020 / 117373), both of which are incorporated by reference herein in their entireties.

[0229] In some embodiments, the auristatin is MMAE (where the wavy line indicates the covalent bond to the linker of the antibody drug conjugate). TIFF2024534012000013.tif32165

[0230] In some embodiments, an exemplary embodiment comprising MMAE and a linker component (described further herein) has the following structure: (wherein L represents an antibody (e.g., an anti-Nectin-4 antibody or an antigen-binding fragment thereof) and p ranges from 1 to 12): TIFF2024534012000014.tif43165

[0231] In some embodiments of the formula set forth in the preceding paragraph, p ranges from 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments of the formula set forth in the preceding paragraph, p ranges from 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments of the formulas set forth in the preceding paragraph, p is in the range of 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, or 3 to 4. In some embodiments of the formulas set forth in the preceding paragraph, p is about 1. In some embodiments of the formulas set forth in the preceding paragraph, p is about 2. In some embodiments of the formulas set forth in the preceding paragraph, p is about 3. In some embodiments of the formulas set forth in the preceding paragraph, p is about 4. In some embodiments of the formulas set forth in the preceding paragraph, p is about 3.8. In some embodiments of the formulas set forth in the preceding paragraph, p is about 5. In some embodiments of the formulas set forth in the preceding paragraph, p is about 6. In some embodiments of the formulas set forth in the preceding paragraph, p is about 7. In some embodiments of the formulas set forth in the preceding paragraph, p is about 8. In some embodiments of the formulas set forth in the preceding paragraph, p is about 9. In some embodiments of the formulas set forth in the preceding paragraph, p is about 10. In some embodiments of the formula set forth in the preceding paragraph, p is about 11. In some embodiments of the formula set forth in the preceding paragraph, p is about 12. In some embodiments of the formula set forth in the preceding paragraph, p is about 13. In some embodiments of the formula set forth in the preceding paragraph, p is about 14. In some embodiments of the formula set forth in the preceding paragraph, p is about 15. In some embodiments of the formula set forth in the preceding paragraph, p is about 16. In some embodiments of the formula set forth in the preceding paragraph, p is about 17. In some embodiments of the formula set forth in the preceding paragraph, p is about 18. In some embodiments of the formula set forth in the preceding paragraph, p is about 19.In some embodiments of the formula described in the previous paragraph, p is about 20.

[0232] Typically, peptide-based drug units can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to solution-phase synthesis methods well known in the field of peptide chemistry (see E. Schroder and K. Lubke, "The Peptides," volume 1, pp. 76-136, 1965, Academic Press). Auristatin / dolastatin Drug Units can be prepared according to the methods of US 5,635,483; US 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, GR, et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784.

[0233] Additional embodiments of cytotoxic agents are described in U.S. Pat. No. 8,637,642 and International Application No. PCT / US2019 / 056214 (Publication No. WO2020 / 117373), both of which are incorporated by reference herein in their entireties.

[0234] 5.3.3 Linker Typically, an antibody-drug conjugate comprises a linker unit between the drug unit (e.g., MMAE) and the antibody unit (e.g., an anti-191P4D12 antibody or antigen-binding fragment thereof). In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In yet other embodiments, the linker unit is not cleavable, and the drug is released, for example, by antibody degradation. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolae). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, etc.) that is hydrolyzable in the lysosome can be used. In yet other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). Various disulfide linkers are known in the art, including those that can be formed using, for example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB, and SMPT.

[0235] A "Linker Unit" (LU) is a bifunctional compound that can be used to link a Drug Unit and an Antibody Unit to form an antibody drug conjugate. In some embodiments, the Linker Unit has the formula: -Aa -W w -Y y - and where -A- is an extender unit; a is 0 or 1, each -W- is independently an amino acid unit; w is an integer ranging from 0 to 12, -Y- is a self-immolative spacer unit; y is 0, 1, or 2.

[0236] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1, or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, when w is 1-12, y is 1 or 2. In some embodiments, w is 2-12, and y is 1 or 2. In some embodiments, a is 1, and w and y are 0. Linkers and each of the extender units, amino acid units, and spacer units are described in U.S. Pat. No. 8,637,642 and International Application No. PCT / US2019 / 056214 (Publication No. WO2020 / 117373), both of which are incorporated by reference herein in their entireties.

[0237] Embodiments of the antibody-drug conjugate may include: TIFF2024534012000015.tif25165 (wherein w and y are 0, 1, or 2, respectively); TIFF2024534012000016.tif26165 (wherein w and y are each 0), TIFF2024534012000017.tif162165.

[0238] 5.3.4 Drug burden Drug loading is represented by p and is the average number of drug units per antibody in the molecule. Drug loading can range from 1 to 20 drug units (D) per antibody. The ADCs provided herein comprise a population of antibodies or antigen-binding fragments conjugated with, for example, 1 to 20 drug units. The average number of drug units per antibody in ADC preparations from conjugation reactions can be characterized by conventional means, such as mass spectrometry and ELISA assays. The quantitative distribution of ADCs with respect to p can also be determined. In some cases, separation, purification, and characterization of homogeneous ADCs with a particular value of p from ADCs with other drug loads can be achieved by means such as electrophoresis.

[0239] In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 20. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 18. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 15. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 12. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 10. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 9. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 8. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 7. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 6. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 5. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 4. In certain embodiments, the drug loading of the ADCs provided herein ranges from 1 to 3. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 12. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 10. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 9. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 8. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 7. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 6. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 5. In certain embodiments, the drug loading of the ADCs provided herein ranges from 2 to 4. In certain embodiments, the drug loading of the ADCs provided herein ranges from 3 to 12. In certain embodiments, the drug loading of the ADCs provided herein ranges from 3 to 10. In certain embodiments, the drug loading of the ADCs provided herein ranges from 3 to 9. In certain embodiments, the drug loading of the ADCs provided herein ranges from 3 to 8.In certain embodiments, the drug loading of the ADCs provided herein ranges from 3 to 7. In certain embodiments, the drug loading of the ADCs provided herein ranges from 3 to 6. In certain embodiments, the drug loading of the ADCs provided herein ranges from 3 to 5. In certain embodiments, the drug loading of the ADCs provided herein ranges from 3 to 4.

[0240] In certain embodiments, the drug loading of the ADCs provided herein is in the range of 1 to about 8, about 2 to about 6, about 3 to about 5, about 3 to about 4, about 3.1 to about 3.9, about 3.2 to about 3.8, about 3.2 to about 3.7, about 3.2 to about 3.6, about 3.3 to about 3.8, or about 3.3 to about 3.7.

[0241] In certain embodiments, the drug loading of the ADCs provided herein is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or more. In certain embodiments, the drug loading of the ADCs provided herein is about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9.

[0242] In some embodiments, the drug loading of the ADCs provided herein is in the range of 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, or 2 to 13. In some embodiments, the drug loading of the ADCs provided herein is in the range of 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, or 3 to 13. In some embodiments, the drug loading of the ADCs provided herein is about 1. In some embodiments, the drug loading of the ADCs provided herein is about 2. In some embodiments, the drug loading of the ADCs provided herein is about 3. In some embodiments, the drug loading of the ADCs provided herein is about 4. In some embodiments, the drug loading of the ADCs provided herein is about 3.8. In some embodiments, the drug loading of the ADCs provided herein is about 5. In some embodiments, the drug loading of the ADCs provided herein is about 6. In some embodiments, the drug loading of the ADCs provided herein is about 7. In some embodiments, the drug loading of the ADCs provided herein is about 8. In some embodiments, the drug loading of the ADCs provided herein is about 9. In some embodiments, the drug loading of the ADCs provided herein is about 10. In some embodiments, the drug loading of the ADCs provided herein is about 11. In some embodiments, the drug loading of the ADCs provided herein is about 12. In some embodiments, the drug loading of the ADCs provided herein is about 13. In some embodiments, the drug loading of the ADCs provided herein is about 14. In some embodiments, the drug loading of the ADCs provided herein is about 15. In some embodiments, the drug loading of the ADCs provided herein is about 16. In some embodiments, the drug loading of the ADCs provided herein is about 17. In some embodiments, the drug loading of the ADCs provided herein is about 18. In some embodiments, the drug loading of the ADCs provided herein is about 19. In some embodiments, the drug loading of the ADCs provided herein is about 20.

[0243] In certain embodiments, fewer drug units than the theoretical maximum are conjugated to the antibody during the conjugation reaction. The antibody may contain, for example, lysine residues that do not react with either the drug-linker intermediate or the linker reagent. Generally, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be linked to a drug unit; in fact, most cysteine ​​thiol residues in antibodies exist as disulfide bridges. In certain embodiments, antibodies can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or complete reducing conditions to generate reactive cysteine ​​thiol groups. In certain embodiments, antibodies are subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine. In some embodiments, a linker unit or drug unit is conjugated via a lysine residue on the antibody unit. In some embodiments, a linker unit or drug unit is conjugated via a cysteine ​​residue on the antibody unit.

[0244] In some embodiments, the amino acid attached to the linker unit or Drug unit is in the heavy chain of the antibody or antigen-binding fragment thereof. In some embodiments, the amino acid attached to the linker unit or Drug unit is in the light chain of the antibody or antigen-binding fragment thereof. In some embodiments, the amino acid attached to the linker unit or Drug unit is in the hinge region of the antibody or antigen-binding fragment thereof. In some embodiments, the amino acid attached to the linker unit or Drug unit is in the Fc region of the antibody or antigen-binding fragment thereof. In other embodiments, the amino acid attached to the linker unit or Drug unit is in the constant region of the antibody or antigen-binding fragment thereof (e.g., CH1, CH2, or CH3 of the heavy chain, or CH1 of the light chain). In still other embodiments, the amino acid attached to the linker unit or Drug unit is in the VH framework region of the antibody or antigen-binding fragment thereof. In still other embodiments, the amino acid attached to the linker unit or Drug unit is in the VL framework region of the antibody or antigen-binding fragment thereof.

[0245] The loading (drug / antibody ratio) of an ADC can be controlled in various ways, for example, by (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the reaction time or temperature of conjugation, (iii) partial or limited reduction conditions for cysteine ​​thiol modification, or (iv) recombinantly manipulating the amino acid sequence of the antibody such that the number and position of cysteine ​​residues are altered to control the number and / or position of linker-drug linkages (such as in a ThioMab or ThioFab prepared as disclosed herein and in WO 2006 / 034488, which is incorporated herein by reference in its entirety).

[0246] It should be understood that when multiple nucleophilic groups react with a drug-linker intermediate or linker reagent and subsequently with a drug unit reagent, the resulting product is a mixture of ADC compounds with a distribution of one or more drug units attached to an antibody unit. The average number of drugs per antibody can be calculated from the mixture by a dual antibody-specific and drug-specific ELISA antibody assay. Individual ADC molecules can be identified in a mixture by mass spectrometry and separated by HPLC, e.g., hydrophobic interaction chromatography (e.g., Hamblett, KJ, et al., "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate," Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, SC, et al., "Controlling the location of drug attachment in antibody-drug conjugates," Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, homogeneous ADCs with a single loading value can be isolated from complex mixtures by electrophoresis or chromatography.

