CN116710483A - Methods of Treating Cancer Using Antibody Drug Conjugates (ADCs) Binding to 191P4D12 Protein - Google Patents

Abstract

Provided herein are methods of treating cancer with antibody drug conjugates (ADCs) that bind to 191P4D12 protein.

Description

Method for treating cancer using an antibody drug conjugate (ADC) bound to 191P4D12 protein

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application No. 63/080,013, filed September 17, 2020, U.S. Application No. 63/196,641, filed June 3, 2021, and U.S. Application No. 63/240,794, filed September 3, 2021, the disclosures of each of which are incorporated herein by reference in their entirety.

This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII format sequence listing with the file name "14369-274-228_SEQ_LISTING.txt" and a creation date of September 13, 2021 and a size of 39,755 bytes. The sequence listing submitted via EFS-Web is part of the present specification and is incorporated herein by reference in its entirety.

1. Technical Field

Provided herein are methods for treating cancer using antibody drug conjugates (ADCs) that bind to the 191P4D12 protein (connexin-4).

2. Background technology

Cancer is the main cause of death of people aged 35 to 65 in the U.S., and it is the second leading cause of death of people worldwide. It is estimated that in 2019, the U.S. will have about 1.7 million newly added cancer cases and about 610,000 deaths from cancer (National Cancer Institute.2019.Cancer Stat Facts:Cancer of AnySite.seer.cancer.gov/statfacts/html/all.html.2019 June 5 acquisition). In 2018, the world estimated that there were 18.1 million newly added cancer cases and about 9.6 million deaths from cancer in 2018 (World Health Organization.Press Release.2018 September.who.int/cancer/PRGlobocanFinal.pdf.2019 June 5 acquisition). Most deaths now occur in patients with metastatic cancer. In fact, in the past 20 years, the progress of treatment, including surgery, radiotherapy and adjuvant chemotherapy has cured most of the patients with local cancer. Patients whose cancer presents or relapses as metastatic disease derive modest benefit from conventional therapy only in terms of overall survival (OS), and cure is rare.

New therapeutic strategies for advanced and/or metastatic cancers include targeting molecular pathways important for cancer cell survival and novel cytotoxic compounds. The benefits of these novel drugs are reflected in long-term survival; however, a large proportion of patients with distant metastases still have poor outcomes and novel therapies are needed.

191P4D12 (also known as connexin-4) is a type I transmembrane protein and a member of the family of related immunoglobulin-like adhesion molecules involved in cell-cell adhesion. 191P4D12 belongs to the connexin family of adhesion molecules. 191P4D12 consists of an extracellular domain (ECD) containing 3 Ig-like domains, a transmembrane helix, and an intracellular region (Takai Y et al., Annu Rev Cell Dev Biol 2008; 24: 309-42). It is believed that connexin mediates Ca2 ⁺ -independent cell-cell adhesion via both homophilic and heterophilic trans interactions at adherens junctions, where it can recruit cadherins and regulate cytoskeletal rearrangement (Rikitake and Takai, Cell Mol Life Sci. 2008; 65 (2): 253-63). The sequence identity of 191P4D12 to other connexin family members is low and ranges between 25% and 30% in the ECD (Reymond N et al., J Biol Chem 2001; 43205-15). Adhesion promoted by connexins supports several biological processes, such as immune regulation, host-pathogen interactions and immune evasion (Sakisaka T et al., Current Opinion in Cell Biology 2007; 19: 593-602).

Urothelial carcinoma

According to the International Agency for Research on Cancer (IARC), urothelial cancer kills more than 165,000 patients each year and is the ninth most common cancer worldwide. Approximately 151,000 new cases of urothelial cancer are diagnosed each year in Europe, with 52,000 deaths each year. More than 22,000 new cases are diagnosed each year in Japan, with 7,600 deaths each year. Cancer Fact Sheets: All cancers excluding Non-Melanoma Skin. International Agency for Research on Cancer 2017. Retrieved from gco.iarc.fr/today/fact-sheets-cancers? cancer=29&type=0&sex=0. Accessed on December 19, 2017. According to the National Cancer Institute, more than 79,000 new cases of urothelial cancer were diagnosed in 2017, and more than 16,000 people died of the disease in the United States (the United States/US). SEER Cancer Stat Facts: Bladder Cancer. National Cancer Institute. Bethesda, MD, seer.cancer.govstatfacts/html/urinb.html. Accessed December 19, 2017.

First-line therapy for metastatic urothelial carcinoma in patients with adequate renal function consists of cisplatin-based combinations such as methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC); or gemcitabine plus cisplatin, which demonstrates an overall response rate of up to 50%, including approximately 10-15% complete response (CR). Bellmunt J et al., N Engl J Med. 2017; 376: 1015-26. DOI: 10.1056/NEJMoa1613683. Regardless of initial chemosensitivity, patients are not cured and the outcome of metastatic urothelial carcinoma after these regimens is poor: the median time to progression is only 7 months and the median overall survival (OS) is 14 months. Approximately 15% of patients survive at least 5 years, and the prognosis is particularly poor in patients with visceral metastases, with a 5-year OS rate of 7%. von der Maase H et al., J Clin Oncol. 2005; 23: 4602-8.

For second-line treatment, the small molecule tubulin inhibitor vinflunine is only approved in Europe. The median OS was 6.9 months, compared to a median OS of 4.6 months with best supportive care. Bellmunt J et al., J Clin Oncol. 2009; 27: 4454-61. For decades, the treatment landscape with only available cytotoxic chemotherapy had not changed significantly until the recent approval of immune checkpoint inhibitors (CPIs) targeting programmed death 1/programmed death-ligand 1 (PD-1/PD-L1). As of May 2016, several CPIs have been approved by the FDA in the United States for urothelial carcinoma in patients pretreated with platinum, starting with the PD-L1 inhibitor atezolizumab. Most approvals have been based on single arm phase II data. See Tecentriq Prescribing Information, Genentech, April 2017, Opdivo Prescribing Information, Bristol-Myers Squibb, September 2017, Imfinzi Prescribing Information, AstraZeneca, May 2017, and Bavencio Prescribing Information, EMD Serono, March 2017. However, in 2017, the results of the Phase III trial KEYNOTE-045 confirmed that patients treated with pembrolizumab had significantly longer survival when compared to standard second-line chemotherapy. Bellmunt J et al., N Engl J Med. 2017; 376: 1015-26. DOI: 10.1056/NEJMoa1613683. This led to the routine approval of pembrolizumab as a second-line treatment for patients with locally advanced or metastatic urothelial carcinoma (mUC; Keytruda Prescribing Information, Merck, September 2017). The approval was based on a median OS of 10.3 months for pembrolizumab compared to a median OS of 7.4 months for taxane chemotherapy or vinflunine. Bellmunt J et al., N Engl J Med. 2017; 376: 1015-26. DOI: 10.1056/NEJMoa1613683. CPI was subsequently approved for sale in Europe and is expected to be approved in Asia. Other PD-1 and PD-L1 inhibitors are currently being evaluated in clinical trials for urothelial carcinoma as first-line and second-line therapies. Mullane SA and Bellmunt J. Curr Opin Urol. 2016; 26: 556-63.

Currently, there are no therapies approved for patients with locally advanced or mUC previously treated with CPIs.

Bladder cancer

Bladder cancer accounts for approximately 5% of all new cancer cases in the United States in men (fifth most common neoplasm) and 3% in women (eighth most common neoplasm). The incidence is slowly increasing with the aging population. The American Cancer Society (cancer.org) estimates that there are 81,400 new cases each year, including 62,100 in men and 19,300 in women, accounting for 4.5% of all cancer cases. The age-adjusted incidence rate is 20/100,000 for both men and women in the United States. An estimated 17,980 deaths are attributed to bladder cancer each year (13,050 in men and 4,930 in women), accounting for 3% of cancer-related deaths. Bladder cancer incidence and mortality increase substantially with age and will become an increasing problem as the population becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is treated with a combination of transurethral resection (TUR) of the bladder and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points to the limitations of TUR. TUR alone cannot cure most muscle-invasive cancers. Radical cystectomy and urinary tract diversion are the most effective means of eliminating cancer, but have an undeniable impact on urinary and sexual function. There continues to be a considerable need for treatment modalities that benefit bladder cancer patients.

There is a considerable need for additional therapeutic approaches for urothelial carcinoma and bladder cancer. These include the use of antibodies and antibody-drug conjugates as therapeutic modalities.

3. Summary of the invention

Provided herein are methods of using antibody drug conjugates (ADCs) that bind 191P4D12 to treat various cancers in a subject, including a subject with previously treated locally advanced or metastatic urothelial carcinoma.

In some embodiments, prior treatment includes platinum-based chemotherapy. In certain embodiments, prior treatment includes immune checkpoint inhibitors (CPIs). In other embodiments, prior treatment includes both platinum-based chemotherapy and CPIs.

Embodiment 1. A method of treating urothelial carcinoma or bladder cancer in a human subject having liver metastases, the method comprising administering to the subject having liver metastases an effective amount of an antibody drug conjugate,

wherein the subject has received immune checkpoint inhibitor (CPI) therapy, and

The antibody-drug conjugate comprises an antibody or an antigen-binding fragment thereof bound to 191P4D12 coupled to one or more monomethyl auristatin E (MMAE) units, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a complementarity determining region (CDR) having an amino acid sequence of the heavy chain variable region as shown in SEQ ID NO: 22; and a light chain variable region comprising a CDR having an amino acid sequence of the CDR of the light chain variable region as shown in SEQ ID NO: 23.

Embodiment 2. A method of treating urothelial carcinoma or bladder cancer in a human subject having a primary tumor site in the upper urinary tract, the method comprising administering to the subject having a primary tumor site in the upper urinary tract an effective amount of an antibody drug conjugate,

The subject has received immune checkpoint inhibitor (CPI) therapy, and

The antibody-drug conjugate comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 coupled to one or more monomethyl auristatin E (MMAE) units, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a complementarity determining region (CDR) having an amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 22; and a light chain variable region comprising a CDR having an amino acid sequence of the CDR of the light chain variable region shown in SEQ ID NO: 23.

Embodiment 3. A method of treating urothelial carcinoma or bladder cancer in a human subject, the method comprising administering to the subject an effective amount of an antibody drug conjugate,

The subject has received immune checkpoint inhibitor (CPI) therapy.

wherein the subject has progression or recurrence of the cancer during or after the CPI therapy, and

The antibody-drug conjugate comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 coupled to one or more monomethyl auristatin E (MMAE) units, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a complementarity determining region (CDR) having an amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 22; and a light chain variable region comprising a CDR having an amino acid sequence of the CDR of the light chain variable region shown in SEQ ID NO: 23.

Embodiment 4. The method of any one of Embodiments 1 to 3, wherein the subject has a duration of response of at least or about 7 months following the treatment.

Embodiment 5. The method of any one of Embodiments 1 to 3, wherein the subject has a duration of response in the range of 5 to 9 months following said treatment.

Embodiment 6. The method of embodiment 1, wherein the subject has a progression-free survival of at least or about 4 months after the treatment.

Embodiment 7. The method of embodiment 2 or 3, wherein the subject has a progression-free survival of at least or about 5 months after the treatment.

Embodiment 8. The method of embodiment 1, wherein the subject has a progression-free survival in the range of 4 to 9 months following the treatment.

Embodiment 9. The method of embodiment 2 or 3, wherein the subject has a progression-free survival in the range of 5 to 9 months after said treatment.

Embodiment 10. The method of embodiment 1, wherein the subject has an overall survival of at least or about 9 months following the treatment.

Embodiment 11. The method of embodiment 2, wherein the subject has an overall survival of at least or about 12 months following the treatment.

Embodiment 12. The method of embodiment 3, wherein the subject has an overall survival of at least or about 11 months following the treatment.

Embodiment 13. The method of any one of Embodiments 1 to 3, wherein the subject has an overall survival in the range of 9 to 19 months following said treatment.

Embodiment 14. The method of any one of Embodiments 1 to 3, wherein the population of subjects is treated by the method, and wherein the percentage of subjects in the treated population with a complete response is at least or about 4%.

Embodiment 15. The method of any one of Embodiments 1 to 3, wherein the population of subjects is treated by the method, and wherein the percentage of subjects in the treated population having a partial response is at least or about 35%.

Embodiment 16. The method of embodiment 1, wherein the population of subjects is treated by the method, and wherein the overall response rate in the treated population is at least or about 35%.

Embodiment 17. The method of embodiment 2, wherein the population of subjects is treated by the method, and wherein the overall response rate in the treated population is at least or about 43%.

Embodiment 18. The method of embodiment 3, wherein the population of subjects is treated by the method, and wherein the overall response rate in the treated population is at least or about 39%.