[0247] Methods for preparing, screening, and characterizing antibody-drug conjugates are known to those of skill in the art, for example, as described in US Pat. No. 8,637,642, which is incorporated herein by reference in its entirety.

[0248] In some embodiments, the antibody-drug conjugate for the methods provided herein is AGS-22M6E, prepared according to the methods described in U.S. Pat. No. 8,637,642 and having the following formula: TIFF2024534012000018.tif40165In the formula, L is Ha22-2(2,4)6.1 and p is 1 to 20.

[0249] In some embodiments, p is in the range of 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is in the range of 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is about 1. In other embodiments, p is about 2. In other embodiments, p is about 3. In other embodiments, p is about 4. In other embodiments, p is about 5. In other embodiments, p is about 6. In other embodiments, p is about 7. In other embodiments, p is about 8. In other embodiments, p is about 9. In other embodiments, p is about 10. In some embodiments, p is about 3.1. In some embodiments, p is about 3.2. In some embodiments, p is about 3.3. In some embodiments, p is about 3.4. In some embodiments, p is about 3.5. In other embodiments, p is about 3.6. In some embodiments, p is about 3.7. In some embodiments, p is about 3.8. In some embodiments, p is about 3.9. In some embodiments, p is about 4.0. In some embodiments, p is about 4.1. In some embodiments, p is about 4.2. In some embodiments, p is about 4.3. In some embodiments, p is about 4.4. In some embodiments, p is about 4.5. In other embodiments, p is about 4.6. In some embodiments, p is about 4.7. In some embodiments, p is about 4.8. In some embodiments, p is about 4.9. In some embodiments, p is about 5.0.

[0250] In some embodiments, the ADC used in the methods provided herein is enfortumab vedotin. Enfortumab vedotin is a fully human immunoglobulin G1 kappa (IgG1) conjugated to a microtubule disrupting agent (MMAE) via a protease-cleavable linker. Κ Enfortumab vedotin is an ADC composed of the 191P4D12 antibody (Challita-Eid PM et al., Cancer Res. 2016;76(10):3003-13). Enfortumab vedotin binds to the 191P4D12 protein on the cell surface, leading to internalization of the ADC-191P4D12 complex, which is then transported to the lysosomal compartment, where MMAE is released via proteolytic cleavage of the linker, thereby inducing antitumor activity. The intracellular release of MMAE subsequently disrupts tubulin polymerization, leading to G2 / M cell cycle arrest and apoptotic cell death (Francisco JA et al., Blood. 2003 Aug 15;102(4):1458-65).

[0251] As described above and in U.S. Patent No. 8,637,642, AGS-22M6E is an ADC derived from a murine hybridoma cell line. Enfortumab vedotin is a Chinese hamster ovary (CHO) cell line-derived equivalent of the AGS-22M6E ADC and is an exemplary product used for human therapy. Enfortumab vedotin has the same amino acid sequence, linker, and cytotoxic drug as AGS-22M6E. The equivalence between enfortumab vedotin and AGS-22M6E was confirmed through extensive analytical and biological characterization studies, including binding affinity to 191P4D12, in vitro cytotoxicity, and in vivo antitumor activity.

[0252] In one embodiment, an ADC provided herein is enfortumab vedotin, also known as EV, PADCEV, AGS-22M6E, AGS-22C3E, or AGS-22C3E. Enfortumab vedotin comprises an anti-191P4D12 antibody, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising amino acid residue 20 to amino acid residue 466 of SEQ ID NO:7 and a light chain comprising amino acid residue 23 to amino acid residue 236 of SEQ ID NO:8.

[0253] Enfortumab vedotin is an antibody-drug conjugate (ADC) against nectin-4, composed of a fully human anti-nectin-4 IgG1 kappa monoclonal antibody (AGS-22C3) conjugated to the small molecule microtubule-disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable maleimidocaproylvaline-citrulline (vc) linker (SGD-1006). Conjugation occurs at the cysteine ​​residues that make up the antibody's interchain disulfide bond, generating a drug-to-antibody ratio of approximately 3.8:1. The molecular weight is approximately 152 kDa.

[0254] Enfortumab vedotin has the following structural formula: TIFF2024534012000019.tif58165

[0255] Approximately four molecules of MMAE are attached to each antibody molecule. Enfortumab vedotin is produced by chemical conjugation of an antibody with a small molecule component. The antibody is produced in mammalian (Chinese hamster ovary) cells, and the small molecule component is produced by chemical synthesis.

[0256] Enfortumab vedotin injection is supplied as a preservative-free, white to off-white, sterile, lyophilized powder in single-dose vials for intravenous injection. Enfortumab vedotin is available in 20 mg and 30 mg vials, which must be reconstituted with 2.3 mL and 3.3 mL, respectively, of Sterile Water for Injection, USP, to yield a clear to slightly opaque, colorless to pale yellow solution at a final concentration of 10 mg / mL. After reconstitution, 2 mL (20 mg) and 3 mL (30 mg) can be withdrawn from each vial. Each mL of the reconstituted solution contains 10 mg of enfortumab vedotin, 1.4 mg of histidine, 2.31 mg of histidine hydrochloride monohydrate, 0.2 mg of polysorbate 20, and 55 mg of trehalose dihydrate, with a pH of 6.0.

[0257] 5.4 Pharmaceutical Compositions In certain embodiments of the methods provided herein, the ADC used in the methods is provided as a "pharmaceutical composition." Such a pharmaceutical composition comprises an antibody-drug conjugate provided herein and one or more pharmaceutically or physiologically acceptable excipients. In certain embodiments, the antibody-drug conjugate is provided in combination with or separately from one or more additional agents. Compositions comprising such one or more additional agents and one or more pharmaceutically or physiologically acceptable excipients are also provided. In certain embodiments, the antibody-drug conjugate and additional agent(s) are present in therapeutically acceptable amounts. Pharmaceutical compositions can be used in accordance with the methods and uses provided herein. Thus, for example, pharmaceutical compositions can be administered ex vivo or in vivo to a subject to practice the therapeutic methods and uses provided herein. The pharmaceutical compositions provided herein can be formulated to be compatible with the intended method or route of administration, exemplary routes of administration are described herein.

[0258] In some embodiments, pharmaceutical compositions of cancer or tumor modulating antibody drug conjugates are provided.

[0259] In certain embodiments of the methods provided herein, the pharmaceutical compositions comprising the ADCs may further comprise other therapeutically active agents or compounds disclosed herein or known to those of skill in the art that can be used in the treatment or prevention of various diseases and disorders (e.g., cancer) described herein. As noted above, the additional therapeutically active agents or compounds may be present in separate pharmaceutical composition(s).

[0260] Pharmaceutical compositions typically comprise a therapeutically effective amount of at least one of the antibody-drug conjugates provided herein and one or more pharmaceutically acceptable formulating agents. In certain embodiments, the pharmaceutical composition further comprises one or more additional agents described herein.

[0261] In one embodiment, a pharmaceutical composition comprises an antibody-drug conjugate provided herein. In some embodiments, a pharmaceutical composition comprises a therapeutically effective amount of an antibody-drug conjugate provided herein. In certain embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0262] In some embodiments, the antibody-drug conjugate in the pharmaceutical compositions provided herein is selected from the antibody-drug conjugates described in Section 5.3, above.

[0263] In certain embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of 0.1 mg / mL to 100 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of 1 mg / mL to 20 mg / mL. In other embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of 5 mg / mL to 15 mg / mL. In other embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of 8 mg / mL to 12 mg / mL. In other embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of 9 mg / mL to 11 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of about 9.5 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of about 9.6 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of about 9.7 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of about 9.8 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of about 9.9 mg / mL. In still other embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of about 10 mg / mL. In still other embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of about 10.1 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of about 10.2 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of about 10.3 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of about 10.3 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of about 10.4 mg / mL. In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of about 10.5 mg / mL.

[0264] In some embodiments, the pharmaceutical compositions provided herein comprise:

[0265] L-histidine, TWEEN-20, and at least one of trehalose dihydrate or sucrose. In some embodiments, the pharmaceutical compositions provided herein further comprise hydrochloric acid (HCl) or succinic acid.

[0266] In some embodiments, the concentration of L-histidine useful in the pharmaceutical compositions provided herein ranges from 5 mM to 50 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein ranges from 10 mM to 40 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein ranges from 15 mM to 35 mM.

[0267] In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein ranges from 15 mM to 30 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein ranges from 15 mM to 25 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein ranges from 15 mM to 35 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 16 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 17 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 18 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 19 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 20 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 21 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 22 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 23 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 24 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 25 mM.

[0268] In some embodiments, the concentration of TWEEN-20 useful in the pharmaceutical compositions provided herein ranges from 0.001% to 0.1% (v / v). In another embodiment, the concentration of TWEEN-20 ranges from 0.0025% to 0.075% (v / v). In one embodiment, the concentration of TWEEN-20 ranges from 0.005% to 0.05% (v / v). In another embodiment, the concentration of TWEEN-20 ranges from 0.0075% to 0.025% (v / v). In another embodiment, the concentration of TWEEN-20 ranges from 0.0075% to 0.05% (v / v). In another embodiment, the concentration of TWEEN-20 ranges from 0.01% to 0.03% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.01% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.015% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.016% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.017% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.018% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.019% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.02% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.021% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.022% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.023% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.024% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.025% (v / v).

[0269] In one embodiment, the concentration of trehalose dihydrate useful in the pharmaceutical compositions provided herein ranges from 1% to 20% (w / v). In another embodiment, the concentration of trehalose dihydrate ranges from 2% to 15% (w / v). In one embodiment, the concentration of trehalose dihydrate ranges from 3% to 10% (w / v). In another embodiment, the concentration of trehalose dihydrate ranges from 4% to 9% (w / v). In another embodiment, the concentration of trehalose dihydrate ranges from 4% to 8% (w / v). In another embodiment, the concentration of trehalose dihydrate ranges from 4% to 7% (w / v). In another embodiment, the concentration of trehalose dihydrate ranges from 4% to 6% (w / v). In another embodiment, the concentration of trehalose dihydrate ranges from 4.5% to 6% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 4.6% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 4.7% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 4.8% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 4.9% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.0% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.1% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.2% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.3% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.4% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.5% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.6% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.7% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.8% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 5.9% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 6.0% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 6.1% (w / v). In another embodiment, the concentration of trehalose dihydrate is about 6.2% (w / v).In another embodiment, the concentration of trehalose dihydrate is about 6.3% (w / v), in another embodiment, the concentration of trehalose dihydrate is about 6.4% (w / v), and in another embodiment, the concentration of trehalose dihydrate is about 6.5% (w / v).

[0270] In certain embodiments, the molar concentration of trehalose dihydrate is 50 mM to 300 mM. In other embodiments, the molar concentration of trehalose dihydrate is 75 mM to 250 mM. In some embodiments, the molar concentration of trehalose dihydrate is 100 mM to 200 mM. In other embodiments, the molar concentration of trehalose dihydrate is 130 mM to 150 mM. In some embodiments, the molar concentration of trehalose dihydrate is 135 mM to 150 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 135 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 136 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 137 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 138 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 139 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 140 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 141 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 142 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 143 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 144 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 145 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 146 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 150 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 151 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 151 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 152 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 153 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 154 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 155 mM.

[0271] In one embodiment, the concentration of sucrose useful in the pharmaceutical compositions provided herein ranges from 1% to 20% (w / v). In another embodiment, the concentration of sucrose ranges from 2% to 15% (w / v). In one embodiment, the concentration of sucrose ranges from 3% to 10% (w / v). In another embodiment, the concentration of sucrose ranges from 4% to 9% (w / v). In another embodiment, the concentration of sucrose ranges from 4% to 8% (w / v). In another embodiment, the concentration of sucrose ranges from 4% to 7% (w / v). In another embodiment, the concentration of sucrose ranges from 4% to 6% (w / v). In another embodiment, the concentration of sucrose ranges from 4.5% to 6% (w / v). In another embodiment, the concentration of sucrose is about 4.6% (w / v). In another embodiment, the concentration of sucrose is about 4.7% (w / v). In another embodiment, the sucrose concentration is about 4.8% (w / v). In another embodiment, the sucrose concentration is about 4.9% (w / v). In another embodiment, the sucrose concentration is about 5.0% (w / v). In another embodiment, the sucrose concentration is about 5.1% (w / v). In another embodiment, the sucrose concentration is about 5.2% (w / v). In another embodiment, the sucrose concentration is about 5.3% (w / v). In another embodiment, the sucrose concentration is about 5.4% (w / v). In another embodiment, the sucrose concentration is about 5.5% (w / v). In another embodiment, the sucrose concentration is about 5.6% (w / v). In another embodiment, the sucrose concentration is about 5.7% (w / v). In another embodiment, the sucrose concentration is about 5.8% (w / v). In another embodiment, the sucrose concentration is about 5.9% (w / v). In another embodiment, the sucrose concentration is about 6.0% (w / v). In another embodiment, the sucrose concentration is about 6.1% (w / v). In another embodiment, the sucrose concentration is about 6.2% (w / v). In another embodiment, the sucrose concentration is about 6.3% (w / v). In another embodiment, the sucrose concentration is about 6.4% (w / v). In another embodiment, the sucrose concentration is about 6.5% (w / v).

[0272] In certain embodiments, the molarity of sucrose is between 50 mM and 300 mM. In other embodiments, the molarity of sucrose is between 75 mM and 250 mM. In some embodiments, the molarity of sucrose is between 100 mM and 200 mM. In other embodiments, the molarity of sucrose is between 130 mM and 150 mM. In some embodiments, the molarity of sucrose is between 135 mM and 150 mM. In certain embodiments, the molarity of sucrose is about 135 mM. In certain embodiments, the molarity of sucrose is about 136 mM. In certain embodiments, the molarity of sucrose is about 137 mM. In certain embodiments, the molarity of sucrose is about 138 mM. In certain embodiments, the molarity of sucrose is about 139 mM. In certain embodiments, the molarity of sucrose is about 140 mM. In certain embodiments, the molarity of sucrose is about 141 mM. In certain embodiments, the molarity of sucrose is about 142 mM. In certain embodiments, the molarity of sucrose is about 143 mM. In certain embodiments, the molarity of sucrose is about 144 mM. In certain embodiments, the molarity of sucrose is about 145 mM. In certain embodiments, the molarity of sucrose is about 146 mM. In certain embodiments, the molarity of sucrose is about 150 mM. In certain embodiments, the molarity of sucrose is about 151 mM. In certain embodiments, the molarity of sucrose is about 151 mM. In certain embodiments, the molarity of sucrose is about 152 mM. In certain embodiments, the molarity of sucrose is about 153 mM. In certain embodiments, the molarity of sucrose is about 154 mM. In a particular embodiment, the molarity of the sucrose is about 155 mM.

[0273] In some embodiments, the pharmaceutical compositions provided herein comprise HCl. In other embodiments, the pharmaceutical compositions provided herein comprise succinic acid.

[0274] In some embodiments, the pharmaceutical compositions provided herein have a pH in the range of 5.5 to 6.5. In other embodiments, the pharmaceutical compositions provided herein have a pH in the range of 5.7 to 6.3. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 5.7. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 5.8. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 5.9. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 6.0. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 6.1. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 6.2. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 6.3.

[0275] In some embodiments, the pH is measured at room temperature. In other embodiments, the pH is measured between 15°C and 27°C. In yet other embodiments, the pH is measured at 4°C. In yet other embodiments, the pH is measured at 25°C.

[0276] In some embodiments, the pH is adjusted with HCl. In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH in the range of 5.5 to 6.5 at room temperature. In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH in the range of 5.7 to 6.3 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.7 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.8 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.9 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.0 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.1 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.2 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.3 at room temperature.

[0277] In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH ranging from 5.5 to 6.5 at 15°C to 27°C. In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH ranging from 5.7 to 6.3 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.7 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.8 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.9 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.0 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.1 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.2 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 6.3 between 15°C and 27°C.

[0278] In some embodiments, the pH is adjusted with succinic acid. In some embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH in the range of 5.5 to 6.5 at room temperature. In some embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH in the range of 5.7 to 6.3 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.7 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.8 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.9 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.0 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.1 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.2 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.3 at room temperature.

[0279] In some embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH ranging from 5.5 to 6.5 at 15°C to 27°C. In some embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH ranging from 5.7 to 6.3 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.7 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.8 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.9 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.0 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.1 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 6.2 at 15° C. to 27° C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 6.3 at 15° C. to 27° C.

[0280] In some specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, and at least one of about 5.5% (w / v) trehalose dihydrate or about 5% (w / v) sucrose. In some embodiments, the pharmaceutical compositions provided herein further comprise HCl or succinic acid. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25° C.

[0281] In some specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and HCl. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25° C.

[0282] In some specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5% (w / v) sucrose, and HCl. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25° C.

[0283] In other specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and succinic acid. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25° C.

[0284] In some specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5% (w / v) sucrose and succinic acid. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25° C.

[0285] In specific embodiments, provided herein are (a) the following structure: TIFF2024534012000020.tif43165, wherein L- represents an antibody or antigen-binding fragment thereof (e.g., an anti-Nectin-4 antibody or antigen-binding fragment thereof) and p is 1 to 10; and (b) A pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and HCl, wherein the antibody-drug conjugate has a concentration of about 10 mg / mL and a pH of about 6.0 at 25°C.

[0286] In another specific embodiment, the pharmaceutical compositions provided herein comprise: (a) the following structure: TIFF2024534012000021.tif44165, wherein L- represents an antibody or antigen-binding fragment thereof (e.g., an anti-Nectin-4 antibody or antigen-binding fragment thereof) and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and succinic acid; The antibody drug conjugate has a concentration of about 10 mg / mL and a pH of about 6.0 at 25°C.

[0287] In yet another specific embodiment, the pharmaceutical compositions provided herein comprise: (a) the following structure: TIFF2024534012000022.tif39165, wherein L- represents an antibody or antigen-binding fragment thereof (e.g., an anti-Nectin-4 antibody or antigen-binding fragment thereof) and p is 1 to 10; and (b) a pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.0% (w / v) sucrose, and HCl; The antibody drug conjugate has a concentration of about 10 mg / mL and a pH of about 6.0 at 25°C.

[0288] Although specific numbers (and ranges thereof) are provided, it is understood that in certain embodiments, numbers within, for example, 2%, 5%, 10%, 15%, or 20% of the above number (or range) are also contemplated.

[0289] The primary solvent in the vehicle can be either aqueous or non-aqueous in nature. Additionally, the vehicle can contain other pharmaceutically acceptable excipients to modify or maintain the pH, osmolality, viscosity, sterility, or stability of the pharmaceutical composition. In certain embodiments, the pharmaceutically acceptable vehicle is an aqueous buffer. In other embodiments, the vehicle contains, for example, sodium chloride and / or sodium citrate.

[0290] The pharmaceutical compositions provided herein may contain additional pharmaceutically acceptable formulating agents to modify or maintain the release rate of the antibody-drug conjugate and / or additional agent, as described herein. Such formulating agents include substances known to those skilled in the art for preparing sustained-release formulations. For further references regarding pharmaceutically and physiologically acceptable formulating agents, see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pp. 1435-1712; The Merck Index, 12th Ed. (1996, Merck Publishing Group, Whitehouse, NJ); and Pharmaceutical Principles of Solid Dosage Forms (1993, Technonic Publishing Co., Inc., Lancaster, Pa.). Additional pharmaceutical compositions suitable for administration are known in the art and may be applicable to the methods and compositions provided herein.

[0291] In some embodiments, the pharmaceutical compositions provided herein are in liquid form. In other embodiments, the pharmaceutical compositions provided herein are lyophilized.

[0292] A pharmaceutical composition can be formulated to be compatible with its intended route of administration. Thus, the pharmaceutical composition comprises excipients suitable for administration by routes including parenteral (e.g., subcutaneous (sc), intravenous, intramuscular, or intraperitoneal), intradermal, oral (e.g., ingestion), inhalation, intracavity, intracranial, and transdermal (topical). Other exemplary routes of administration are described herein.

[0293] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and suspending agents disclosed herein or known to those skilled in the art. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable diluents, solvents, and dispersion media that may be used include water, Ringer's solution, isotonic sodium chloride solution, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS), ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Additionally, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Additionally, fatty acids, such as oleic acid, are used in the preparation of injectables. Prolonged absorption of certain injectable formulations can be achieved by including an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0294] In one embodiment, the pharmaceutical compositions provided herein can be administered parenterally by injection, infusion, or implantation for local or systemic administration. As used herein, parenteral administration includes intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration.

[0295] In one embodiment, the pharmaceutical compositions provided herein may be formulated in any dosage form suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solution or suspension in liquid prior to injection. Such dosage forms may be prepared according to conventional methods known to those skilled in the art of pharmacy (see, e.g., Remington, The Science and Practice of Pharmacy, supra).

[0296] In one embodiment, a pharmaceutical composition intended for parenteral administration may comprise one or more pharmaceutically acceptable excipients including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against microbial growth, stabilizers, solubility enhancers, isotonicity agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.

[0297] In one embodiment, suitable aqueous vehicles include, but are not limited to, water, saline, saline or phosphate-buffered saline (PBS), sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, medium-chain triglycerides of coconut oil, and palm seed oil. Water-miscible vehicles include, but are not limited to, ethanol, 1,3-butanediol, liquid polyethylene glycols (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N-methyl-2-pyrrolidone, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0298] In one embodiment, suitable antimicrobial agents or preservatives include, but are not limited to, phenol, cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate, thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl- and propyl-paraben, and sorbic acid. Suitable isotonicity agents include, but are not limited to, sodium chloride, glycerin, and dextrose. Suitable buffers include, but are not limited to, phosphates and citrates. Suitable antioxidants include those described herein, including bisulfites and sodium metabisulfite. Suitable local anesthetics include, but are not limited to, procaine hydrochloride. Suitable suspending and dispersing agents include those described herein, including sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Suitable emulsifying agents include those described herein, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate. Suitable sequestering or chelating agents include, but are not limited to, EDTA. Suitable pH adjusters include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Suitable complexing agents include, but are not limited to, cyclodextrins, such as α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, and sulfobutylether 7-β-cyclodextrin (CAPTISOL®, CyDex, Lenexa, KS).