Embodiment 19. The method of any one of Embodiments 1 to 3, wherein the population of subjects is treated by the method, and wherein the percentage of subjects with stable disease in the treated population is at least or about 30%.

Embodiment 20. The method of any one of Embodiments 1 to 3, wherein the population of subjects is treated by the method, and wherein the median duration of response in the treated population is at least or about 7 months.

Embodiment 21. The method of any one of Embodiments 1 to 3, wherein the population of subjects is treated by the method, and wherein the duration of response in the treated population ranges from 5 to 9 months.

Embodiment 22. The method of embodiment 1, wherein the population of subjects is treated by the method, and wherein the median progression-free survival in the treated population is at least or about 4 months.

Embodiment 23. The method of embodiment 1, wherein the population of subjects is treated by the method, and wherein the progression-free survival in the treated population is in the range of 4 to 9 months.

Embodiment 24. The method of embodiment 2 or 3, wherein the population of subjects is treated by the method, and wherein the median progression-free survival in the treated population is at least or about 5 months.

Embodiment 25. The method of embodiment 2 or 3, wherein the population of subjects is treated by the method, and wherein the progression-free survival in the treated population is in the range of 5 to 9 months.

Embodiment 26. The method of embodiment 1, wherein the population of subjects is treated by the method, and wherein the median overall survival in the treated population is at least or about 9 months.

Embodiment 27. The method of embodiment 2, wherein the population of subjects is treated by the method, and wherein the median overall survival in the treated population is at least or about 12 months.

Embodiment 28. The method of embodiment 3, wherein the population of subjects is treated by the method, and wherein the median overall survival in the treated population is at least or about 11 months.

Embodiment 29. The method of any one of Embodiments 1 to 3, wherein the population of subjects is treated by the method, and wherein the overall survival in the treated population is in the range of 9 to 19 months.

Embodiment 30. The method of any one of embodiments 1 to 3, wherein the complete response rate in a population of subjects treated with said method is at least or about 4%.

Embodiment 31. The method of any one of Embodiments 1 to 3, wherein the partial response rate in a population of subjects treated with said method is at least or about 35%.

Embodiment 32. The method of embodiment 1, wherein the overall response rate in a population of subjects treated with the method is at least or about 35%.

Embodiment 33. The method of embodiment 2, wherein the overall response rate in a population of subjects treated with the method is at least or about 43%.

Embodiment 34. The method of embodiment 3, wherein the overall response rate in a population of subjects treated with the method is at least or about 39%.

Embodiment 35. The method of any one of Embodiments 1 to 3, wherein the median duration of response in a population of subjects treated with said method is at least or about 7 months.

Embodiment 36. The method of any one of embodiments 1 to 3, wherein the duration of response in the subject population treated with said method is 5 to 9 months.

Embodiment 37. The method of embodiment 1, wherein the median progression-free survival in a population of subjects treated with the method is at least or about 4 months.

Embodiment 38. The method of any one of Embodiments 1 to 3, wherein the progression-free survival of the subject population treated with said method is 4 to 9 months.

Embodiment 39. The method of embodiment 2 or 3, wherein the median progression-free survival in a population of subjects treated with the method is at least or about 5 months.

Embodiment 40. The method of embodiment 2 or 3, wherein the progression-free survival of the subject population treated with said method is 5 to 9 months.

Embodiment 41. The method of embodiment 1, wherein the median overall survival of a population of subjects treated with said method is at least or about 9 months.

Embodiment 42. The method of embodiment 2, wherein the median overall survival of a population of subjects treated with the method is at least or about 12 months.

Embodiment 43. The method of embodiment 3, wherein the median overall survival of a population of subjects treated with the method is at least or about 11 months.

Embodiment 44. The method of any one of embodiments 1 to 3, wherein the overall survival of the subject population treated with said method is 9 to 19 months.

Embodiment 45. The method of any one of Embodiments 1 to 44, wherein the subject is a subject receiving platinum-based chemotherapy.

Embodiment 46. The method of any one of Embodiments 1 to 45, wherein the cancer is urothelial carcinoma, and wherein the human subject has locally advanced or metastatic urothelial carcinoma.

Embodiment 47. The method of any one of embodiments 1 to 46, wherein the subject has one or more conditions selected from the group consisting of:

(i) Absolute neutrophil count (ANC) not less than 1500/mm ³ ;

(ii) platelet count not less than 100 × 109 cells/L;

(iii) hemoglobin not less than 9 g/dL;

(iv) serum bilirubin not exceeding 1.5 times the upper limit of normal (ULN) or not exceeding 3 times the ULN for patients with Gilbert's disease;

(v) CrCl not less than 30 mL/min, and

(vi) Alanine aminotransferase and aspartate aminotransferase not exceeding 3 times ULN.

Embodiment 48. The method of embodiment 47, wherein the subject has all of conditions (i) to (vi) as described in embodiment 47.

Embodiment 49. The method of embodiment 47 or 48, wherein the CrCl is measured by 24-hour urine collection or estimated by the Cockcroft-Gault criteria.

Embodiment 50. The method of any one of Embodiments 1 to 49, wherein the subject has no more than grade 2 sensory or motor neuropathy.

Embodiment 51. The method of any one of Embodiments 1 to 50, wherein the subject does not have active central nervous system metastases.

Embodiment 52. The method of any one of Embodiments 1 to 51, wherein the subject does not have uncontrolled diabetes.

Embodiment 53. The method of embodiment 52, wherein the uncontrolled diabetes is determined by a hemoglobin A1c (HbA1c) of not less than 8% or a HbA1c between 7% and 8% accompanied by related diabetic symptoms not otherwise explained.

Embodiment 54. The method of embodiment 53, wherein the associated diabetes symptoms comprise or consist of polyuria, polydipsia, or both polyuria and polydipsia.

Embodiment 55. The method of any one of Embodiments 1 to 54, wherein the CPI therapy is therapy with a programmed death receptor-1 (PD-1) inhibitor.

Embodiment 56. The method of any one of Embodiments 1 to 54, wherein the CPI therapy is therapy with a programmed death-ligand 1 (PD-L1) inhibitor.

Embodiment 57. The method of embodiment 55, wherein the PD-1 inhibitor is nivolumab or pembrolizumab.

Embodiment 58. The method of embodiment 56, wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

Embodiment 59. The method of any one of Embodiments 1 to 58, wherein the antibody or antigen-binding fragment thereof comprises: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 12, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 13, and a 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, and 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.

Embodiment 60. The method of any one of embodiments 1 to 58, wherein the antibody or antigen-binding fragment thereof comprises: a CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 9, a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 10, a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 11; a CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 12, a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 13, and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 14, or

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.

Embodiment 61. A method as described in any one of Embodiments 1 to 60, 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.

Embodiment 62. A method as described in any one of embodiments 1 to 61, 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.

Embodiment 63. A method as described in any one of Embodiments 1 to 61, wherein the antigen binding fragment is Fab, F(ab')2, Fv or scFv.

Embodiment 64. The method of any one of Embodiments 1 to 62, wherein the antibody is a fully human antibody.

Embodiment 65. The method of any one of Embodiments 1 to 62 and 64, wherein the antibody is IgG1 and the light chain is a kappa light chain.

Embodiment 66. The method of any one of Embodiments 1 to 65, wherein the antibody or antigen-binding fragment thereof is recombinantly produced.

Embodiment 67. The method of any one of Embodiments 1 to 66, wherein the antibody or antigen-binding fragment is coupled to each unit of MMAE via a linker.

Embodiment 68. The method of Embodiment 67, wherein the linker is an enzyme-cleavable linker, and wherein the linker forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof.

Embodiment 69. A method as described in embodiment 67 or 68, wherein the linker has the following formula: -Aa-Ww-Yy-; wherein -A- is a stretcher unit, a is 0 or 1; -W- is an amino acid unit, w is an integer ranging from 0 to 12; and -Y- is a spacer unit, y is 0, 1 or 2.

Embodiment 70. A method as described in Embodiment 69, wherein the stretcher unit has a structure of the following formula (1); the amino acid unit is valine-citrulline; and the spacer unit is a PAB group comprising a structure of the following formula (2):

Embodiment 71. A method as described in Embodiment 69 or 70, wherein the stretcher unit forms a bond with the sulfur atom of the antibody or antigen-binding fragment thereof; and wherein the spacer unit is linked to MMAE via a carbamate group.

Embodiment 72. The method of any one of Embodiments 1 to 71, wherein the ADC comprises 1 to 20 MMAE units per antibody or antigen-binding fragment thereof.

Embodiment 73. The method of any one of Embodiments 1 to 72, wherein the ADC comprises 1 to 10 MMAE units per antibody or antigen-binding fragment thereof.

Embodiment 74. The method of any one of Embodiments 1 to 73, wherein the ADC comprises 2 to 8 MMAE units per antibody or antigen-binding fragment thereof.

Embodiment 75. The method of any one of Embodiments 1 to 74, wherein the ADC comprises 3 to 5 MMAE units per antibody or antigen-binding fragment thereof.

Embodiment 76. The method of any one of Embodiments 1 to 73, wherein the ADC has the following structure:

wherein L- represents the antibody or antigen-binding fragment thereof, and p is 1 to 10.

Embodiment 77. A method as described in Embodiment 76, wherein p is 2 to 8.

Embodiment 78. A method as described in Embodiment 76 or 77, wherein p is 3 to 5.

Embodiment 79. The method of any one of Embodiments 76 to 78, wherein p is 3 to 4.

Embodiment 80. The method of any one of Embodiments 77 to 79, wherein p is about 4.

Embodiment 81. The method of any one of Embodiments 76 to 79, wherein the effective amount of the antibody drug conjugate has an average p-value of about 3.8.

Embodiment 82. The method of any one of Embodiments 1 to 81, wherein the ADC is administered at a dose of about 1 to about 10 mg/kg of the subject's body weight, about 1 to about 5 mg/kg of the subject's body weight, about 1 to about 2.5 mg/kg of the subject's body weight, or about 1 to about 1.25 mg/kg of the subject's body weight.

Embodiment 83. The method of any one of Embodiments 1 to 82, wherein the ADC is administered at a dose of about 0.25 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1.0 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, about 1.75 mg/kg, about 2.0 mg/kg, about 2.25 mg/kg, or about 2.5 mg/kg of the subject's body weight.

Embodiment 84. The method of any one of Embodiments 1 to 83, wherein the ADC is administered at a dose of about 1 mg/kg body weight of the subject.

Embodiment 85. The method of any one of Embodiments 1 to 83, wherein the ADC is administered at a dose of about 1.25 mg/kg body weight of the subject.

Embodiment 86. The method of any one of Embodiments 1 to 85, wherein the ADC is administered by intravenous (IV) injection or infusion.

Embodiment 87. The method of any one of Embodiments 1 to 86, wherein the ADC is administered by three IV injections or infusions per four-week cycle.

Embodiment 88. The method of any one of Embodiments 1 to 87, wherein the ADC is administered by IV injection or infusion on Day 1, Day 8, and Day 15 of each four-week cycle.

Embodiment 89. The method of any one of Embodiments 1 to 88, wherein the ADC is administered by three approximately 30-minute IV injections or infusions per four-week cycle.

Embodiment 90. The method of any one of Embodiments 1 to 89, wherein the ADC is administered by IV injection or infusion over about 30 minutes on Days 1, 8, and 15 of each four-week cycle.

Embodiment 91. The method of any one of Embodiments 1 to 90, wherein the ADC is formulated in a pharmaceutical composition comprising L-histidine, polysorbate-20 (TWEEN-20), and trehalose dehydrate.

Embodiment 92. The method of any one of Embodiments 1 to 91, 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 wherein the pharmaceutical composition has a pH of about 6.0 at 25°C.

Embodiment 93. The method of any one of Embodiments 1 to 91, 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, and wherein the pharmaceutical composition has a pH of about 6.0 at 25°C.

Embodiment 94. A method as described in any one of Embodiments 1 to 93, wherein the ADC is enfortumab vedotin (EV) or a biosimilar thereof, wherein the EV is administered at a dose of about 1.25 mg/kg body weight of the subject, and wherein the dose is administered by IV injection or infusion over about 30 minutes on Days 1, 8, and 15 of each four-week cycle.

Embodiment 95. The method of any one of Embodiments 1 to 94, wherein the population of subjects has a complete response following said treatment.

Embodiment 96. The method of any one of Embodiments 1 to 94, wherein the population of subjects has a partial response following the treatment.

Embodiment 97. The method of any one of Embodiments 1 to 94, wherein the population of subjects has a complete response or a partial response following the treatment.

Embodiment 98. The method of any one of Embodiments 1 to 94, wherein the population of subjects has stable disease following said treatment.

4. Description of the Figures

Figures 1A to 1E depict the nucleotide and amino acid sequences of the connexin-4 protein (Figure 1A), the nucleotide and amino acid sequences of the heavy chain (Figure 1B) and light chain (Figure 1C) of Ha22-2(2.4)6.1, and the amino acid sequences of the heavy chain (Figure 1D) and light chain (Figure 1E) of Ha22-2(2.4)6.1.

Figure 2A depicts the overall study design for the clinical study described in Section 6.1. Figure 2B depicts the study protocol for the clinical study described in Section 6.1. Figure 2C depicts the EuroQOL 5 Dimensions (EQ-5D-5L) described in Section 6.1.

Figure 3 depicts the analytical efficacy boundaries of the clinical study described in Section 6.1. #Note: The predetermined efficacy boundary IDMC for IA (=0.00661) for OS was adjusted using 299 deaths observed at the initial data snapshot for each initial cutoff value. Two more deaths were reported after the initial data snapshot, and therefore 301 deaths are shown in the primary analysis table for OS in TLR.

Figure 4 depicts the overall survival Kaplan Meier curves - FAS for the clinical study described in Section 6.1.

Figure 5 depicts the overall survival subcohort results of the clinical study described in Section 6.1.

Figure 6 depicts the PFS Kaplan Meier curves - FAS for the clinical study described in Section 6.1.

FIG. 7 depicts the results of subgroup analyses, which show that there is a progression-free survival benefit of enroku vedotin (EV) in multiple subgroups.

FIG8 depicts the results of subgroup analyses of overall response rate.

Figure 9 depicts the Kaplan-Meier estimates of investigator-assessed duration of response in all patients with a confirmed complete or partial response, based on treatment group.

Figures 10A to 10D depict Kaplan-Meier estimates of overall survival based on subgroups. Figure 10A depicts a subgroup aged ≥65 years. Figure 10B depicts a subgroup with liver metastases. Figure 10C depicts a subgroup with primary upper urinary tract disease. Figure 10D depicts a subgroup with no response to a prior PD-1/L1 inhibitor. Abbreviations: CI, confidence interval; HR, hazard ratio; PD-1/L1, programmed cell death protein-1 or programmed death-ligand 1.

Figures 11A to 11D depict Kaplan-Meier estimates of progression-free survival based on subgroups. Figure 11A depicts a subgroup aged ≥65 years. Figure 11B depicts a subgroup with liver metastases. Figure 11C depicts a subgroup with primary upper urinary tract disease. Figure 11D depicts a subgroup with no response to a prior PD-1/L1 inhibitor. Abbreviations: CI, confidence interval; HR, hazard ratio; PD-1/L1, programmed cell death protein-1 or programmed death-ligand 1.

Figure 12 depicts treatment-related adverse events (safety population). Abbreviations: EV, enroku vedotin; PD-1/L1, programmed cell death protein-1 or programmed death-ligand 1; SC, standard chemotherapy.

Figure 13 depicts exposure-adjusted grade ≥3 treatment-related adverse events in the difficult-to-treat subgroup. Abbreviations: EV, enrofloxacin; PD-1/L1, programmed cell death protein-1 or programmed death-ligand 1; SC, standard chemotherapy.

5. Specific implementation methods

Before the present disclosure is further described, it is to be understood that the present disclosure is not limited to the particular embodiments set forth herein and it is to be further understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

5.1 Definitions

The techniques and procedures described or referred to herein include those generally well understood and/or commonly employed by those skilled in the art using conventional methods, such as 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 the widely used methods described in Antibody Engineering, Vols. 1 and 2 (Kontermann and Dübel eds., 2nd ed. 2010).

Unless otherwise defined herein, the technical and scientific terms used in this specification should have the meanings commonly understood by those of ordinary skill in the art. For the purpose of interpreting this specification, the following terminology descriptions will apply and, whenever appropriate, terms used in the singular will also include the plural form and vice versa. In the event of a conflict between any description of the term set forth and any document incorporated herein by reference, the term description set forth below shall prevail.

The terms "antibody", "immunoglobulin" or "Ig" are used interchangeably herein and are used in the broadest sense and specifically encompass, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with multi-epitope or mono-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, as long as they exhibit the desired biological activity), single-chain antibodies, and fragments thereof, as described below. The antibody may be a human antibody, a humanized antibody, a chimeric antibody, and/or an affinity matured antibody, as well as antibodies from other species (e.g., mice and rabbits, etc.). The term "antibody" is intended to include polypeptide products of B cells within the class of immunoglobulin polypeptides, which are capable of binding to specific molecular antigens and are composed of two pairs of identical polypeptide chains, each pair having a heavy chain (about 50-70 kDa) and a light chain (about 25 kDa), each amino terminal portion of each chain including a variable region having about 100 to about 130 or more amino acids, and each carboxyl terminal portion of each chain including a constant region. See, for example, Antibody Engineering (Borrebaeck, ed., 2nd ed., 1995); and Kuby, Immunology (3rd ed., 1997). In a specific embodiment, a specific molecular antigen can be bound by an antibody (including a polypeptide or epitope) provided herein. Antibodies also include, but are not limited to, synthetic antibodies, antibodies produced in a recombinant manner, camelized antibodies, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, which refers to a portion of an antibody heavy chain 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 Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab) ₂ fragments, F(ab') ₂ fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies and minibodies. In particular, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen binding domains or molecules containing an antigen binding site that binds to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found in, for example, 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; Plückthun and Skerra, 1989, Meth. Enzymol. 178: 497-515; and Day, Advanced Immunochemistry (2nd ed. 1990). The antibodies provided herein may belong to any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. The antibody may be an agonist antibody or an antagonist antibody.

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 naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single antigenic site. In contrast to polyclonal antibody preparations, which may include different antibodies for different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

An "antigen" is a structure to which an antibody can selectively bind. The target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, such as present on or in a cell (e.g., a cancer cell).

A "complete" antibody is an antibody that comprises an antigen binding site as well as CL and at least heavy chain constant regions CH1, CH2 and CH3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In certain embodiments, the complete antibody has one or more effector functions.

The terms "antigen binding fragment", "antigen binding domain", "antigen binding region" and similar terms refer to the portion of an antibody that contains the amino acid residues that interact with the antigen and imparts its specificity and affinity to the antigen (e.g., CDR) to the binding agent. As used herein, "antigen binding fragment" includes "antibody fragments" that contain a portion of an intact antibody, such as an antigen binding region or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , and Fv fragments; bifunctional antibodies and di-bifunctional antibodies (see, 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., 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.

The term "binds/binding" refers to the interaction between molecules, including, for example, the formation of a complex. The interaction may be, for example, a non-covalent interaction, including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. The complex may also include the combination of two or more molecules bound together by covalent or non-covalent bonds, interactions, or forces. The total non-covalent interaction strength between a single antigen binding site on an antibody and a single epitope of a target molecule (e.g., an antigen) is the affinity of the antibody or functional fragment for the epitope. The ratio (koff/kon) of the dissociation rate (koff) of a binding molecule (e.g., an antibody) to a monovalent antigen is the dissociation constant _KD , which is negatively correlated with affinity. The lower the _KD value, the higher the antibody affinity. _{The KD} value varies due to different complexes of the antibody and the antigen and depends on both _kon and _koff . The dissociation constant _KD of the antibodies provided herein can be determined using any of the methods provided herein or any other method well known to those skilled in the art. Affinity at one binding site does not always reflect the true strength of interaction between antibody and antigen. When a complex antigen containing multiple, repeated antigenic determinants (e.g., a multivalent antigen) contacts an antibody containing multiple binding sites, the interaction between antibody and antigen at one site will increase the probability of reaction at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called avidity.

In conjunction with the antibodies or antigen-binding fragments thereof described herein, terms such as "bind to,""that specifically binds to," and similar terms are also used interchangeably herein and refer to a binding molecule having an antigen binding domain that specifically binds to an antigen (e.g., a polypeptide). An antibody or antigen-binding fragment that binds to or specifically binds to an antigen may have cross-reactivity with related antigens. In certain embodiments, an antibody or antigen-binding fragment that binds to or specifically binds to an antigen does not have cross-reactivity with other antigens. An antibody or antigen-binding fragment that binds to or specifically binds to an antigen may be detected, for example, by immunoassay, Or other techniques known to those skilled in the art to identify. In some embodiments, when the antibody or antigen-binding fragment binds to the antigen with a higher affinity than any cross-reactive antigen, as determined using experimental techniques (e.g., radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA)), the antibody or antigen-binding fragment binds to or specifically binds to the antigen. Typically, a specific or selective reaction will be at least twice the background signal or noise and may exceed 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul, ed., 2nd edition, 1989). In certain embodiments, the degree of binding of an antibody or antigen-binding fragment to a "non-target" protein is less than about 10% of the binding of a binding molecule or antigen-binding domain to its specific target antigen, such as determined by fluorescence activated cell sorting (fluorescence activated cell sorting; FACS) analysis or RIA. For example, the term "specific binding", "specifically binding to" or "specific for..." means a binding that is measurably different from a nonspecific interaction. Specific binding can be measured, for example, by comparing the binding of the assay molecule to the binding of a control molecule, which is typically 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 (e.g., an excess of unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, specific binding is indicated. Antibodies or antigen-binding fragments that bind to an antigen include antibodies or antigen-binding fragments that can bind to the antigen with an affinity sufficient to make the binding molecule suitable for use as, for example, a diagnostic agent in targeting the antigen. In certain embodiments, the antibody or antigen-binding fragment binds to an antigen with a dissociation constant ( _KD ) less than or equal to 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. In certain embodiments, the antibody or antigen-binding fragment binds to an epitope of the antigen that is conserved between antigens from different species (e.g., between human and cynomolgus species).

"Binding affinity" generally refers to the total strength 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 can generally be represented by a dissociation constant ( _KD ). Affinity can be measured by common methods known in the art (including those described herein). Low-affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigen faster and tend to remain bound for a longer time. A variety of methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure. Specific illustrative embodiments include the following. In one embodiment, " _KD " or " _KD value" can be measured by an assay known in the art, such as by a binding assay. _KD can be measured using RIA, for example, using a Fab form of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81). _KD or _KD values can also be determined by using biolayer interferometry (BLI) or surface plasmon resonance (SPR), by Use e.g. QK384 system, or through Use e.g. TM-2000 or The "on-rate" or "association rate" or "kon" can also be measured using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, using, for example, QK384, TM-2000 or TM-3000 system for determination.

In certain embodiments, the antibodies or antigen-binding fragments may comprise "chimeric" sequences in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived 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).

In certain embodiments, an antibody or antigen-binding fragment may include a portion of a "humanized" form of a non-human (e.g., murine) antibody, which is a chimeric antibody (e.g., a receptor antibody) comprising a human immunoglobulin, wherein the native CDR residues are replaced by residues from a non-human species (e.g., mouse, rat, rabbit, or non-human primate) (e.g., a donor antibody) with the desired specificity, affinity, and ability. In some cases, one or more FR region residues of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may be included in residues not found in a receptor antibody or a donor antibody. These modifications are made to further optimize antibody performance. A humanized antibody heavy chain or light chain may include substantially all at least one or more variable regions, wherein all or substantially all CDRs correspond to the CDRs of non-human immunoglobulins, and all or substantially all FRs are FRs of human immunoglobulin sequences. In certain embodiments, a humanized antibody will include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For additional 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. Pat. Nos. 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

In certain embodiments, an antibody or antigen-binding fragment may include a portion of a "fully human antibody" or "human antibody", wherein the terms are used interchangeably herein and refer to antibodies comprising human variable regions and, for example, human constant regions. In specific embodiments, the term refers to antibodies comprising variable regions and constant regions of human origin. In certain embodiments, a "fully human" antibody may also encompass antibodies that bind to a polypeptide and are encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies comprising variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or has been prepared using any technique for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using various 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). 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. The methods described can also be used to prepare human monoclonal antibodies. Human antibodies can be prepared by administering an antigen to a transgenic animal (e.g., mouse) that has been modified to produce such antibodies in response to antigenic stimulation, but whose endogenous loci have been disabled (see, e.g., Jakobovits, 1995, Curr. Opin. Biotechnol. 6(5):561-66; Brüggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; and U.S. Pat. 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 produced via human B cell hybridoma technology.

In certain embodiments, the antibody or antigen-binding fragment may include a portion of a "recombinant human antibody", wherein the phrase includes human antibodies prepared, expressed, generated or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial human antibody library; antibodies isolated from transgenic and/or transchromosomal animals (e.g., mice or cattle) of human immunoglobulin genes (see, e.g., Taylor, L.D. et al. (1992) Nucl. Acids Res. 20: 6287-6295) or antibodies prepared, expressed, generated or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies may 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, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when an animal transgenic for human Ig sequences is used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, do not naturally exist within the human antibody germline antibody repertoire in vivo.