[0299] In one embodiment, the pharmaceutical compositions provided herein can be formulated for single or multiple doses. Single dose formulations are packaged in ampoules, vials, or syringes. Multi-dose parenteral formulations can contain antibacterial agents at bacteriostatic or fungistatic concentrations. All parenteral formulations must be sterile, as is known and practiced in the art.

[0300] In one embodiment, the pharmaceutical composition is provided as a ready-to-use sterile solution. In another embodiment, the pharmaceutical composition is provided as a sterile dry soluble product, including lyophilized powders and hypodermic tablets, to be reconstituted with a vehicle before use. In yet another embodiment, the pharmaceutical composition is provided as a ready-to-use sterile suspension. In yet another embodiment, the pharmaceutical composition is provided as a sterile dry insoluble product, to be reconstituted with a vehicle before use. In yet another embodiment, the pharmaceutical composition is provided as a ready-to-use sterile emulsion.

[0301] In one embodiment, the pharmaceutical compositions provided herein may be formulated as immediate or modified release dosage forms, including delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed-release forms.

[0302] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.

[0303] Pharmaceutical compositions can also contain excipients to protect the composition from rapid degradation or elimination from the body, such as controlled-release formulations, including implants, liposomes, hydrogels, prodrugs, and microencapsulated delivery systems. For example, time-delay materials such as glyceryl monostearate or glyceryl stearate can be used alone or in combination with wax. Prolonged absorption of injectable pharmaceutical compositions can be achieved by including an agent that delays absorption, such as aluminum monostearate or gelatin. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.

[0304] The pharmaceutical compositions provided herein may be stored at -80°C, 4°C, 25°C, or 37°C.

[0305] Lyophilized compositions can be produced by freeze-drying the liquid pharmaceutical compositions provided herein. In specific embodiments, the pharmaceutical compositions provided herein are freeze-dried pharmaceutical compositions. In some embodiments, the pharmaceutical formulations are freeze-dried powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. They can also be reconstituted and formulated as solids or gels.

[0306] In some embodiments, preparation of the lyophilized formulations provided herein comprises batch processing of the formulated bulk solution for lyophilization, sterile filtration, filling into vials, freezing the vials in a lyophilization chamber, followed by lyophilization, stoppering, and capping.

[0307] A freeze-dryer can be used to prepare lyophilized formulations. For example, a VirTis Genesis Model EL pilot unit can be used. The unit incorporates a chamber with three working shelves (total usable shelf area approximately 0.4 square meters), an external condenser, and a mechanical vacuum pump system. Cascaded mechanical refrigeration can cool the shelves to -70°C or below and the external condenser to -90°C or below. Shelf temperature and chamber pressure were automatically controlled to + / - 0.5°C and + / - 2 microns (milliTorr), respectively. The unit was equipped with a capacitance manometer vacuum gauge, a Pirani vacuum gauge, a pressure transducer (to measure 0 to 1 atmosphere), and a relative humidity sensor.

[0308] The lyophilized powder can be prepared by dissolving the antibody-drug conjugate provided herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some embodiments, the lyophilized powder is sterilized. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial contains a single dose or multiple doses of the antibody-drug conjugate. The lyophilized powder can be stored under appropriate conditions, for example, at about 4°C to room temperature.

[0309] This lyophilized powder is reconstituted with water for injection to provide a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable excipients. Such amounts can be empirically determined and adjusted according to specific needs.

[0310] An exemplary reconstitution procedure is as follows: (1) Attach an 18- or 20-gauge needle to a 5 mL or 3 mL syringe and fill the syringe with water for injection (WFI)-grade water; (2) Measure the appropriate amount of WFI using the syringe's scale, ensuring there are no air bubbles in the syringe; (3) Insert the needle into the rubber stopper; (4) Dispense the entire contents of the syringe down the vial wall into a container, remove the syringe and needle, and place in a sharps container; (4) Carefully solubilize the entire contents of the vial by continuously swirling the vial until completely reconstituted (e.g., about 20 seconds to about 40 seconds), minimizing excessive agitation of the protein solution, which may result in foaming.

[0311] In some embodiments, the pharmaceutical compositions provided herein are supplied as a dry, sterile, lyophilized powder or water-free concentrate in a sealed container, which can be reconstituted, for example, with water or saline, to the appropriate concentration for administration to a subject. In certain embodiments, the antibody-drug conjugate is supplied as a dry, sterile, lyophilized powder in a sealed container in a unit dose of at least 0.1 mg, at least 0.5 mg, at least 1 mg, at least 2 mg, at least 3 mg, at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 60 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg. The lyophilized antibody-drug conjugate can be stored in its original container at 2-8°C, and the antibody-drug conjugate can be administered within 12 hours, e.g., within 6 hours, within 5 hours, within 3 hours, or within 1 hour, after reconstitution. In alternative embodiments, pharmaceutical compositions comprising the antibody-drug conjugates provided herein are supplied in liquid form in a hermetically sealed container indicating the amount and concentration of the antibody-drug conjugate. In certain embodiments, the liquid form of the antibody-drug conjugate is supplied in a hermetically sealed container at at least 0.1 mg / ml, at least 0.5 mg / ml, at least 1 mg / ml, at least 5 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 30 mg / ml, at least 40 mg / ml, at least 50 mg / ml, at least 60 mg / ml, at least 70 mg / ml, at least 80 mg / ml, at least 90 mg / ml, or at least 100 mg / ml.

[0312] Additional embodiments of pharmaceutical compositions are described in U.S. Pat. No. 8,637,642 and International Application No. PCT / US2019 / 056214 (Publication No. WO2020 / 117373), both of which are incorporated by reference herein in their entireties.

[0313] 5.5 Combination therapy methods The methods for inhibiting tumor cell growth using the pharmaceutical compositions provided herein can be used in combination with chemotherapy or radiation therapy, or both, and include administering the pharmaceutical compositions of the present invention before, during, or after chemotherapy or radiation therapy, as well as any combination thereof (i.e., before and during, before and after, during and after, or before, during, and after chemotherapy and / or radiation therapy). Depending on the treatment protocol and the needs of the particular patient, the methods are implemented in a manner that provides the most effective treatment and ultimately extends the patient's lifespan. Additional embodiments of such combination therapies are described in U.S. Patent No. 8,637,642 and International Application No. PCT / US2019 / 056214 (Publication No. WO2020 / 117373), both of which are incorporated herein by reference in their entireties.

[0314] 5.6 ADC dosage for each method In some embodiments, the amount of a prophylactic or therapeutic agent (e.g., an antibody-drug conjugate provided herein), or a pharmaceutical composition provided herein, that is effective in the prevention and / or treatment of cancer can be determined by standard clinical techniques. In some embodiments, the effective amount can be extrapolated from dose-response curves derived from in vitro or animal model test systems. It will be understood that the precise dose to be employed in the formulation will also depend on the route of administration and the severity of the cancer in question, and should be decided according to the judgment of the practitioner and each patient's circumstances.

[0315] In some embodiments, the ADC for the methods for which various dosages are described in this section (Section 5.6) is enfortumab vedotin (EV).

[0316] In some embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition provided herein is administered to a patient via the nasal, intramuscular, intravenous, or a combination thereof, although other routes described herein are also acceptable. Each dose may or may not be administered via the same administration route. In some embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition provided herein can be administered via multiple administration routes simultaneously with or after other doses of one or more additional therapeutic agents. In some embodiments, the pharmaceutical composition comprising the antibody-drug conjugate provided herein is administered intravesically.

[0317] For pharmaceutical compositions comprising an antibody-drug conjugate provided herein, an effective amount of the ADC is a dose of about 10 mg to about 1000 mg in an injection volume of about 10 mL to about 100 mL. In some embodiments, an effective amount of the ADC is a dose of about 125 mg to about 950 mg in an injection volume of about 10 mL to about 100 mL. In some embodiments, an effective amount of the ADC is a dose of about 125 mg to about 900 mg in an injection volume of about 10 mL to about 100 mL. In some embodiments, an effective amount of the ADC is a dose of about 125 mg to about 850 mg in an injection volume of about 10 mL to about 100 mL. In some embodiments, an effective amount of the ADC is a dose of about 125 mg to about 800 mg in an injection volume of about 10 mL to about 100 mL. In some embodiments, an effective amount of the ADC is a dose of about 125 mg to about 750 mg in an injection volume of about 10 mL to about 100 mL. In some embodiments, an effective amount of the ADC is a dose of about 125 mg to about 750 mg in an injection volume of about 25 mL.

[0318] In some embodiments, the effective amount of ADC is a dose of about 10 mg to about 1000 mg. In some embodiments, the effective amount of ADC is a dose of about 50 mg to about 1000 mg. In some embodiments, the effective amount of ADC is a dose of about 100 mg to about 900 mg. In some embodiments, the effective amount of ADC is a dose of about 125 mg to about 900 mg. In some embodiments, the effective amount of ADC is a dose of about 125 mg to about 850 mg. In some embodiments, the effective amount of ADC is a dose of about 125 mg to about 800 mg. In some embodiments, the effective amount of ADC is a dose of about 125 mg to about 750 mg.

[0319] In some embodiments, the effective amount of the ADC is about a 100 mg dose. In some embodiments, the effective amount of the ADC is about a 125 mg dose. In some embodiments, the effective amount of the ADC is about a 150 mg dose. In some embodiments, the effective amount of the ADC is about a 200 mg dose. In some embodiments, the effective amount of the ADC is about a 250 mg dose. In some embodiments, the effective amount of the ADC is about a 300 mg dose. In some embodiments, the effective amount of the ADC is about a 350 mg dose. In some embodiments, the effective amount of the ADC is about a 400 mg dose. In some embodiments, the effective amount of the ADC is about a 450 mg dose. In some embodiments, the effective amount of the ADC is about a 500 mg dose. In some embodiments, the effective amount of the ADC is about a 550 mg dose. In some embodiments, the effective amount of the ADC is about a 600 mg dose. In some embodiments, the effective amount of the ADC is about a 650 mg dose. In some embodiments, the effective amount of the ADC is a dose of about 700 mg. In some embodiments, the effective amount of the ADC is a dose of about 750 mg. In some embodiments, the effective amount of the ADC is a dose of about 800 mg. In some embodiments, the effective amount of the ADC is a dose of about 850 mg. In some embodiments, the effective amount of the ADC is a dose of about 900 mg.