In certain embodiments, the antibody or antigen-binding fragment may comprise a portion of a "monoclonal antibody", wherein as used herein, the term refers to an antibody obtained from a population of substantially homogeneous antibodies (e.g., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts), and each monoclonal antibody will generally recognize a single epitope on the antigen. In specific embodiments, as used herein, a "monoclonal antibody" is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method for preparing an antibody. For example, monoclonal antibodies suitable for the present disclosure may be prepared by the hybridoma method first described by Kohler et al., 1975, Nature 256:495, or may be prepared using recombinant DNA methods in bacteria or eukaryotic or plant cells (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those 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 monoclonal antibodies expressed therefrom are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al., ed., 5th ed. 2002).

The typical 4-chain antibody unit is a heterotetrameric glycoprotein, which is composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds depending on the H chain isotype. Each H chain and L chain also have regularly separated intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) of each α and γ chain and four CH domains of μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at the other end. VL is compared with VH, and CL is compared with the first constant domain (CH1) of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain. VH and VL are paired together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al., eds., 8th ed. 1994); and Immunobiology (Janeway et al., ed., 5th ed. 2001).

The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. Conventional IgGs typically include two Fab regions, each residing on one of the two arms of a Y-shaped IgG structure. Each Fab region is typically composed of a variable region and a constant region of each of a heavy chain and a light chain. More specifically, the heavy chain variable region and constant region in the Fab region are VH and CH1 regions, and the light chain variable region and constant region in the Fab region are VL and CL regions. The VH, CH1, VL, and CL in the Fab region can be arranged in various ways to confer antigen binding ability according to the present disclosure. For example, the VH and CH1 regions can be located on one polypeptide, and the VL and CL regions can be located on separate polypeptides, similar to the Fab regions of conventional IgGs. 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.

The term "variable region", "variable domain", "V region" or "V domain" refers to a portion of the light chain or heavy chain of an antibody, which is usually located at the amino terminus of the light chain or heavy chain and is about 120 to 130 amino acids in length in the heavy chain and about 100 to 110 amino acids in length in the light chain, and is used for the binding and specificity of each specific antibody to its specific antigen. The variable region of the heavy chain may be referred to as "VH". The variable region of the light chain may be referred to as "VL". The term "variable" refers to the fact that certain segments of the variable region are widely different in sequence among antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed within the 110 amino acid span of the variable region. In practice, the V region consists of a weakly variable (e.g., relatively constant) extension of about 15-30 amino acids, called a framework region (FR), which is separated by a shorter region of greater variability (e.g., extreme variability), called a "hypervariable region", each of which is about 9-12 amino acids in length. The variable regions of the heavy and light chains each contain four FRs that largely adopt a β-folded configuration, and the FRs are connected by three hypervariable regions, which form a loop connecting the β-folded structure and form a part of the β-folded structure in some cases. The hypervariable regions in each chain are tightly combined with the hypervariable regions from other chains through the FRs to promote the formation of the antigen binding site of the antibody (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest (5th Edition, 1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as causing the antibody to participate in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable region is widely different in terms of sequence between different antibodies. In a specific embodiment, the variable region is a human variable region.

The term "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system used by Kabat et al. (see above) to compile antibody heavy chain variable regions or light chain variable regions. Using this numbering system, the actual linear amino acid sequence may contain a shortening of the FR or CDR corresponding to the variable domain or fewer or additional amino acids inserted therein. For example, the heavy chain variable domain may include a single amino acid insertion (residue 52a, according to Kabat) after residue 52 and three insertion residues (e.g., residues 82a, 82b, and 82c, etc., according to Kabat) after residue 82. For a given antibody, the Kabat numbering of the residues can be determined by aligning the homologous region of the antibody sequence with the "standard" Kabat numbering sequence. The Kabat numbering system is typically 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., see above). The "EU numbering system" or "EU index" (e.g., the EU index as reported in Kabat et al., supra) is often used when referring to residues in the constant region of an immunoglobulin heavy chain. The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

When used with reference to an antibody, the term "heavy chain" refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids, and the carboxyl-terminal portion includes a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types (e.g., isotypes), referred to as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The different heavy chains are of different sizes: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with a light chain, these different types of heavy chains produce the five well-known classes (e.g., isotypes) of antibodies, namely IgA, IgD, IgE, IgG, and IgM, including the four subclasses of IgG, i.e., IgG1, IgG2, IgG3, and IgG4.

When used in reference to antibodies, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion includes a variable region of about 100 to about 110 or more amino acids, and the carboxyl terminal portion includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called kappa (κ) or lambda (λ).

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 the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of the three hypervariable regions (L1, L2 or L3) within the non-framework region of the VL β-sheet framework of an antibody. Thus, CDRs are variable region sequences interspersed within framework region sequences.

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 most commonly used (see, e.g., Kabat et al., supra). Chothia actually refers to the position of the structural loop (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17). When numbering using the Kabat numbering convention, 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 the insertion at H35A and H35B; if neither 35A nor 35B exists, the loop end is at 32; if only 35A exists, the loop end is at 33; if both 35A and 35B exist, the loop end is at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Volume 2 (Kontermann and Dübel, ed., 2nd ed., 2010)). The "contact" hypervariable regions are based on 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 dedicated to immunoglobulins (IG), T-cell receptors (TCRs) and major histocompatibility complexes (MHCs) of humans and other vertebrates. Herein, CDRs are referred to with respect to amino acid sequences and positions within light or heavy chains. Since the "position" of CDRs within the immunoglobulin variable domain structure is conserved between species and is present in structures called loops, CDRs and framework residues are easily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into a receptor framework typically from human antibodies. Another numbering system (AHon) has been developed by Honegger and Plückthun, 2001, J. Mol. Biol. 309:657-70. The correspondence between numbering systems (including, for example, Kabat numbering and the IMGT unique numbering system) is well known to those skilled in the art (see, for example, Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are indicated in Table 1 below.

Table 1

The boundaries of a given CDR may vary according to the scheme used for identification. Therefore, unless otherwise specified, the term "CDR" and "complementarity determining region" or its region, such as a variable region, and individual CDRs (such as CDR-H1, CDR-H2) or its region of a given antibody should be understood to encompass the complementary determining region defined by any one of the known schemes described above. In some cases, it is specified to identify the scheme for one or more specific CDRs, such as the CDRs defined by the Kabat, Chothia or Contact methods. In other cases, the specific amino acid sequence of the CDR is given.

The hypervariable regions may comprise the following "extended hypervariable regions": 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.

The term "constant region" or "constant domain" refers to the carboxyl terminal portion of the light and heavy chains that is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as interaction with Fc receptors. The term refers to the portion of the immunoglobulin molecule that contains a more conserved amino acid sequence relative to the rest of the immunoglobulin (the variable region, which contains the antigen binding site). The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

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

Herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region may be removed, for example, during the preparation or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody. Thus, a composition of a complete antibody may include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residue has not been removed, and an antibody population comprising 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), etc. Such effector functions generally require an Fc region in combination with a binding region or binding domain (e.g., an antibody variable region or domain), and can be assessed using various assays known to those skilled in the art. A "variant Fc region" comprises an amino acid sequence that differs from 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 in the native sequence Fc region or in the Fc region of the parent polypeptide, such as about one to about ten amino acid substitutions or about one to about five amino acid substitutions, compared to the native sequence Fc region or compared to the Fc region of the parent polypeptide. The variant Fc region herein may have at least about 80% homology with the native sequence Fc region and/or the Fc region of the parent polypeptide, or at least about 90% homology therewith, such as at least about 95% homology therewith.

As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody) can specifically bind. An epitope may be a linear epitope or may be a conformational, nonlinear, or discontinuous epitope. For example, in the case of a polypeptide antigen, an epitope may be a continuous amino acid of a polypeptide (a "linear" epitope), or an epitope may comprise amino acids from two or more discontinuous regions of a polypeptide (a "conformational", "non-linear", or "discontinuous" epitope). It will be appreciated by those skilled in the art that, in general, a linear epitope may or may not be dependent on a secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule is bound to a group of amino acids, regardless of whether it is folded in a native three-dimensional protein structure. In other embodiments, the binding molecule requires that the amino acid residues constituting the epitope present a specific conformation (e.g., bend, twist, turn, or fold) to recognize and bind to the epitope.

The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be straight or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification). Also included within the definition are, for example, polypeptides containing one or more amino acid analogs (including but not limited to non-natural amino acids) and other modifications known in the art. It should be understood that because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in certain embodiments, a "polypeptide" may appear in a single chain or in two or more related chains.

As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopia, the European Pharmacopia, or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

"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 accelerators, antioxidants, binders, buffers, carriers, coatings, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavorings, humectants, lubricants, spices, preservatives, propellants, release agents, sterilants, sweeteners, solubilizers, wetting agents and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., Freunds' adjuvant) (complete or incomplete) or vehicle.

In one embodiment, each component is "pharmaceutically acceptable" in the following sense: compatible with the other ingredients of the pharmaceutical formulation and suitable for contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, for example, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th edition; Rowe et al., eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd edition; Ash and Ash, eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd edition; 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 used. In some embodiments, a pharmaceutically acceptable excipient is an aqueous pH buffer solution.

The abbreviation "MMAE" refers to monomethylauristatin E.

Unless the context indicates otherwise, a hyphen (-) indicates a point of attachment to a side molecule.

The term "chemotherapeutic agent" refers to all compounds that are effective in inhibiting tumor growth. Non-limiting examples of chemotherapeutic agents include: alkylating agents, such as nitrogen mustards, ethyleneimine compounds, and alkyl sulfonates; antimetabolites, such as folic acid, purine or pyrimidine antagonists; mitotic inhibitors, such as anti-tubulin 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, biomolecules, and small molecules.

As used herein, the term "conservative substitution" refers to amino acid substitutions that are known to those skilled in the art and can generally be performed without changing the biological activity of the resulting molecule. It is recognized by those skilled in the art that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially change biological activity (see, e.g., Watson et al., MOLECULARBIOLOGY OF THE GENE, The Benjamin/Cummings Pub. Co., p. 224 (4th edition 1987)). Such exemplary substitutions are preferably carried out according to those substitutions set forth in Tables 2 and 3. For example, such changes include substitutions of any one of isoleucine (I), valine (V) and leucine (L) for any other amino acid in these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Depending on the environment of a particular amino acid and its role in the three-dimensional structure of a protein, other substitutions may also be considered conservative. For example, glycine (G) and alanine (A) are often interchangeable, and alanine (A) and valine (V) are also interchangeable. Methionine (M), which is relatively hydrophobic, is often interchangeable with leucine and isoleucine, and is sometimes interchangeable with valine. Lysine (K) and arginine (R) are often interchangeable in positions where the significant feature of the amino acid residue is its charge, and the pK difference of the two amino acid residues is not significant. In a specific environment, there are still other changes that can be considered "conservative" (see, for example, Table 3 herein; "Biochemistry" 2nd edition, Lubert Stryer, ed. (Stanford University), pp. 13-15; 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 also permissible and can be determined empirically or based on known conservative substitutions.

Table 2 Amino Acid Abbreviations

Single letter Three letters Full name F Phe Phenylalanine L Leu Leucine S Ser Serine Y Tyr Tyrosine C Cys Cysteine W Trp Tryptophan P Pro Proline H His Histidine Q Gln Glutamine R Arg Arginine I Ile Isoleucine M Met Methionine T Thr Threonine N Asn Asparagine K Lys Lysine V Val Valine A Ala Alanine D Asp Aspartic acid E Glu Glutamate G Gly Glycine

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 to be found in the related natural protein.

The term "homology" or "homologous" is intended to refer to the sequence similarity between two polynucleotides or between two polypeptides. Similarity can be determined by comparing the position in each sequence compared for comparison purposes. If the given position of two polypeptide sequences is not the same, the similarity or conservation of the position can be determined by assessing the similarity of the amino acid at the position, for example, according to Table 3, assessing the similarity of the amino acid at the position. The degree of similarity between sequences varies with the number of matching or homologous positions shared by the sequences. Comparing two sequences to determine their percentage sequence similarity can be performed using software programs known in the art, such as Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999) described in the software program. Preferably, the comparison is performed using default parameters, and an example thereof is described as follows. A well-known alignment program that can be used in the art is BLAST set to default parameters. In particular, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; chain = two; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sorting mode = HIGH SCORE; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + Swissprotein + SPupdate + PIR. Details of these programs can be found in the National Center for Biotechnology Information.

The term "homolog" of a given amino acid sequence or nucleic acid sequence is intended to indicate that the corresponding sequence of the "homolog" has substantial identity or homology with the given amino acid sequence or nucleic acid sequence.