[0320] In some embodiments, the effective amount of the ADC is a 100 mg dose. In some embodiments, the effective amount of the ADC is a 125 mg dose. In some embodiments, the effective amount of the ADC is a 150 mg dose. In some embodiments, the effective amount of the ADC is a 200 mg dose. In some embodiments, the effective amount of the ADC is a 250 mg dose. In some embodiments, the effective amount of the ADC is a 300 mg dose. In some embodiments, the effective amount of the ADC is a 350 mg dose. In some embodiments, the effective amount of the ADC is a 400 mg dose. In some embodiments, the effective amount of the ADC is a 450 mg dose. In some embodiments, the effective amount of the ADC is a 500 mg dose. In some embodiments, the effective amount of the ADC is a 550 mg dose. In some embodiments, the effective amount of the ADC is a 600 mg dose. In some embodiments, the effective amount of the ADC is a 650 mg dose. In some embodiments, the effective amount of the ADC is a 700 mg dose. In some embodiments, the effective amount of ADC is a 750 mg dose. In some embodiments, the effective amount of ADC is an 800 mg dose. In some embodiments, the effective amount of ADC is an 850 mg dose. In some embodiments, the effective amount of ADC is a 900 mg dose.

[0321] In some embodiments, the injection volume is about 10 mL to about 100 mL. In some embodiments, the injection volume is about 10 mL to about 50 mL. In some embodiments, the injection volume is about 15 mL to about 30 mL. In some embodiments, the injection volume is about 10 mL. In some embodiments, the injection volume is about 15 mL. In some embodiments, the injection volume is about 20 mL. In some embodiments, the injection volume is about 25 mL. In some embodiments, the injection volume is about 30 mL. In some embodiments, the injection volume is about 35 mL. In some embodiments, the injection volume is about 40 mL. In some embodiments, the injection volume is about 45 mL. In some embodiments, the injection volume is about 50 mL. In some embodiments, the injection volume is about 55 mL. In some embodiments, the injection volume is about 60 mL. In some embodiments, the injection volume is about 65 mL. In some embodiments, the injection volume is about 70 mL. In some embodiments, the injection volume is about 75 mL. In some embodiments, the injection volume is about 80 mL. In some embodiments, the injection volume is about 85 mL. In some embodiments, the injection volume is about 90 mL. In some embodiments, the injection volume is about 95 mL. In some embodiments, the injection volume is about 100 mL.

[0322] In some embodiments, the injection volume is 10 mL. In some embodiments, the injection volume is 15 mL. In some embodiments, the injection volume is 20 mL. In some embodiments, the injection volume is 25 mL. In some embodiments, the injection volume is 30 mL. In some embodiments, the injection volume is 35 mL. In some embodiments, the injection volume is 40 mL. In some embodiments, the injection volume is 45 mL. In some embodiments, the injection volume is 50 mL. In some embodiments, the injection volume is 55 mL. In some embodiments, the injection volume is 60 mL. In some embodiments, the injection volume is 65 mL. In some embodiments, the injection volume is 70 mL. In some embodiments, the injection volume is 75 mL. In some embodiments, the injection volume is 80 mL. In some embodiments, the injection volume is 85 mL. In some embodiments, the injection volume is 90 mL. In some embodiments, the injection volume is 95 mL. In some embodiments, the injection volume is 100 mL.

[0323] In some embodiments, the effective amount of the ADC is about a 100 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 125 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 150 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 200 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 250 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 300 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 350 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 400 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 450 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 500 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 550 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 600 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 650 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 700 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 750 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about an 800 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about an 850 mg dose in an injection volume of about 10 mL. In some embodiments, the effective amount of the ADC is about a 900 mg dose in an injection volume of about 10 mL.

[0324] In some embodiments, the effective amount of ADC is a 100 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of ADC is a 125 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of ADC is a 150 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of ADC is a 200 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of ADC is a 250 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of ADC is a 300 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of ADC is a 350 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of ADC is a 400 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of ADC is a 450 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of the ADC is a 500 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of the ADC is a 550 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of the ADC is a 600 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of the ADC is a 650 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of the ADC is a 700 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of the ADC is a 750 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of the ADC is an 800 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of the ADC is an 850 mg dose in a 10 mL injection volume. In some embodiments, the effective amount of the ADC is a 900 mg dose in a 10 mL injection volume.

[0325] In some embodiments, the effective amount of ADC is about a 100 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of ADC is about a 125 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of ADC is about a 150 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of ADC is about a 200 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of ADC is about a 250 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of ADC is about a 300 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of ADC is about a 350 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of ADC is about a 400 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of ADC is about a 450 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of the ADC is about a 500 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of the ADC is about a 550 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of the ADC is about a 600 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of the ADC is about a 650 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of the ADC is about a 700 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of the ADC is about a 750 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of the ADC is about an 800 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of the ADC is about an 850 mg dose in an injection volume of about 15 mL. In some embodiments, the effective amount of the ADC is about a 900 mg dose in an injection volume of about 15 mL.

[0326] In some embodiments, the effective amount of ADC is a 100 mg dose in an injection volume of 15 mL. In some embodiments, the effective amount of ADC is a 125 mg dose in an injection volume of 15 mL. In some embodiments, the effective amount of ADC is a 150 mg dose in an injection volume of 15 mL. In some embodiments, the effective amount of ADC is a 200 mg dose in an injection volume of 15 mL. In some embodiments, the effective amount of ADC is a 250 mg dose in an injection volume of 15 mL. In some embodiments, the effective amount of ADC is a 300 mg dose in an injection volume of 15 mL. In some embodiments, the effective amount of ADC is a 350 mg dose in an injection volume of 15 mL. In some embodiments, the effective amount of ADC is a 400 mg dose in an injection volume of 15 mL. In some embodiments, the effective amount of ADC is a 450 mg dose in an injection volume of 15 mL. In some embodiments, the effective amount of ADC is a 500 mg dose in an infusion volume of 15 mL. In some embodiments, the effective amount of ADC is a 550 mg dose in an infusion volume of 15 mL. In some embodiments, the effective amount of ADC is a 600 mg dose in an infusion volume of 15 mL. In some embodiments, the effective amount of ADC is a 650 mg dose in an infusion volume of 15 mL. In some embodiments, the effective amount of ADC is a 700 mg dose in an infusion volume of 15 mL. In some embodiments, the effective amount of ADC is a 750 mg dose in an infusion volume of 15 mL. In some embodiments, the effective amount of ADC is an 800 mg dose in an infusion volume of 15 mL. In some embodiments, the effective amount of ADC is an 850 mg dose in an infusion volume of 15 mL. In some embodiments, the effective amount of ADC is a 900 mg dose in an infusion volume of 15 mL.

[0327] In some embodiments, the effective amount of ADC is about a 100 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of ADC is about a 125 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of ADC is about a 150 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of ADC is about a 200 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of ADC is about a 250 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of ADC is about a 300 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of ADC is about a 350 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of ADC is about a 400 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of ADC is about a 450 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of the ADC is about a 500 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of the ADC is about a 550 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of the ADC is about a 600 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of the ADC is about a 650 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of the ADC is about a 700 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of the ADC is about a 750 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of the ADC is about an 800 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of the ADC is about an 850 mg dose in an injection volume of about 20 mL. In some embodiments, the effective amount of the ADC is about a 900 mg dose in an injection volume of about 20 mL.

[0328] In some embodiments, the effective amount of ADC is a 100 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 125 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 150 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 200 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 250 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 300 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 350 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 400 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 450 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 500 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 550 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 600 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 650 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 700 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 750 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is an 800 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is an 850 mg dose in a 20 mL injection volume. In some embodiments, the effective amount of ADC is a 900 mg dose in a 20 mL injection volume.

[0329] In some embodiments, the effective amount of ADC is about a 100 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 125 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 150 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 200 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 250 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 300 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 350 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 400 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 450 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 500 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 550 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 600 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 650 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 700 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 750 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about an 800 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about an 850 mg dose in an injection volume of about 25 mL. In some embodiments, the effective amount of ADC is about a 900 mg dose in an injection volume of about 25 mL.

[0330] In some embodiments, the effective amount of ADC is a 100 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 125 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 150 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 200 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 250 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 300 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 350 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 400 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 450 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 500 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 550 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 600 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 650 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 700 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 750 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is an 800 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is an 850 mg dose in an infusion volume of 25 mL. In some embodiments, the effective amount of ADC is a 900 mg dose in an infusion volume of 25 mL.

[0331] In some embodiments, the effective amount of ADC is about a 100 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 125 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 150 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 200 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 250 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 300 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 350 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 400 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 450 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 500 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 550 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 600 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 650 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 700 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 750 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about an 800 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about an 850 mg dose in an injection volume of about 30 mL. In some embodiments, the effective amount of ADC is about a 900 mg dose in an injection volume of about 30 mL.

[0332] In some embodiments, the effective amount of ADC is a 100 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 125 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 150 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 200 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 250 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 300 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 350 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 400 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 450 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 500 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 550 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 600 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 650 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 700 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 750 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is an 800 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is an 850 mg dose in an infusion volume of 30 mL. In some embodiments, the effective amount of ADC is a 900 mg dose in an infusion volume of 30 mL.

[0333] In some embodiments, the effective amount of ADC is about a 100 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of ADC is about a 125 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of ADC is about a 150 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of ADC is about a 200 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of ADC is about a 250 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of ADC is about a 300 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of ADC is about a 350 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of ADC is about a 400 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of ADC is about a 450 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of the ADC is about a 500 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of the ADC is about a 550 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of the ADC is about a 600 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of the ADC is about a 650 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of the ADC is about a 700 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of the ADC is about a 750 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of the ADC is about an 800 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of the ADC is about an 850 mg dose with an injection volume of about 35 mL. In some embodiments, the effective amount of the ADC is about a 900 mg dose with an injection volume of about 35 mL.

[0334] In some embodiments, the effective amount of ADC is a 100 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 125 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 150 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 200 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 250 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 300 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 350 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 400 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 450 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 500 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 550 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 600 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 650 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 700 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 750 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is an 800 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is an 850 mg dose in an infusion volume of 35 mL. In some embodiments, the effective amount of ADC is a 900 mg dose in an infusion volume of 35 mL.

[0335] In some embodiments, the effective amount of ADC is about a 100 mg dose in an injection volume of about 40 mL. In some embodiments, the effective amount of ADC is about a 125 mg dose in an injection volume of about 40 mL. In some embodiments, the effective amount of ADC is about a 150 mg dose in an injection volume of about 40 mL. In some embodiments, the effective amount of ADC is about a 200 mg dose in an injection volume of about 40 mL. In some embodiments, the effective amount of ADC is about a 250 mg dose in an injection volume of about 40 mL. In some embodiments, the effective amount of ADC is about a 300 mg dose in an injection volume of about 40 mL. In some embodiments, the effective amount of ADC is about a 350 mg dose in an injection volume of about 40 mL. In some embodiments, the effective amount of ADC is about a 400 mg dose in an injection volume of about 40 mL. In some embodiments, the effective amount of ADC is about a 450 mg dose in an injection volume of about 40 mL. In some embodiments, the effective amount of the ADC is about a 500 mg dose with an injection volume of about 40 mL. In some embodiments, the effective amount of the ADC is about a 550 mg dose with an injection volume of about 40 mL. In some embodiments, the effective amount of the ADC is about a 600 mg dose with an injection volume of about 40 mL. In some embodiments, the effective amount of the ADC is about a 650 mg dose with an injection volume of about 40 mL. In some embodiments, the effective amount of the ADC is about a 700 mg dose with an injection volume of about 40 mL. In some embodiments, the effective amount of the ADC is about a 750 mg dose with an injection volume of about 40 mL. In some embodiments, the effective amount of the ADC is about an 800 mg dose with an injection volume of about 40 mL. In some embodiments, the effective amount of the ADC is about an 850 mg dose with an injection volume of about 40 mL. In some embodiments, the effective amount of the ADC is about a 900 mg dose with an injection volume of about 40 mL.