The determination of the percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 2264 2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 5873 5877. Such algorithms are incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215: 403. BLAST nucleotide searches can be performed using the NBLAST nucleotide program parameter set (e.g., for score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program parameter set, e.g., for a score of 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To achieve gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI BLAST can be used to perform an iterated search, which detects long-range relationships between molecules (supra). When utilizing BLAST, gapped BLAST, and PSI Blast programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm 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.

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

The term "cytotoxic agent" refers to a substance that inhibits or prevents cell expression activity, cell function and/or causes cell destruction. The term is intended to include radioactive isotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants or animals, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to, auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), aureomycin, maytansinoids, ricin, ricin A chain, combrestatin, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxols, cisplatin, cc1065, ethidium bromide, chloramphenicol ... tablets, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraquinone dione, actinomycin, diphtheria toxin, Pseudomonas aeruginosa exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain A chain), α-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitors and glucocorticoids and other chemotherapeutic agents, and radioisotopes such as At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² or Bi ²¹³ , P ³² and radioisotopes of Lu, including Lu ¹⁷⁷ . The antibody can also be coupled to an anticancer prodrug activating enzyme capable of converting the prodrug into its active form.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of a binding molecule (eg, antibody) or pharmaceutical composition provided herein sufficient to produce a desired result.

The terms "subject" and "patient" are used interchangeably. As used herein, in certain embodiments, the subject is a mammal, such as a non-primate (e.g., a cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., a monkey and a human). In a specific embodiment, the subject is a human. In one embodiment, the subject is a mammal, such as a human, diagnosed with a disease or condition. In another embodiment, the subject is a mammal, such as a human, at risk of developing a disease or condition.

"Administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body into a patient's body, such as by transmucosal, intradermal, intravenous, intramuscular delivery, and/or any other physical delivery method described herein or known in the art.

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

The terms "prevent", "preventing" and "prevention" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition or related symptoms (eg, cancer).

The term "cancer" or "cancer cell" is used herein to refer to tissues or cells found in neoplasms that possess characteristics that distinguish them from normal tissues or tissue cells. Such characteristics include, but are not limited to, degree of anaplasia, irregularity of shape, unclear cell outlines, nuclear size, changes in nuclear or cytoplasmic structure, other phenotypic changes, the presence of cellular proteins indicative of a cancerous or precancerous state, increased number of mitosis and metastatic capacity. Words related to "cancer" include carcinomas, sarcomas, tumors, epithelioma, leukemias, lymphomas, polyps and scirrhosis, transformations, neoplasms, and the like.

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

As used herein, "metastatic" cancer refers to cancer that has spread from where it started to different parts of the body.

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 stated value or range.

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

It should be understood that when an embodiment is described herein with the term "comprising", other similar embodiments described with the terms "consisting of" and/or "consisting essentially of" are also provided. It should also be understood that when an embodiment is described herein with the phrase "consisting essentially of", other similar embodiments described with the term "consisting of" are also provided.

The term "and/or" as used herein in phrases such as "A and/or B" is intended to include 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).

The term "variant" refers to a molecule that exhibits differences from a described type or specification, such as a protein having one or more different amino acid residues in corresponding positions of a specific described protein (e.g., the 191P4D12 protein shown in FIG. 1A ). Analogs are examples of variant proteins. Splicing isoforms and single nucleotide polymorphisms (SNPs) are other examples of variants.

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

5.2 Methods for treating cancer

5.2.1 General Approach to Treating Cancer and Approach to Patients Who Have Previously Received Cancer Treatment

Provided herein are methods of using antibody drug conjugates (ADCs) that bind 191P4D12 to treat various cancers in a subject, including a subject with previously treated locally advanced or metastatic urothelial carcinoma.

In one aspect, provided herein is a method for treating a subject's urothelial carcinoma using an antibody drug conjugate (ADC) in conjunction with 191P4D12. In certain embodiments, urothelial carcinoma has previously been treated with platinum-based chemotherapy and CPI. In some embodiments, urothelial carcinoma has previously been treated with platinum-based chemotherapy. In other embodiments, urothelial carcinoma has previously been treated with CPI. In one embodiment, the subject has previously been treated with platinum-based chemotherapy and CPI. In another embodiment, the subject has previously been treated with platinum-based chemotherapy. In another embodiment, the subject has previously been treated with CPI.

On the other hand, provided herein is a method for treating locally advanced urothelial carcinoma of a subject using an antibody drug conjugate (ADC) in conjunction with 191P4D12. In certain embodiments, locally advanced urothelial carcinoma has previously been treated with chemotherapy based on platinum and CPI. In some embodiments, locally advanced urothelial carcinoma has previously been treated with chemotherapy based on platinum. In other embodiments, locally advanced urothelial carcinoma has previously been treated with CPI. In one embodiment, the subject has previously been treated with chemotherapy based on platinum and CPI. In another embodiment, the subject has previously been treated with chemotherapy based on platinum. In another embodiment, the subject has previously been treated with CPI.

On the other hand, provided herein is a method for treating metastatic urothelial carcinoma of a subject using an antibody drug conjugate (ADC) in conjunction with 191P4D12. In certain embodiments, metastatic urothelial carcinoma has previously been treated with platinum-based chemotherapy and CPI. In some embodiments, metastatic urothelial carcinoma has previously been treated with platinum-based chemotherapy. In other embodiments, metastatic urothelial carcinoma has previously been treated with CPI. In one embodiment, the subject has previously been treated with platinum-based chemotherapy and CPI. In another embodiment, the subject has previously been treated with platinum-based chemotherapy. In another embodiment, the subject has previously been treated with CPI.

In one aspect, provided herein is a method for treating urothelial carcinoma of a subject using an antibody drug conjugate (ADC) in conjunction with 191P4D12. In certain embodiments, urothelial carcinoma has previously been treated with platinum-based chemotherapy and PD-1 inhibitors. In some embodiments, urothelial carcinoma has previously been treated with platinum-based chemotherapy. In other embodiments, urothelial carcinoma has previously been treated with PD-1 inhibitors. In one embodiment, the subject has previously been treated with platinum-based chemotherapy and PD-1 inhibitors. In another embodiment, the subject has previously been treated with platinum-based chemotherapy. In another embodiment, the subject has previously been treated with PD-1 inhibitors.

On the other hand, provided herein is a method for treating locally advanced urothelial carcinoma of a subject using an antibody drug conjugate (ADC) in conjunction with 191P4D12. In certain embodiments, locally advanced urothelial carcinoma has previously been treated with chemotherapy based on platinum and PD-1 inhibitors. In some embodiments, locally advanced urothelial carcinoma has previously been treated with chemotherapy based on platinum. In other embodiments, locally advanced urothelial carcinoma has previously been treated with PD-1 inhibitors. In one embodiment, the subject has previously been treated with chemotherapy based on platinum and PD-1 inhibitors. In another embodiment, the subject has previously been treated with chemotherapy based on platinum. In another embodiment, the subject has previously been treated with PD-1 inhibitors.

On the other hand, provided herein is a method for treating metastatic urothelial carcinoma of a subject using an antibody drug conjugate (ADC) in conjunction with 191P4D12. In certain embodiments, metastatic urothelial carcinoma has previously been treated with platinum-based chemotherapy and PD-1 inhibitors. In some embodiments, metastatic urothelial carcinoma has previously been treated with platinum-based chemotherapy. In other embodiments, metastatic urothelial carcinoma has previously been treated with PD-1 inhibitors. In one embodiment, the subject has previously been treated with platinum-based chemotherapy and PD-1 inhibitors. In another embodiment, the subject has previously been treated with platinum-based chemotherapy. In another embodiment, the subject has previously been treated with PD-1 inhibitors.

In one aspect, provided herein is a method for treating urothelial carcinoma of a subject using an antibody drug conjugate (ADC) in combination with 191P4D12. In certain embodiments, urothelial carcinoma has previously been treated with platinum-based chemotherapy and PD-L1 inhibitors. In some embodiments, urothelial carcinoma has previously been treated with platinum-based chemotherapy. In other embodiments, urothelial carcinoma has previously been treated with PD-L1 inhibitors. In one embodiment, the subject has previously been treated with platinum-based chemotherapy and PD-L1 inhibitors. In another embodiment, the subject has previously been treated with platinum-based chemotherapy. In another embodiment, the subject has previously been treated with PD-L1 inhibitors.

On the other hand, provided herein is a method for treating locally advanced urothelial carcinoma of a subject using an antibody drug conjugate (ADC) in combination with 191P4D12. In certain embodiments, locally advanced urothelial carcinoma has previously been treated with platinum-based chemotherapy and PD-L1 inhibitors. In some embodiments, locally advanced urothelial carcinoma has previously been treated with platinum-based chemotherapy. In other embodiments, locally advanced urothelial carcinoma has previously been treated with PD-L1 inhibitors. In one embodiment, the subject has previously been treated with platinum-based chemotherapy and PD-L1 inhibitors. In another embodiment, the subject has previously been treated with platinum-based chemotherapy. In another embodiment, the subject has previously been treated with PD-L1 inhibitors.

On the other hand, provided herein is a method for treating metastatic urothelial carcinoma of a subject using an antibody drug conjugate (ADC) in combination with 191P4D12. In certain embodiments, metastatic urothelial carcinoma has previously been treated with platinum-based chemotherapy and PD-L1 inhibitors. In some embodiments, metastatic urothelial carcinoma has previously been treated with platinum-based chemotherapy. In other embodiments, metastatic urothelial carcinoma has previously been treated with PD-L1 inhibitors. In one embodiment, the subject has previously been treated with platinum-based chemotherapy and PD-L1 inhibitors. In another embodiment, the subject has previously been treated with platinum-based chemotherapy. In another embodiment, the subject has previously been treated with PD-L1 inhibitors.

In some embodiments of the methods provided herein, the subject has been treated with one or more other cancer therapies. In certain embodiments of the methods provided herein, urothelial carcinoma (including locally advanced or metastatic urothelial carcinoma) has been treated with one or more other cancer therapies.

In some embodiments, the CPI provided for use in the methods may comprise or consist of any CPI as described in this section (Section 5.2.1).

In all methods provided herein, and in particular the methods described in the preceding six paragraphs: ADCs that can be used are described in Sections 3, 5.2, 5.3, 5.4, 5.5, and 6; selection of patients 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 this section (Section 5.2), infra Sections 5.6, 5.7, and 6; biomarkers that can be used to identify therapeutic agents, select patients, determine the results of these methods, and/or serve as criteria for these methods in any way are described herein and exemplified in this section (Section 5.2, including 5.2.1 and 5.2.2) and Section 6; biomarkers can be determined as described in Section 5.8 or as known in the art; the treatment outcomes of the methods provided herein can be improvements of the biomarkers described herein, such as those described and exemplified in this section (Section 5.2, including 5.2.2) and Section 6. Thus, it will be understood by those skilled in the art that the methods provided herein include all permutations and combinations of patients, therapeutic agents, dosing regimens, biomarkers, and treatment outcomes as described above and below.

In certain embodiments, the methods provided herein are used to treat subjects with urothelial cancer that expresses 191P4D12 RNA, 191P4D12 protein, or both 191P4D12 RNA and 191P4D12 protein. In some embodiments, the methods provided herein are used to treat subjects with urothelial cancer and subjects previously treated with platinum-based chemotherapy and CPIs, and the urothelial cancer expresses 191P4D12 RNA, 191P4D12 protein, or both 191P4D12 RNA and 191P4D12 protein. In one embodiment, the methods provided herein are used to treat subjects with urothelial cancer and subjects previously treated with platinum-based chemotherapy, and the urothelial cancer expresses 191P4D12 RNA, 191P4D12 protein, or both 191P4D12 RNA and 191P4D12 protein. In another embodiment, the methods provided herein are used to treat a subject having a urothelial cancer that expresses 191P4D12 RNA, expresses 191P4D12 protein, or expresses both 191P4D12 RNA and 191P4D12 protein and a subject that has been previously treated with a CPI.

In certain embodiments, the methods provided herein are used to treat subjects with locally advanced urothelial cancer, which expresses 191P4D12 RNA, 191P4D12 protein, or both 191P4D12 RNA and 191P4D12 protein. In some embodiments, the methods provided herein are used to treat subjects with locally advanced urothelial cancer and subjects previously treated with platinum-based chemotherapy and CPIs, which express 191P4D12 RNA, 191P4D12 protein, or both 191P4D12 RNA and 191P4D12 protein. In one embodiment, the methods provided herein are used to treat subjects with locally advanced urothelial cancer and subjects previously treated with platinum-based chemotherapy, which express 191P4D12 RNA, 191P4D12 protein, or both 191P4D12 RNA and 191P4D12 protein. In another embodiment, the methods provided herein are used to treat a subject having locally advanced urothelial cancer that expresses 191P4D12 RNA, expresses 191P4D12 protein, or expresses both 191P4D12 RNA and 191P4D12 protein and a subject that has been previously treated with a CPI.

In certain embodiments, the methods provided herein are used to treat subjects with metastatic cancer that express 191P4D12 RNA, 191P4D12 protein, or both 191P4D12 RNA and 191P4D12 protein. In some embodiments, the methods provided herein are used to treat subjects with metastatic urothelial cancer and subjects previously treated with platinum-based chemotherapy and CPIs, and the metastatic urothelial cancer expresses 191P4D12 RNA, 191P4D12 protein, or both 191P4D12 RNA and 191P4D12 protein. In one embodiment, the methods provided herein are used to treat subjects with metastatic urothelial cancer and subjects previously treated with platinum-based chemotherapy, and the metastatic urothelial cancer expresses 191P4D12 RNA, 191P4D12 protein, or both 191P4D12 RNA and 191P4D12 protein. In another embodiment, the methods provided herein are used to treat a subject having metastatic urothelial cancer that expresses 191P4D12 RNA, expresses 191P4D12 protein, or expresses both 191P4D12 RNA and 191P4D12 protein and a subject that has been previously treated with a CPI.

In some embodiments, 191P4D12 RNA expression in cancer is determined by polynucleotide hybridization, sequencing (assessing the relative abundance of the sequence) and/or PCR (including RT-PCR). In some embodiments, 191P4D12 protein expression in cancer is determined by analysis and/or Western blot in IHC, fluorescence activated cell sorting (FACS). 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 IHC methods.

In some embodiments, locally advanced or metastatic urothelial cancer is confirmed histologically, cytologically, or both histologically and cytologically. In some embodiments, locally advanced or metastatic bladder cancer is confirmed histologically, cytologically, or both histologically and cytologically.

In some embodiments, the treatable subject in the methods provided herein includes a subject who has received one or more other cancer treatments. In some embodiments, the treatable subject in the methods provided herein includes a subject who has received one or more other cancer treatments and whose cancer progresses or relapses after one or more treatments. Such one or more treatments include, for example, first-line or multi-line immune checkpoint inhibitor therapy, chemotherapy, and immune checkpoint inhibitor therapy and chemotherapy. In some embodiments, the treatable subject in the methods provided herein includes a subject whose cancer progresses or relapses after CPI treatment. In some embodiments, the treatable subject in the methods provided herein includes a subject whose cancer progresses or relapses after platinum-containing chemotherapy. In some embodiments, the treatable subject in the methods provided herein includes a subject whose cancer progresses or relapses after platinum-containing chemotherapy in a neoadjuvant setting. In some embodiments, the treatable subject in the methods provided herein includes a subject whose cancer progresses or relapses after platinum-containing chemotherapy in an adjuvant setting. In some embodiments, the treatable subject in the methods provided herein includes a subject whose cancer progresses or relapses after platinum-containing chemotherapy in an adjuvant setting. In some embodiments, the treatable subject in the methods provided herein includes a subject whose cancer progresses or relapses after platinum-containing chemotherapy in a neoadjuvant, locally advanced setting. In some embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after platinum-containing chemotherapy in a neoadjuvant, metastatic setting. In some embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after platinum-containing chemotherapy in an adjuvant, locally advanced setting. In some embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after platinum-containing chemotherapy in an adjuvant, metastatic setting. In some embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after platinum-containing chemotherapy in a metastatic setting. In some embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after platinum-containing chemotherapy in a locally advanced setting.

In some specific embodiments, the subject that can be treated in the method provided herein includes the subject whose cancer progresses or relapses after treatment with CPI and platinum-containing chemotherapy. In some specific embodiments, the subject that can be treated in the method provided herein includes the subject whose cancer progresses or relapses after treatment with CPI and platinum-containing chemotherapy in the neoadjuvant background. In some specific embodiments, the subject that can be treated in the method provided herein includes the subject whose cancer progresses or relapses after treatment with CPI and platinum-containing chemotherapy in the auxiliary background. In some specific embodiments, the subject that can be treated in the method provided herein includes the subject whose cancer progresses or relapses after treatment with CPI and platinum-containing chemotherapy in the local advanced background. In some specific embodiments, the subject that can be treated in the method provided herein includes the subject whose cancer progresses or relapses after treatment with CPI and platinum-containing chemotherapy in the metastatic background. In some specific embodiments, the subject that can be treated in the method provided herein includes the subject whose cancer progresses or relapses after treatment with CPI and platinum-containing chemotherapy in the neoadjuvant, local advanced background. In some specific embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after treatment with CPI and platinum-containing chemotherapy in a neoadjuvant, metastatic setting. In some specific embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after treatment with CPI and platinum-containing chemotherapy in an adjuvant, locally advanced setting. In some specific embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after treatment with CPI and platinum-containing chemotherapy in an adjuvant, metastatic setting.

In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a CPI. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a CPI in a neoadjuvant setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a CPI in an auxiliary setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a CPI in a locally advanced setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a CPI in a metastatic setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a CPI in a neoadjuvant, locally advanced setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a CPI in a neoadjuvant, locally advanced setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a CPI in a neoadjuvant, metastatic setting. In some embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after treatment with a CPI in an adjuvant, locally advanced setting. In some embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after treatment with a CPI in an adjuvant, metastatic setting.

In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a PD-1 inhibitor and platinum-containing chemotherapy. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a PD-1 inhibitor and platinum-containing chemotherapy in a neoadjuvant setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a PD-1 inhibitor and platinum-containing chemotherapy in an auxiliary setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a PD-1 inhibitor and platinum-containing chemotherapy in a locally advanced setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a PD-1 inhibitor and platinum-containing chemotherapy in a metastatic setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a PD-1 inhibitor and platinum-containing chemotherapy in a neoadjuvant, locally advanced setting. In some specific embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after treatment with a PD-1 inhibitor and platinum-containing chemotherapy in a neoadjuvant, metastatic setting. In some specific embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after treatment with a PD-1 inhibitor and platinum-containing chemotherapy in an adjuvant, locally advanced setting. In some specific embodiments, the subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses after treatment with a PD-1 inhibitor and platinum-containing chemotherapy in an adjuvant, metastatic setting.

In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-1 inhibitors. In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-1 inhibitors in a neoadjuvant setting. In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-1 inhibitors in an auxiliary setting. In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-1 inhibitors in a locally advanced setting. In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-1 inhibitors in a metastatic setting. In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-1 inhibitors in a neoadjuvant, locally advanced setting. In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-1 inhibitors in a neoadjuvant, metastatic setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses following treatment with a PD-1 inhibitor in the adjuvant, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or relapses following treatment with a PD-1 inhibitor in the adjuvant, metastatic setting.

In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a PD-L1 inhibitor and platinum-containing chemotherapy. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a PD-L1 inhibitor and platinum-containing chemotherapy in a neoadjuvant setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a PD-L1 inhibitor and platinum-containing chemotherapy in an adjuvant setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a PD-L1 inhibitor and platinum-containing chemotherapy in a locally advanced setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a PD-L1 inhibitor and platinum-containing chemotherapy in a metastatic setting. In some specific embodiments, the subject that can be treated in the methods provided herein includes a subject whose cancer progresses or relapses after treatment with a PD-L1 inhibitor and platinum-containing chemotherapy in a neoadjuvant, locally advanced setting. In some specific embodiments, the subjects treatable in the methods provided herein include subjects whose cancer has progressed or relapsed after treatment with a PD-L1 inhibitor and platinum-containing chemotherapy in a neoadjuvant, metastatic setting. In some specific embodiments, the subjects treatable in the methods provided herein include subjects whose cancer has progressed or relapsed after treatment with a PD-L1 inhibitor and platinum-containing chemotherapy in an adjuvant, locally advanced setting. In some specific embodiments, the subjects treatable in the methods provided herein include subjects whose cancer has progressed or relapsed after treatment with a PD-L1 inhibitor and platinum-containing chemotherapy in an adjuvant, metastatic setting.

In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-L1 inhibitors. In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-L1 inhibitors in a neoadjuvant setting. In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-L1 inhibitors in an auxiliary setting. In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-L1 inhibitors in a locally advanced setting. In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-L1 inhibitors in a metastatic setting. In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-L1 inhibitors in a neoadjuvant, locally advanced setting. In some embodiments, the treatable subjects in the methods provided herein include subjects whose cancer progresses or relapses after treatment with PD-L1 inhibitors in a neoadjuvant, metastatic setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed following treatment with a PD-L1 inhibitor in the adjuvant, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed following treatment with a PD-L1 inhibitor in the adjuvant, metastatic setting.

In certain embodiments, subjects treatable in the methods provided herein include those whose cancer progresses or recurs within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months after other treatments, including, for example, but not limited to, any one or any combination of the treatments described in the preceding paragraphs. In some specific embodiments, the subject's cancer progresses or recurs within 6 months after the platinum-based therapy. In other embodiments, the subject's cancer progresses or recurs within 12 months after the platinum-based therapy.

In some embodiments, the subject that can be treated in the methods provided herein has certain phenotypic or genotypic characteristics. In some embodiments, the subject has any permutation and combination of the phenotypic or genotypic characteristics described herein.

In some embodiments, histology, cytology or both histology and cytology determine phenotype or genotype characteristics. In some embodiments of the method provided herein, the histology and/or cytology of phenotype and/or genotype characteristics are determined as described in the American Society of Clinical Oncology/College of American Pathologists; ASCO/CAP guidelines, based on the most recently analyzed tissue execution, which is incorporated herein by reference as a whole. In some embodiments, phenotype or genotype characteristics are determined by including next generation sequencing (e.g., from Illumina, Inc's NGS), DNA hybridization and/or RNA hybridization.

In various aspects or embodiments of the methods provided herein (including the methods provided in this section (Section 5.2), such as the methods provided in this paragraph and the preceding paragraphs), the methods involve prior treatment with an immune checkpoint inhibitor as provided in the method. As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that reduces, inhibits, interferes with, or regulates one or more checkpoint proteins in whole or in part. A variety of checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 and its ligands PD-L1 and PD-L2 (Pardoll, Nature Reviews Cancer, 2012, 12, 252-264). Other exemplary checkpoint proteins include LAG-3, B7, TIM3 (HAVCR2), OX40 (CD134), GITR, CD137, CD40, VTCN1, IDO1, CD276, PVRIG, TIGIT, CD25 (IL2RA), IFNAR2, IFNAR1, CSF1R, VSIR (VISTA) or HLA. These proteins appear to be responsible for the co-stimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins appear to regulate and maintain the duration and amplitude of self-tolerance and physiological immune responses. Immune checkpoint inhibitors include antibodies or are derived from antibodies.

In certain embodiments, the checkpoint inhibitor of the method provided herein may be an inhibitor or activator for a checkpoint protein upregulated in cancer. In some specific embodiments, the checkpoint inhibitor of the method provided herein may be an inhibitor or activator for a checkpoint protein including the following: LAG-3, B7, TIM3 (HAVCR2), OX40 (CD134), GITR, CD137, CD40, VTCN1, IDO1, CD276, PVRIG, TIGIT, CD25 (IL2RA), IFNAR2, IFNAR1, CSF1R, VSIR (VISTA) or HLA. In some embodiments, the checkpoint inhibitor of the method provided herein may be an inhibitor or activator selected from the group consisting of: PD-1 inhibitor, PD-L1 inhibitor, PD-L2 inhibitor, CTLA-4 inhibitor, LAG-3 inhibitor, B7 inhibitor, TIM3 (HAVCR2) inhibitor, OX40 (CD134) inhibitor, GITR agonist, CD137 agonist, or CD40 agonist, VTCN1 inhibitor, IDO1 inhibitor, CD276 inhibitor, PVRIG inhibitor, TIGIT inhibitor, CD25 (IL2RA) inhibitor, IFNAR2 inhibitor, IFNAR1 inhibitor, CSF1R inhibitor, VSIR (VISTA) inhibitor or therapeutic agent targeting HLA. Such inhibitors, activators or therapeutic agents are further provided below.

In some embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor. In one embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. Examples of anti-CTLA-4 antibodies include, but are not limited to, those described in the following: U.S. Patent Nos. 5,811,097, 5,811,097, 5,855,887, 6,051,227, 6,207,157, 6,682,736, 6,984,720 and 7,605,238, all of which are incorporated herein in their entirety. In one embodiment, the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP-675,206). In another embodiment, the anti-CTLA-4 antibody is ipilimumab (also known as MDX-010 or MDX-101). Ipilimumab is a fully human monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is sold under the trade name Yervoy ^™ .

In certain embodiments, the checkpoint inhibitor is a PD-1/PD-L1 inhibitor. Examples of PD-L/PD-L1 inhibitors include, but are not limited to, those described in U.S. Patent Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, and PCT Patent Application Publication Nos. WO2003042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699, all of which are incorporated herein in their entirety.