[0336] In some embodiments, the effective amount of ADC is a 100 mg dose in an infusion volume of 40 mL. In some embodiments, the effective amount of ADC is a 125 mg dose in an infusion volume of 40 mL. In some embodiments, the effective amount of ADC is a 150 mg dose in an infusion volume of 40 mL. In some embodiments, the effective amount of ADC is a 200 mg dose in an infusion volume of 40 mL. In some embodiments, the effective amount of ADC is a 250 mg dose in an infusion volume of 40 mL. In some embodiments, the effective amount of ADC is a 300 mg dose in an infusion volume of 40 mL. In some embodiments, the effective amount of ADC is a 350 mg dose in an infusion volume of 40 mL. In some embodiments, the effective amount of ADC is a 400 mg dose in an infusion volume of 40 mL. In some embodiments, the effective amount of ADC is a 450 mg dose in an infusion volume of 40 mL. In some embodiments, the effective amount of ADC is a 500 mg dose in a 40 mL infusion volume. In some embodiments, the effective amount of ADC is a 550 mg dose in a 40 mL infusion volume. In some embodiments, the effective amount of ADC is a 600 mg dose in a 40 mL infusion volume. In some embodiments, the effective amount of ADC is a 650 mg dose in a 40 mL infusion volume. In some embodiments, the effective amount of ADC is a 700 mg dose in a 40 mL infusion volume. In some embodiments, the effective amount of ADC is a 750 mg dose in a 40 mL infusion volume. In some embodiments, the effective amount of ADC is an 800 mg dose in a 40 mL infusion volume. In some embodiments, the effective amount of ADC is an 850 mg dose in a 40 mL infusion volume. In some embodiments, the effective amount of ADC is a 900 mg dose in a 40 mL infusion volume.

[0337] In some embodiments, the effective amount of ADC is about a 100 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of ADC is about a 125 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of ADC is about a 150 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of ADC is about a 200 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of ADC is about a 250 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of ADC is about a 300 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of ADC is about a 350 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of ADC is about a 400 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of ADC is about a 450 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of the ADC is about a 500 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of the ADC is about a 550 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of the ADC is about a 600 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of the ADC is about a 650 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of the ADC is about a 700 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of the ADC is about a 750 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of the ADC is about an 800 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of the ADC is about an 850 mg dose with an injection volume of about 45 mL. In some embodiments, the effective amount of the ADC is about a 900 mg dose with an injection volume of about 45 mL.

[0338] In some embodiments, the effective amount of ADC is a 100 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 125 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 150 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 200 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 250 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 300 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 350 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 400 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 450 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 500 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 550 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 600 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 650 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 700 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 750 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is an 800 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is an 850 mg dose in an infusion volume of 45 mL. In some embodiments, the effective amount of ADC is a 900 mg dose in an infusion volume of 45 mL.

[0339] In some embodiments, the effective amount of ADC is about a 100 mg dose in an injection volume of about 50 mL. In some embodiments, the effective amount of ADC is about a 125 mg dose in an injection volume of about 50 mL. In some embodiments, the effective amount of ADC is about a 150 mg dose in an injection volume of about 50 mL. In some embodiments, the effective amount of ADC is about a 200 mg dose in an injection volume of about 50 mL. In some embodiments, the effective amount of ADC is about a 250 mg dose in an injection volume of about 50 mL. In some embodiments, the effective amount of ADC is about a 300 mg dose in an injection volume of about 50 mL. In some embodiments, the effective amount of ADC is about a 350 mg dose in an injection volume of about 50 mL. In some embodiments, the effective amount of ADC is about a 400 mg dose in an injection volume of about 50 mL. In some embodiments, the effective amount of ADC is about a 450 mg dose in an injection volume of about 50 mL. In some embodiments, the effective amount of the ADC is about a 500 mg dose with an injection volume of about 50 mL. In some embodiments, the effective amount of the ADC is about a 550 mg dose with an injection volume of about 50 mL. In some embodiments, the effective amount of the ADC is about a 600 mg dose with an injection volume of about 50 mL. In some embodiments, the effective amount of the ADC is about a 650 mg dose with an injection volume of about 50 mL. In some embodiments, the effective amount of the ADC is about a 700 mg dose with an injection volume of about 50 mL. In some embodiments, the effective amount of the ADC is about a 750 mg dose with an injection volume of about 50 mL. In some embodiments, the effective amount of the ADC is about an 800 mg dose with an injection volume of about 50 mL. In some embodiments, the effective amount of the ADC is about an 850 mg dose with an injection volume of about 50 mL. In some embodiments, the effective amount of the ADC is about a 900 mg dose with an injection volume of about 50 mL.

[0340] In some embodiments, the effective amount of ADC is a 100 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 125 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 150 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 200 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 250 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 300 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 350 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 400 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 450 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 500 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 550 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 600 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 650 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 700 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 750 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is an 800 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is an 850 mg dose in a 50 mL infusion volume. In some embodiments, the effective amount of ADC is a 900 mg dose in a 50 mL infusion volume.

[0341] In some embodiments, the maximum residence time of each intravesical administration is about 120 minutes. In some embodiments, the maximum residence time of each intravesical administration is about 90 minutes. In some embodiments, the maximum residence time of each intravesical administration is the subject's tolerable residence time. In some embodiments, the maximum residence time of each intravesical administration is about 30 minutes to about 120 minutes. In some embodiments, the maximum residence time of each intravesical administration is about 30 minutes to about 90 minutes. In some embodiments, the residence time of each intravesical administration is about 30 minutes. In some embodiments, the residence time of each intravesical administration is about 40 minutes. In some embodiments, the residence time of each intravesical administration is about 50 minutes. In some embodiments, the residence time of each intravesical administration is about 60 minutes. In some embodiments, the residence time of each intravesical administration is about 70 minutes. In some embodiments, the residence time of each intravesical administration is about 80 minutes. In some embodiments, the residence time of each intravesical administration is about 90 minutes. In some embodiments, the residence time of each intravesical administration is about 100 minutes. In some embodiments, the residence time of each intravesical administration is about 110 minutes. In some embodiments, the residence time of each intravesical administration is about 120 minutes. In some embodiments, the residence time of each intravesical administration is 30 minutes. In some embodiments, the residence time of each intravesical administration is 40 minutes. In some embodiments, the residence time of each intravesical administration is 50 minutes. In some embodiments, the residence time of each intravesical administration is 60 minutes. In some embodiments, the residence time of each intravesical administration is 70 minutes. In some embodiments, the residence time of each intravesical administration is 80 minutes. In some embodiments, the residence time of each intravesical administration is 90 minutes. In some embodiments, the residence time of each intravesical administration is 100 minutes. In some embodiments, the residence time of each intravesical administration is 110 minutes. In some embodiments, the residence time of each intravesical administration is 120 minutes.

[0342] In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically during an induction phase. In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically during a maintenance phase. In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically in two phases, wherein the two phases are an induction phase and a maintenance phase. In some embodiments, the maintenance phase begins after the induction phase. In some embodiments, the maintenance phase begins 6 to 10 weeks, 6 to 9 weeks, or 6 to 8 weeks after the induction phase. In some embodiments, the maintenance phase begins 10 weeks after the induction phase. In some embodiments, the maintenance phase begins 9 weeks after the induction phase. In some embodiments, the maintenance phase begins 8 weeks after the induction phase. In some embodiments, the maintenance phase begins 7 weeks after the induction phase. In some embodiments, the maintenance phase begins 6 weeks after the induction phase.

[0343] In some embodiments, pharmaceutical compositions comprising antibody-drug conjugates provided herein are administered from about 1 to about 25 times, and the dose may be administered as needed, for example, weekly, biweekly, monthly, bimonthly, quarterly, etc., as determined by a physician. In some embodiments, pharmaceutical compositions comprising antibody-drug conjugates provided herein are administered weekly. In some embodiments, pharmaceutical compositions comprising antibody-drug conjugates provided herein are administered biweekly. In some embodiments, pharmaceutical compositions comprising antibody-drug conjugates provided herein are administered monthly. In some embodiments, pharmaceutical compositions comprising antibody-drug conjugates provided herein are administered bimonthly. In some embodiments, pharmaceutical compositions comprising antibody-drug conjugates provided herein are administered every quarter. In some embodiments, pharmaceutical compositions comprising an antibody-drug conjugate provided herein are administered 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time to treat NMIBC, and the dose is from about 10 mg to about 1000 mg in an injection volume of from about 10 mL to about 100 mL.

[0344] In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically once a week for four weeks during the induction phase. In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically once a week for five weeks during the induction phase. In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically once a week for six weeks during the induction phase. In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically once a week for seven weeks during the induction phase. In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically once a week for eight weeks during the induction phase.

[0345] In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically once a month for six months during the maintenance phase. In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically once a month for seven months during the maintenance phase. In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically once a month for eight months during the maintenance phase. In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically once a month for nine months during the maintenance phase. In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically once a month for ten months during the maintenance phase. In some embodiments, a pharmaceutical composition comprising an antibody-drug conjugate provided herein is administered intravesically once a month for eleven months during the maintenance phase.

[0346] In some embodiments, a pharmaceutical composition comprising an antibody drug conjugate provided herein is administered intravesically once weekly for 6 weeks during an induction phase and once monthly for 9 months during a maintenance phase, with the maintenance phase beginning 6-10 weeks, 6-9 weeks, or 6-8 weeks after the induction phase.

[0347] In more specific embodiments of the methods provided herein, the ADC has the following structure: TIFF2024534012000023.tif46165 wherein L- represents an antibody or antigen-binding fragment thereof, p is about 3 to about 4, and the antibody comprises a heavy chain comprising an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7, and a light chain comprising an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8, wherein the ADC is administered intravesically at a dose of about 125 mg, with an instillation volume of about 25 mL and a maximum residence time of 90 minutes, the dose being administered intravesically once weekly for 6 weeks during an induction phase and once monthly for 9 months during a maintenance phase, the maintenance phase starting 6 to 10 weeks after the induction phase.

[0348] In more specific embodiments of the methods provided herein, the ADC has the following structure: TIFF2024534012000024.tif41165 wherein L- represents an antibody or antigen-binding fragment thereof, p is about 3 to about 4, and the antibody comprises a heavy chain comprising an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7, and a light chain comprising an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8, wherein the ADC is administered intravesically at a dose of about 250 mg, with an instillation volume of about 25 mL and a maximum residence time of 90 minutes, the dose being administered intravesically once weekly for 6 weeks during an induction phase and once monthly for 9 months during a maintenance phase, the maintenance phase starting 6 to 10 weeks after the induction phase.