In some embodiments, the checkpoint inhibitor is a PD-1 inhibitor. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody. In one embodiment, the anti-PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558 or MDX1106) or pembrolizumab (also known as MK-3475, SCH900475 or lambrolizumab). In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab is a human IgG4 anti-PD-1 monoclonal antibody and is sold under the trade name Opdivo ^TM . In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 antibody and is sold under the trade name Keytruda ^TM . In another embodiment, the anti-PD-1 antibody is a humanized antibody CT-011. Administration of CT-011 alone in the setting of relapse failed to show a response in the treatment of acute myeloid leukemia (AML). In another embodiment, the anti-PD-1 antibody is the fusion protein AMP-224. In another embodiment, the PD-1 antibody is BGB-A317. BGB-A317 is a monoclonal antibody in which the ability to bind to Fcγ receptor I is specifically engineered, and it has the characteristics of uniquely binding to PD-1 with high affinity and excellent target specificity. In one embodiment, the PD-1 antibody is cemiplimab. In another embodiment, the PD-1 antibody is camrelizumab. In another embodiment, the PD-1 antibody is sintilimab. In some embodiments, the PD-1 antibody is tislelizumab. In certain embodiments, the PD-1 antibody is TSR-042. In another embodiment, the PD-1 antibody is PDR001. In another embodiment, the PD-1 antibody is toripalimab.

In certain embodiments, the checkpoint inhibitor is a PD-L1 inhibitor. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one embodiment, the anti-PD-L1 antibody is MEDI4736 (durvalumab). In another embodiment, the anti-PD-L1 antibody is BMS-936559 (also known as MDX-1105-01). In another embodiment, the PD-L1 inhibitor is atezolizumab (also known as MPDL3280A and ). In another embodiment, the PD-L1 inhibitor is avelumab.

In one embodiment, the checkpoint inhibitor is a PD-L2 inhibitor. In one embodiment, the PD-L2 inhibitor is an anti-PD-L2 antibody. In one embodiment, the anti-PD-L2 antibody is rHIgM12B7A.

In one embodiment, the checkpoint inhibitor is a lymphocyte activation gene-3 (LAG-3) inhibitor. In one embodiment, the LAG-3 inhibitor is a soluble Ig fusion protein IMP321 (Brignone et al., J. Immunol., 2007, 179, 4202-4211). In another embodiment, the LAG-3 inhibitor is BMS-986016.

In one embodiment, the checkpoint inhibitor is a B7 inhibitor. In one embodiment, the B7 inhibitor is a B7-H3 inhibitor or a B7-H4 inhibitor. In one embodiment, the B7-H3 inhibitor is the anti-B7-H3 antibody MGA271 (Loo et al., Clin. Cancer Res., 2012, 3834).

In one embodiment, the checkpoint inhibitor is a TIM3 (T cell immunoglobulin domain and mucin domain 3) inhibitor (Fourcade et al., J. Exp. Med., 2010, 207, 2175-86; Sakuishi et al., J. Exp. Med., 2010, 207, 2187-94).

In one embodiment, the checkpoint inhibitor is an OX40 (CD134) agonist. In one embodiment, the checkpoint inhibitor is an anti-OX40 antibody. In one embodiment, the anti-OX40 antibody is anti-OX-40. In another embodiment, the anti-OX40 antibody is MEDI6469.

In one embodiment, the checkpoint inhibitor is a GITR agonist. In one embodiment, the checkpoint inhibitor is an anti-GITR antibody. In one embodiment, the anti-GITR antibody is TRX518.

In one embodiment, the checkpoint inhibitor is a CD137 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD137 antibody. In one embodiment, the anti-CD137 antibody is urelumab. In another embodiment, the anti-CD137 antibody is PF-05082566.

In one embodiment, the checkpoint inhibitor is a CD40 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD40 antibody. In one embodiment, the anti-CD40 antibody is CF-870,893.

In one embodiment, the checkpoint inhibitor is recombinant human leukocyte factor-15 (rhIL-15).

In one embodiment, the checkpoint inhibitor is a VTCN inhibitor. In one embodiment, the VTCN inhibitor is FPA150.

In one embodiment, the checkpoint inhibitor is an IDO inhibitor. In one embodiment, the IDO inhibitor is INCB024360. In another embodiment, the IDO inhibitor is indoximod. In one embodiment, the IDO inhibitor is epacadostat. In another embodiment, the IDO inhibitor is BMS986205. In another embodiment, the IDO inhibitor is Navoximod. In one embodiment, the IDO inhibitor is PF-06840003. In another embodiment, the IDO inhibitor is KHK2455. In another embodiment, the IDO inhibitor is RG70099. In one embodiment, the IDO inhibitor is IOM-E. In another embodiment, the IDO inhibitor is IOM-D.

In some embodiments, the checkpoint inhibitor is a TIGIT inhibitor. In certain embodiments, the TIGIT inhibitor is an anti-TIGIT antibody. In one embodiment, the TIGIT inhibitor is MTIG7192A. In another embodiment, the TIGIT inhibitor is BMS-986207. In another embodiment, the TIGIT inhibitor is OMP-313M32. In one embodiment, the TIGIT inhibitor is MK-7684. In another embodiment, the TIGIT inhibitor is AB154. In another embodiment, the TIGIT inhibitor is CGEN-15137. In one embodiment, the TIGIT inhibitor is SEA-TIGIT. In another embodiment, the TIGIT inhibitor is ASP8374. In another embodiment, the TIGIT inhibitor is AJUD008.

In some embodiments, the checkpoint inhibitor is a VSIR inhibitor. In certain embodiments, the VSIR inhibitor is an anti-VSIR antibody. In one embodiment, the VSIR inhibitor is MTIG7192A. In another embodiment, the VSIR inhibitor is CA-170. In another embodiment, the VSIR inhibitor is JNJ 61610588. In one embodiment, the VSIR inhibitor is HMBD-002.

In some embodiments, the checkpoint inhibitor is a TIM3 inhibitor. In certain embodiments, the TIM3 inhibitor is an anti-TIM3 antibody. In one embodiment, the TIM3 inhibitor is AJUD009.

In some embodiments, the checkpoint inhibitor is a CD25 (IL2RA) inhibitor. In certain embodiments, the CD25 (IL2RA) inhibitor is an anti-CD25 (IL2RA) antibody. In one embodiment, the CD25 (IL2RA) inhibitor is daclizumab. In another embodiment, the CD25 (IL2RA) inhibitor is basiliximab.

In some embodiments, the checkpoint inhibitor is an IFNAR1 inhibitor. In certain embodiments, the IFNAR1 inhibitor is an anti-IFNAR1 antibody. In one embodiment, the IFNAR1 inhibitor is anifrolumab. In another embodiment, the IFNAR1 inhibitor is sifalimumab.

In some embodiments, the checkpoint inhibitor is a CSF1R inhibitor. In certain embodiments, the CSF1R inhibitor is an anti-CSF1R antibody. In one embodiment, the CSF1R inhibitor is pexidartinib. In another embodiment, the CSF1R inhibitor is emactuzumab. In another embodiment, the CSF1R inhibitor is cabiralizumab. In one embodiment, the CSF1R inhibitor is ARRY-382. In another embodiment, the CSF1R inhibitor is BLZ945. In another embodiment, the CSF1R inhibitor is AJUD010. In one embodiment, the CSF1R inhibitor is AMG820. In another embodiment, the CSF1R inhibitor is IMC-CS4. In another embodiment, the CSF1R inhibitor is JNJ-40346527. In one embodiment, the CSF1R inhibitor is PLX5622. In another embodiment, the CSF1R inhibitor is FPA008.

In some embodiments, the checkpoint inhibitor is a therapeutic agent targeting HLA. In certain embodiments, the therapeutic agent targeting HLA is an anti-HLA antibody. In one embodiment, the therapeutic agent targeting HLA is GSK01. In another embodiment, the therapeutic agent targeting HLA is IMC-C103C. In another embodiment, the therapeutic agent targeting HLA is IMC-F106C. In one embodiment, the therapeutic agent targeting HLA is IMC-G107C. In another embodiment, the therapeutic agent targeting HLA is ABBV-184.

In certain embodiments, the immune checkpoint inhibitors provided herein include two or more of the checkpoint inhibitors described herein (including checkpoint inhibitors of the same or different categories). In addition, the methods described herein may be used in combination with one or more second active agents as described herein, when appropriate, to treat diseases described herein and understood in the art.

In some embodiments, after administering the ADC provided herein, a checkpoint inhibitor is administered. In other embodiments, a checkpoint inhibitor is administered simultaneously with the ADC provided herein (e.g., in the same administration period). In other embodiments, a checkpoint inhibitor is administered after administering the ADC provided herein.

In some embodiments, the amount of the checkpoint inhibitor used in the various methods provided herein can be determined by standard clinical techniques. In certain embodiments, the amount of the checkpoint inhibitor used in the various methods is provided in Section 5.6.

In some embodiments, the subject treatable in the methods provided herein is a mammal. In some embodiments, the subject treatable in the methods provided herein is a human.

5.2.1.1 Other patient demographics

In addition, the human subjects to which the methods provided herein can be used are human subjects with various other conditions. In one embodiment, the human subjects to which the methods provided herein can be used can have a primary tumor site in the lower urinary tract. In some embodiments, the human subjects to which the methods provided herein can be used can have visceral metastases. In certain embodiments, the human subjects to which the methods provided herein can be used can have liver metastases. In other embodiments, the human subjects to which the methods provided herein can be used can have at least 1 Bellmunt risk factor. In other embodiments, the human subjects to which the methods provided herein can be used can have an ECOG performance status score of 0. In one embodiment, the human subjects to which the methods provided herein can be used can have a primary tumor site and visceral metastases in the lower urinary tract. In some embodiments, the human subjects to which the methods provided herein can be used can have a primary tumor site and liver metastases in the lower urinary tract. In certain embodiments, the human subjects to which the methods provided herein can be used can have a primary tumor site in the lower urinary tract and at least 1 Bellmunt risk factor. In other embodiments, the human subjects to which the methods provided herein can be used can have a primary tumor site in the lower urinary tract and an ECOG performance status score of 0. In other embodiments, human subjects for whom the methods provided herein can be used may have visceral metastasis and liver metastasis. In one embodiment, human subjects for whom the methods provided herein can be used may have visceral metastasis and at least 1 Bellmunt risk factor. In some embodiments, human subjects for whom the methods provided herein can be used may have visceral metastasis and an ECOG performance status score of 0. In other embodiments, human subjects for whom the methods provided herein can be used may have liver metastasis and at least 1 Bellmunt risk factor. In other embodiments, human subjects for whom the methods provided herein can be used may have liver metastasis and an ECOG performance status score of 0. In one embodiment, human subjects for whom the methods provided herein can be used may have at least 1 Bellmunt risk factor and an ECOG performance status score of 0. In other embodiments, human subjects for whom the methods provided herein can be used may have primary tumor sites, visceral metastasis, and liver metastasis in the lower urinary tract. In other embodiments, human subjects for whom the methods provided herein can be used may have primary tumor sites, visceral metastasis, and at least 1 Bellmunt risk factor in the lower urinary tract. In other embodiments, human subjects for whom the methods provided herein can be used may have a primary tumor site in the lower urinary tract, visceral metastasis, and an ECOG performance status score of 0. In some embodiments, human subjects for whom the methods provided herein can be used may have a primary tumor site in the lower urinary tract, liver metastasis, and at least 1 Bellmunt risk factor. In certain embodiments, human subjects for whom the methods provided herein can be used may have a primary tumor site in the lower urinary tract, liver metastasis, and an ECOG performance status score of 0. In other embodiments, human subjects for whom the methods provided herein can be used may have a primary tumor site in the lower urinary tract, liver metastasis, and an ECOG performance status score of 0. In some embodiments, human subjects for whom the methods provided herein can be used may have visceral metastasis, liver metastasis, and at least 1 Bellmunt risk factor. In certain embodiments, human subjects for whom the methods provided herein can be used may have visceral metastasis, liver metastasis, and an ECOG performance status score of 0. In other embodiments, human subjects for whom the methods provided herein can be used may have visceral metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0. In other embodiments, human subjects for whom the methods provided herein can be used may have liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0. In other embodiments, human subjects for whom the methods provided herein can be used may have primary tumor sites in the lower urinary tract, visceral metastasis, liver metastasis, and at least 1 Bellmunt risk factor. In other embodiments, human subjects for whom the methods provided herein can be used may have primary tumor sites in the lower urinary tract, visceral metastasis, liver metastasis, and an ECOG performance status score of 0. In some embodiments, human subjects for whom the methods provided herein can be used may have primary tumor sites in the lower urinary tract, visceral metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0. In certain embodiments, human subjects for whom the methods provided herein can be used may have primary tumor sites in the lower urinary tract, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0. In other embodiments, human subjects for whom the methods provided herein can be used may have visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0. In certain embodiments, the human subjects for whom the methods provided herein can be used may have a primary tumor site in the lower urinary tract, visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0. In some embodiments, the human subjects for whom the methods provided herein can be used may have any one of the following: a primary tumor site in the lower urinary tract, visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0. In some embodiments, the human subjects for whom the methods provided herein can be used may have any two of the following: a primary tumor site in the lower urinary tract, visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0, in any combination or arrangement. In some embodiments, the human subjects for whom the methods provided herein can be used may have any three of the following: a primary tumor site in the lower urinary tract, visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0, in any combination or arrangement. In some embodiments, the human subjects for whom the methods provided herein can be used can have any four of the following: a primary tumor site in the lower urinary tract, visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0, in any combination or arrangement. In some embodiments, the human subjects for whom the methods provided herein can be used can have all five of the following: a primary tumor site in the lower urinary tract, visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0.