[0349] In more specific embodiments of the methods provided herein, the ADC has the following structure: TIFF2024534012000025.tif42165 wherein L- represents an antibody or antigen-binding fragment thereof, p is about 3 to about 4, and the antibody comprises a heavy chain comprising an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7, and a light chain comprising an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8, wherein the ADC is administered intravesically at a dose of about 500 mg, with an instillation volume of about 25 mL and a maximum residence time of 90 minutes, the dose being administered intravesically once weekly for 6 weeks during an induction phase and once monthly for 9 months during a maintenance phase, the maintenance phase starting 6 to 10 weeks after the induction phase.

[0350] In more specific embodiments of the methods provided herein, the ADC has the following structure: TIFF2024534012000026.tif41165 wherein L- represents an antibody or antigen-binding fragment thereof, p is about 3 to about 4, and the antibody comprises a heavy chain comprising an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7, and a light chain comprising an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8, wherein the ADC is administered intravesically at a dose of about 750 mg, with an instillation volume of about 25 mL and a maximum residence time of 90 minutes, the dose being administered intravesically once weekly for 6 weeks during an induction phase and once monthly for 9 months during a maintenance phase, the maintenance phase starting 6 to 10 weeks after the induction phase.

[0351] 5.7 Methods for determining biomarkers The present disclosure provides that the expression of any of the markers provided herein can be determined by various methods known in the art. In some embodiments, the expression of a marker can be determined by the amount or relative amount of mRNA transcribed from the marker gene. In one embodiment, the expression of a marker gene can be determined by the amount or relative amount of the protein product encoded by the marker gene. In another embodiment, the expression of a marker gene can be determined by the level of a biological or chemical response induced by the protein product encoded by the marker gene. In addition, in certain embodiments, the expression of a marker gene can be determined by the expression of one or more genes that correlate with the expression of the marker gene.

[0352] As described above, the level or amount of gene transcripts (e.g., mRNA) of a marker gene can be used as a surrogate for the expression level of the marker gene. Numerous different PCR or qPCR protocols are known in the art, including those exemplified herein. In some embodiments, various PCR or qPCR methods are applied or adapted to determine the mRNA levels of various marker genes. Quantitative PCR (qPCR) (also referred to as real-time PCR) is applied and adapted to some embodiments because it not only provides quantitative measurements but also reduces time and contamination. As used herein, "quantitative PCR (or "qPCR")" refers to directly monitoring the progress of PCR amplification as it occurs, without requiring repeated sampling of reaction products. In quantitative PCR, generated and tracked reaction products can be monitored via a signal transduction mechanism (e.g., fluorescence) before the signal exceeds background levels and the reaction plateaus. The number of cycles required to achieve a detectable or "threshold" level of fluorescence is directly proportional to the concentration of amplifiable target at the start of the PCR process, allowing for measurement of signal intensity, which provides a real-time measure of the amount of target nucleic acid in the sample. When applying qPCR to determine mRNA expression level, an additional step of reverse transcription from mRNA to DNA is carried out before qPCR analysis.Examples of PCR methods can be found in literature (Wong et al., BioTechniques 39:75-85(2005); D'haene et al., Methods 50:262-270(2010)), which are incorporated herein by reference in their entirety.Examples of PCR assays can also be found in U.S. Patent No. 6,927,024, which is incorporated herein by reference in its entirety.Examples of RT-PCR methods can be found in U.S. Patent No. 7,122,799, which is incorporated herein by reference in its entirety.The method of fluorescent in situ PCR is described in U.S. Patent No. 7,186,507, which is incorporated herein by reference in its entirety.

[0353] In a specific embodiment, qPCR can be performed to determine or measure the mRNA level of a marker gene as follows: Briefly, the average Ct (cycle threshold) value (or synonymously referred to herein as Cq (quantitation cycle)) of replicate qPCR reactions for the marker gene and one or more housekeeping genes is determined. The average Ct value of the marker gene can then be normalized to the Ct value of the housekeeping gene using the following exemplary formula: marker gene ΔCt = (average Ct of marker gene - average Ct of housekeeping gene A). The relative marker gene ΔCt can then be used to normalize, for example, mRNA expression = 2 -△Ct The relative levels of marker gene mRNA can be determined by using the following formula. For an overview of Ct and Cq values, see the MIQE guidelines (Bustin et al., The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments, Clinical Chemistry 55:4 (2009)).

[0354] Other commonly used methods known in the art can be used to quantify RNA transcripts of marker genes in a sample as a surrogate for marker gene expression, including Northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assay (Hod, Biotechniques 13:852-854 (1992)); microarray (Hoheisel et al., Nature Reviews Genetics 7:200-210 (2006); Jaluria et al., Microbial Cell Factories 6:4 (2007)); and polymerase chain reaction (PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)). RNA in situ hybridization (ISH) is a widely used molecular biology technique for measuring and localizing specific RNA sequences, such as messenger RNA (mRNA), long non-coding RNA (lncRNA), and microRNA (miRNA), within cells, such as circulating tumor cells (CTCs) or tissue sections, while maintaining the cellular and tissue context. ISH is a type of hybridization that uses directly or indirectly labeled complementary DNA or RNA strands, such as probes, to bind to and localize specific nucleic acids, such as DNA or RNA, within a sample, particularly within tissue or cell sections (in situ). Probe types can be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded complementary RNA (sscRNA), messenger RNA (mRNA), microRNA (miRNA), ribosomal RNA, mitochondrial RNA, and / or synthetic oligonucleotides. The term "fluorescence in situ hybridization" or "FISH" refers to a type of ISH that utilizes fluorescent labels. The term "chromogenic in situ hybridization" or "CISH" refers to a type of ISH that uses a chromogenic label.ISH, FISH, and CISH are well known to those skilled in the art (see, for example, Stoler, Clinics in Laboratory Medicine 10(1):215-236 (1990); In situ hybridization. A practical approach, Wilkinson, ed., IRL Press, Oxford (1992); Schwarzacher and Heslop-Harrison, Practical in situ hybridization, BIOS Scientific Publishers Ltd, Oxford (2000)). Thus, RNA ISH allows for spatiotemporal visualization and quantification of gene expression in cells and tissues. It has been widely applied in research and diagnostics (Hu et al., Biomark. Res. 2(1):1-13, doi:10.1186 / 2050-7771-2-3(2014); Ratan et al., Cureus 9(6):e1325.doi:10.7759 / cureus.1325(2017); Weier et al., Expert Rev. Mol. Diagn. 2(2):109-119(2002)). Fluorescent RNA ISH utilizes fluorescent dyes and fluorescence microscopy for RNA labeling and detection, respectively. Fluorescent RNA ISH can enable multiplexing of 4-5 target sequences.

[0355] Alternatively, the RNA transcripts of the marker genes in a sample can be determined by sequencing techniques as a surrogate for the expression of the marker genes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE) and massively parallel signature sequencing (MPSS).

[0356] In some embodiments, the expression of marker genes can be determined by the relative abundance of the RNA transcripts (including, for example, mRNA) of the marker genes in a pool of transcribed total RNA. Such relative abundance of the RNA transcripts of marker genes can be determined by next-generation sequencing, known as RNA-seq. In one example of an RNA-seq procedure, RNA from different sources (blood, tissue, cells) is purified, optionally concentrated (for example, using oligo(dT) primers), converted into cDNA, and fragmented. Millions or billions of short sequence reads are generated from the randomly fragmented cDNA library. See Zhao et al. BMC genomics 16:97 (2015); Zhao et al. Scientific Reports 8:4781 (2018); Shanrong Zhao et al., RNA, April 13, 2020, pre-publication, doi:10.1261 / rna.074922.120 (all of which are incorporated by reference in their entirety). The expression level of each mRNA transcript of a marker gene is determined by the total number of mapped fragments during normalization, which is directly proportional to its abundance level. Several normalization methods are known that facilitate using the abundance of RNA transcripts as a parameter for determining gene expression. Normalization methods include RPKM (Reads Per Kilobase Million), FPKM (Fragments Per Kilobase Million), and / or TPM (Transcripts Per Kilobase Million). Briefly, RPKM can be calculated as follows: Count the total number of reads for a sample and divide that number by 1,000,000. This is the "per million" conversion factor. Divide the number of reads by the "per million" conversion factor. Normalize this to sequencing depth to get reads per million (RPM), then divide the RPM value by the gene depth (in kilobases) to get RPKM. FPKM is closely related to RPKM, except reads are replaced by fragments. RPKM is generated for single-end RNA-seq, where every read corresponds to one sequenced fragment.FPKM was created for paired-end RNA-seq, where two reads correspond to one fragment, or one read corresponds to one fragment if one read in the pair is unmapped. TPM is very similar to RPKM and FPKM and is calculated as follows: The number of reads is divided by the length of each gene (in kilobases) to obtain reads per kilobase (RPK). The RPK value of the sample is counted and divided by 1,000,000 to obtain a "per million" conversion factor. Dividing the RPK value by the "per million" conversion factor gives the TPM. See Zhao et al. BMC genomics 16:97 (2015); Zhao et al. Scientific Reports 8:4781 (2018); Shanrong Zhao et al., RNA, prepublished April 13, 2020, doi:10.1261 / rna.074922.120 (all of which are incorporated by reference in their entireties).

[0357] In one embodiment, the expression of the marker genes is determined by RNA-seq, e.g., TPM, RPKM, and / or FPKM. In some embodiments, the expression of the marker genes is determined by TPM. In some embodiments, the expression of the marker genes is determined by RPKM. In some embodiments, the expression of the marker genes is determined by FPKM.

[0358] As described above, the expression of marker genes can be determined in a sample from a subject. In some embodiments, the sample is a blood sample, a serum sample, a plasma sample, a body fluid (e.g., tissue fluid, including cancer tissue fluid), or a tissue (e.g., cancer tissue or cancer-peripheral tissue). In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a tissue fraction isolated or extracted from a mammal, particularly a human. In some embodiments, the tissue sample is a population of cells isolated or extracted from a mammal, particularly a human. In some embodiments, the tissue sample is a sample obtained from a biopsy. In certain embodiments, samples can be obtained from various organs of a subject, including a human subject. In some embodiments, the sample is obtained from an organ of a subject with cancer. In some embodiments, the sample is obtained from an organ of a subject with cancer that has cancer. In other embodiments, the sample, e.g., a reference sample, is obtained from a normal organ of the patient or a second human subject.

[0359] In certain embodiments of the methods provided herein, the tissue includes tissue from the bladder, ureter, breast, lung, colon, rectum, ovary, fallopian tube, esophagus, cervix, endometrium, skin, larynx, bone marrow, salivary gland, kidney, prostate, brain, spinal cord, placenta, adrenal gland, pancreas, parathyroid gland, pituitary gland, testis, thyroid gland, spleen, tonsils, thymus, heart, stomach, small intestine, liver, skeletal muscle, peripheral nerve, mesothelium, or eye.

[0360] In further embodiments of the methods provided herein, the expression of various marker genes can be detected by various immunoassays known in the art, including immunohistochemistry (IHC) assays, immunoblotting assays, FACS assays, and ELISAs.

[0361] In various IHC assays, the expression of various marker genes can be detected by antibodies directed against the protein products encoded by the marker genes. IHC staining of tissue sections has been shown to be a reliable method for assessing or detecting the presence of proteins in a sample. IHC techniques utilize antibodies to probe and visualize cellular antigens in situ, typically by ch...

Claims

1. A medicament comprising an antibody-drug conjugate (ADC) for use in a method of treating bladder cancer in a human subject, wherein the method comprises intravesical administration of an effective amount of the ADC to the subject, wherein the ADC comprises an antibody or an antigen-binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethyl auristatin E (MMAE), wherein the antibody or the antigen-binding fragment thereof comprises a heavy-chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy-chain variable region set forth in SEQ ID NO: 22, and a light-chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light-chain variable region set forth in SEQ ID NO: 23, wherein the bladder cancer is non-muscle invasive bladder cancer (NMIBC), said medicament.

2. The medicament according to claim 1, wherein the NMIBC is histologically confirmed and is carcinoma in situ (CIS).

3. The medicament according to claim 2, wherein the subject has a papillary disease.

4. The medicament according to claim 2, wherein the subject does not have a papillary disease.

5. The medicament according to claim 1, wherein the NMIBC is histologically confirmed and the main histological component (>50%) is urothelial (transitional cell) carcinoma.

6. The medicament according to claim 1, wherein the subject has a high-risk Mycobacterium bovis BCG non-responsive disease.

7. The medicament according to claim 1, wherein the subject is ineligible for or has refused radical cystectomy.

8. The medicament according to claim 1, wherein all visible papillary Ta / T1 tumors of the subject have been completely resected within 60 days prior to the treatment.

9. The medicament according to claim 8, wherein the subject has residual pure CIS.

10. The medicament according to claim 8, wherein the subject does not have residual pure CIS.

11. (i) The subject has an Eastern Cooperative Oncology Group (ECOG) performance status score of 0, (ii) The subject has an Eastern Cooperative Oncology Group (ECOG) performance status score of 1, or (iii) The subject has an Eastern Cooperative Oncology Group (ECOG) performance status score of 2, The medicament according to claim 1.

12. The subject has an Eastern Cooperative Oncology Group (ECOG) performance status score of 2, and The pharmaceutical according to claim 11, wherein the glomerular filtration rate (GFR) of the subject is 50 mL / min or more, and the subject does not have New York Heart Association (NYHA) class III heart failure.

13. The subject is a. The absolute neutrophil count (ANC) is 1500 / μL or more, b. Hemoglobin (Hgb) is 10 g / dL or more, c. The platelet count is 100,000 / μL or more, d. Serum bilirubin is 1.5 × the upper limit of normal (ULN) or less, or in the case of a patient with Gilbert's disease, 3 × ULN or less, e. The calculated value of creatinine clearance (CrCl) is 30 mL / min or more (GFR can also be used instead of creatinine or CrCl), and CrCl should be calculated using the Cockcroft-Gault method or the Modification of Diet in Renal Disease (MDRD) equation. For a subject with an ECOG performance status of 2, the GFR must be 50 mL / min or more. f. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are 3 × ULN or less, or g. The international normalized ratio (INR), or prothrombin time (PT), activated partial thromboplastin time (aPTT) or partial thromboplastin time (PTT) is 1.5 ULN or less, provided that the subject is not receiving anticoagulant treatment and PT or aPTT is within the therapeutic range for the purpose of using anticoagulants. The pharmaceutical according to claim 1, having one or more of the states selected from the group consisting of, and optionally the subject having all of the states a - g.

14. The pharmaceutical according to claim 1, wherein the estimated mean remaining life of the subject exceeds 2 years.

15. The antibody or antigen-binding fragment thereof comprises CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14, or The antibody or antigen-binding fragment thereof comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

21. The medicament according to any one of claims 1 to 14.

16. The medicament according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

23.

17. The medicament according to any one of claims 1 to 14, wherein the antibody comprises a heavy chain comprising an amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO: 7 and a light chain comprising an amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO:

8.

18. (i) The antigen-binding fragment is Fab, F(ab’)2, Fv or scFv, and / or (ii) The antigen-binding fragment is recombinantly produced. The medicament according to any one of claims 1 to 14.

19. (i) The antibody is a fully human antibody, (ii) The antibody is IgG1 and the light chain is a kappa light chain, and / or (iii) The antibody is recombinantly produced. The medicament according to any one of claims 1 to 14.

20. The medicament according to any one of claims 1 to 14, wherein the antibody or antigen-binding fragment is conjugated to each unit of MMAE via a linker.

21. The medicament according to claim 20, wherein the linker is an enzyme-cleavable linker and forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof.

22. The medicament according to claim 20, wherein the linker has the formula -Aa-Ww-Yy-, wherein -A- is an extension unit, a is 0 or 1, -W- is an amino acid unit, w is an integer in the range of 0 to 12, -Y- is a spacer unit, and y is 0, 1 or 2.

23. The pharmaceutical according to claim 22, wherein the elongation unit has the structure of the following formula (1), the amino acid unit is valine-citrulline, and the spacer unit is a PAB group containing the structure of the following formula (2): 。

24. The pharmaceutical according to claim 22, wherein the elongation unit forms a bond with a sulfur atom of an antibody or an antigen-binding fragment thereof, and the spacer unit is linked to MMAE via a carbamate group.

25. The ADC is (i) 1 to 20 units of MMAE per antibody or antigen-binding fragment thereof, (ii) 1 to 10 units of MMAE per antibody or antigen-binding fragment thereof, (iii) 2 to 8 units of MMAE per antibody or antigen-binding fragment thereof, or (iv) 3 to 5 units of MMAE per antibody or antigen-binding fragment thereof and is the pharmaceutical according to any one of claims 1 to 14.

26. The ADC has the following structure: wherein L− represents an antibody or an antigen-binding fragment thereof, and (i) p is 1 to 10, (ii) p is 2 to 8, (iii) p is 3 to 5, (iv) p is 3 to 4, or (v) p is 4 and is the pharmaceutical according to any one of claims 1 to 14.

27. The pharmaceutical according to claim 26, wherein the average p value of the effective amount of the antibody-drug conjugate is about 3.

8.

28. The pharmaceutical according to claim 14, wherein the ADC is formulated in a pharmaceutical composition containing L-histidine, polysorbate-20, and trehalose dihydrate.

29. The ADC is (i) formulated in a pharmaceutical composition containing about 20 mM of L-histidine, about 0.02% (w / v) of polysorbate-20, about 5.5% (w / v) of trehalose dihydrate, and a hydrochloride salt, and the pH of the pharmaceutical composition is about 6.0 at 25°C, or (ii) formulated in a pharmaceutical composition containing about 9 mM of histidine, about 11 mM of histidine hydrochloride monohydrate, about 0.02% (w / v) of polysorbate-20, and about 5.5% (w / v) of trehalose dihydrate, and the pH of the pharmaceutical composition is about 6.0 at 25°C, and is the pharmaceutical according to any one of claims 1 to 14.

30. The effective amount of the ADC is in a drip infusion volume of about 10 mL to about 100 mL and in a dose of about 100 mg to about 1000 mg, about 125 mg to about 950 mg, about 125 mg to about 900 mg, about 125 mg to about 850 mg, about 125 mg to about 800 mg, or about 125 mg to about 750 mg, and is the pharmaceutical according to any one of claims 1 to 14.

31. The pharmaceutical according to any one of claims 1 to 14, wherein the effective amount of the ADC is a dosage of about 125 mg to about 750 mg with an infusion volume of about 25 mL.

32. The effective amount of the ADC is (i) a dosage of about 125 mg with an infusion volume of about 25 mL, (ii) a dosage of about 250 mg with an infusion volume of about 25 mL, (iii) a dosage of about 500 mg with an infusion volume of about 25 mL, or (iv) a dosage of about 750 mg with an infusion volume of about 25 mL The pharmaceutical according to any one of claims 1 to 14.

33. (i) The maximum residence time for each intravesical administration is about 90 minutes, (ii) The maximum residence time for each intravesical administration is about 120 minutes, or (iii) The residence time for each intravesical administration is about 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, or 120 minutes The pharmaceutical according to any one of claims 1 to 14.

34. The pharmaceutical according to any one of claims 1 to 14, wherein the ADC is administered intravesically during two stages: an induction stage and a maintenance stage.

35. The pharmaceutical according to claim 34, wherein the maintenance stage starts 6 to 10 weeks, 6 to 9 weeks, or 6 to 8 weeks after the induction stage.

36. The pharmaceutical according to claim 34, wherein the ADC is administered intravesically once a week for 6 weeks during the induction stage.

37. The pharmaceutical according to claim 34, wherein the ADC is administered intravesically once a month for 9 months during the maintenance stage.

38. The ADC has the following structure: wherein L− represents an antibody or an antigen-binding fragment thereof, p is about 3 to about 4, the antibody comprises a heavy chain having an amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO: 7, and a light chain having an amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO:

8. Here, the ADC is administered intravesically at a dosage of about 125 mg, with an infusion volume of about 25 mL and a maximum residence time of 90 minutes. The dosage is administered intravesically once a week for 6 weeks during the induction stage and once a month for 9 months during the maintenance stage. The maintenance stage starts 6 to 10 weeks after the induction stage. The pharmaceutical according to any one of claims 1 to 14.

39. The ADC has the following structure: having, wherein L− represents an antibody or an antigen-binding fragment thereof, p is from about 3 to about 4, and the antibody comprises a heavy chain comprising an amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO: 7, and a light chain comprising an amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO: 8, wherein the ADC is intravesically administered at a dose of about 250 mg, with a bolus infusion volume of about 25 mL and a maximum dwell time of 90 minutes, and the dose is administered intravesically once a week for 6 weeks during the induction phase and once a month for 9 months during the maintenance phase, and the maintenance phase starts 6 to 10 weeks after the induction phase, a medicament according to any one of claims 1 to 14. [

40. ] The ADC has the following structure: having, wherein L− represents an antibody or an antigen-binding fragment thereof, p is from about 3 to about 4, and the antibody comprises a heavy chain comprising an amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO: 7, and a light chain comprising an amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO: 8, wherein the ADC is intravesically administered at a dose of about 500 mg, with a bolus infusion volume of about 25 mL and a maximum dwell time of 90 minutes, and the dose is administered intravesically once a week for 6 weeks during the induction phase and once a month for 9 months during the maintenance phase, and the maintenance phase starts 6 to 10 weeks after the induction phase, a medicament according to any one of claims 1 to 14. [

41. ] The ADC has the following structure: having, wherein L− represents an antibody or an antigen-binding fragment thereof, p is from about 3 to about 4, said antibody comprising a heavy chain comprising an amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO: 7, and a light chain comprising an amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO: 8, wherein said ADC is administered intravesically at a dose of about 750 mg, with an infusion volume of about 25 mL and a residence time of up to 90 minutes, said dose being administered intravesically once a week for 6 weeks during said induction phase and once a month for 9 months during said maintenance phase, said maintenance phase starting 6 to 10 weeks after said induction phase, a pharmaceutical according to any one of claims 1 to 14.

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