In other embodiments, the human subjects of the methods provided herein can be used to have a primary tumor site in the upper urinary tract. In one embodiment, the human subjects of the methods provided herein can be used to have a primary tumor site in the upper urinary tract and visceral metastasis. In some embodiments, the human subjects of the methods provided herein can be used to have a primary tumor site in the upper urinary tract and liver metastasis. In certain embodiments, the human subjects of the methods provided herein can be used to have a primary tumor site in the upper urinary tract and at least 1 Bellmunt risk factor. In other embodiments, the human subjects of the methods provided herein can be used to have a primary tumor site in the upper urinary tract and an ECOG performance status score of 0. In other embodiments, the human subjects of the methods provided herein can be used to have a primary tumor site in the upper urinary tract, visceral metastasis and liver metastasis. In other embodiments, the human subjects of the methods provided herein can be used to have a primary tumor site in the upper urinary tract, visceral metastasis and at least 1 Bellmunt risk factor. In other embodiments, human subjects using the methods provided herein may have a primary tumor site in the upper urinary tract, visceral metastasis, and an ECOG performance status score of 0. In some embodiments, human subjects using the methods provided herein may have a primary tumor site in the upper urinary tract, liver metastasis, and at least 1 Bellmunt risk factor. In certain embodiments, human subjects using the methods provided herein may have a primary tumor site in the upper urinary tract, liver metastasis, and an ECOG performance status score of 0. In other embodiments, human subjects using the methods provided herein may have a primary tumor site in the upper urinary tract, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0. In other embodiments, human subjects using the methods provided herein may have a primary tumor site in the upper urinary tract, visceral metastasis, liver metastasis, and at least 1 Bellmunt risk factor. In other embodiments, human subjects using the methods provided herein may have a primary tumor site in the upper urinary tract, visceral metastasis, liver metastasis, and an ECOG performance status score of 0. In some embodiments, the human subjects for whom the methods provided herein can be used may have a primary tumor site in the upper urinary tract, visceral metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0. In certain embodiments, the human subjects for whom the methods provided herein can be used may have a primary tumor site in the upper urinary tract, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0. In certain embodiments, the human subjects for whom the methods provided herein can be used may have a primary tumor site in the upper urinary tract, visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0. In some embodiments, the human subjects for whom the methods provided herein can be used may have any of the following: a primary tumor site in the upper urinary tract, visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0. In some embodiments, the human subjects for whom the methods provided herein can be used may have any two of the following: a primary tumor site in the upper urinary tract, visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0, in any combination or arrangement. In some embodiments, the human subjects for whom the methods provided herein can be used may have any three of the following: a primary tumor site in the upper urinary tract, visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0, in any combination or arrangement. In some embodiments, the human subjects for whom the methods provided herein can be used may have any four of the following: a primary tumor site in the upper urinary tract, visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0, in any combination or arrangement. In some embodiments, the human subjects for whom the methods provided herein can be used may have all five of the following: a primary tumor site in the upper urinary tract, visceral metastasis, liver metastasis, at least 1 Bellmunt risk factor, and an ECOG performance status score of 0.

In other embodiments of the methods provided herein, including the methods of the preceding paragraphs, the human subjects to which the methods provided herein can be applied are human subjects with various other conditions. In one embodiment, the human subjects to which the methods provided herein can be applied also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L. In some embodiments, the human subjects to which the methods provided herein can be applied also have the following conditions: the platelet count is not less than 100×10 ⁹ /L. In certain embodiments, the human subjects to which the methods provided herein can be applied also have the following conditions: hemoglobin is not less than 9 g/dL. In other embodiments, the human subjects to which the methods provided herein can be applied also have the following conditions: serum bilirubin is not more than 1.5 times the upper limit of normal (ULN) or is not more than 3 times ULN for patients with Gilbert's disease. In other embodiments, the human subjects to which the methods provided herein can be applied also have the following conditions: CrCl is not less than 30 mL/min. In another embodiment, the human subjects to which the methods provided herein can be applied also have the following conditions: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are not more than 3 times ULN. In one embodiment, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L and the platelet count is not less than 100×10 ⁹ /L. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L and the hemoglobin is not less than 9g/dL. In certain embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L and the serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L and the CrCl is not less than 30mL/min. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L and ALT and AST are not more than 3 times ULN. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the platelet count is not less than 100×10 ⁹ /L and the hemoglobin is not less than 9g/dL. In one embodiment, the human subjects to whom the methods provided herein can be used also have the following conditions: the platelet count is not less than 100×10 ⁹ /L and the serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the platelet count is not less than 100×10 ⁹ /L and the CrCl is not less than 30mL/min. In certain embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the platelet count is not less than 100×10 ⁹ /L and the ALT and AST are not more than 3 times ULN. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: hemoglobin is not less than 9g/dL and serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: hemoglobin is not less than 9g/dL and CrCl is not less than 30mL/min. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: hemoglobin is not less than 9g/dL and ALT and AST are not more than 3 times ULN. In one embodiment, the human subjects to whom the methods provided herein can be used also have the following conditions: serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease and CrCl is not less than 30mL/min. In another embodiment, the human subjects to whom the methods provided herein can be used also have the following conditions: serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease and ALT and AST are not more than 3 times ULN. In another embodiment, the human subjects to whom the methods provided herein can be used also have the following conditions: CrCl is not less than 30 mL/min and ALT and AST are not more than 3 times ULN. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the platelet count is not less than 100×10 ⁹ /L, and the hemoglobin is not less than 9 g/dL. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the platelet count is not less than 100×10 ⁹ /L, and the serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the platelet count is not less than 100×10 ⁹ /L, and the CrCl is not less than 30 mL/min. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the platelet count is not less than 100×10 ^{9/L, and the ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 9 /} L, the hemoglobin is not less than 9g/dL, and the serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN. In certain embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the hemoglobin is not less than 9g/dL, and the CrCl is not less than 30mL/min. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the hemoglobin is not less than 9 g/dL, and the ALT and AST are not more than 3 times ULN. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the serum bilirubin is not more than 1.5 times ULN, or for patients with Gilbert's disease, it is not more than 3 times ULN, and the CrCl is not less than 30 mL/min. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the serum bilirubin is not more than 1.5 times ULN, or for patients with Gilbert's disease, it is not more than 3 times ULN, and the ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the CrCl is not less than 30 mL/min, and the ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the platelet count is not less than 100×10 ⁹ /L, the hemoglobin is not less than 9 g/dL, and the serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN. In certain embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the platelet count is not less than 100×10 ⁹ /L, the hemoglobin is not less than 9 g/dL, and the CrCl is not less than 30 mL/min. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the platelet count is not less than 100×10 ⁹ /L, the hemoglobin is not less than 9 g/dL, and the ALT and AST are not more than 3 times ULN. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: platelet count is not less than 100×10 ⁹ /L, serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN, and CrCl is not less than 30 mL/min. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: platelet count is not less than 100×10 ⁹ /L, serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN, and ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: platelet count is not less than 100×10 ⁹ /L, CrCl is not less than 30 mL/min, and ALT and AST are not more than 3 times ULN. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: hemoglobin is not less than 9g/dL, serum bilirubin is not more than 1.5 times ULN, or for patients with Gilbert's disease, it is not more than 3 times ULN, and CrCl is not less than 30mL/min. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: hemoglobin is not less than 9g/dL, serum bilirubin is not more than 1.5 times ULN, or for patients with Gilbert's disease, it is not more than 3 times ULN, and ALT and AST are not more than 3 times ULN. In certain embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: hemoglobin is not less than 9g/dL, CrCl is not less than 30mL/min, and ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: serum bilirubin is not more than 1.5 times ULN, or for patients with Gilbert's disease, it is not more than 3 times ULN, CrCl is not less than 30mL/min, and ALT and AST are not more than 3 times ULN. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the platelet count is not less than 100×10 ⁹ /L, the hemoglobin is not less than 9g/dL, and the serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the platelet count is not less than 100×10 ⁹ /L, the hemoglobin is not less than 9g/dL, and the CrCl is not less than 30mL/min. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the platelet count is not less than 100×10 ⁹ /L, the hemoglobin is not less than 9g/dL, and the ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the platelet count is not less than 100×10 ⁹ /L, the serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, and CrCl is not less than 30 mL/min. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the platelet count is not less than 100×10 ⁹ /L, the serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, and ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the platelet count is not less than 100×10 ⁹ /L, the CrCl is not less than 30 mL/min, and the ALT and AST are not more than 3 times ULN. In certain embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the hemoglobin is not less than 9 g/dL, the serum bilirubin is not more than 1.5 times ULN, or for patients with Gilbert's disease, it is not more than 3 times ULN, and the CrCl is not less than 30 mL/min. In certain embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the hemoglobin is not less than 9 g/dL, the serum bilirubin is not more than 1.5 times ULN, or for patients with Gilbert's disease, it is not more than 3 times ULN, and the ALT and AST are not more than 3 times ULN. In certain embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the hemoglobin is not less than 9 g/dL, the CrCl is not less than 30 mL/min, and the ALT and AST are not more than 3 times ULN. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the absolute neutrophil count is not less than 1.0×10 ⁹ /L, the serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN, the CrCl is not less than 30 mL/min, and the ALT and AST are not more than 3 times ULN. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: the platelet count is not less than 100×10 ⁹ /L, the hemoglobin is not less than 9 g/dL, the serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN and the CrCl is not less than 30 mL/min. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: platelet count is not less than 100×10 ⁹ /L, hemoglobin is not less than 9 g/dL, serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN, and ALT and AST are not more than 3 times ULN. In other embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: platelet count is not less than 100×10 ⁹ /L, hemoglobin is not less than 9 g/dL, CrCl is not less than 30 mL/min, and ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: platelet count is not less than 100×10 ⁹ /L, serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN, CrCl is not less than 30 mL/min, and ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: hemoglobin is not less than 9 g/dL, serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, CrCl is not less than 30 mL/min, and ALT and AST are not more than 3 times ULN. In certain embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: absolute neutrophil count is not less than 1.0 × 10 ⁹ / L, platelet count is not less than 100 × 10 ⁹ / L, hemoglobin is not less than 9 g/dL, serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, and CrCl is not less than 30 mL/min. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: absolute neutrophil count is not less than 1.0×10 ⁹ /L, platelet count is not less than 100×10 ⁹ /L, hemoglobin is not less than 9 g/dL, serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN, and ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: absolute neutrophil count is not less than 1.0×10 ⁹ /L, platelet count is not less than 100×10 ⁹ /L, hemoglobin is not less than 9 g/dL, CrCl is not less than 30 mL/min, and ALT and AST are not more than 3 times ULN. In certain embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: absolute neutrophil count is not less than 1.0×10 ⁹ /L, platelet count is not less than 100×10 ⁹ /L, serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, CrCl is not less than 30 mL/min, and ALT and AST are not more than 3 times ULN. In certain embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: absolute neutrophil count is not less than 1.0×10 ⁹ /L, hemoglobin is not less than 9 g/dL, serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, CrCl is not less than 30 mL/min, and ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: platelet count is not less than 100×10 ⁹ /L, hemoglobin is not less than 9 g/dL, serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN, CrCl is not less than 30 mL/min, and ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have the following conditions: absolute neutrophil count is not less than 1.0×10 ⁹ /L, platelet count is not less than 100×10 ⁹ /L, hemoglobin is not less than 9 g/dL, serum bilirubin is not more than 1.5 times ULN or for patients with Gilbert's disease, it is not more than 3 times ULN, CrCl is not less than 30 mL/min, and ALT and AST are not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have any one of the following conditions: an absolute neutrophil count of not less than 1.0×10 ⁹ /L, a platelet count of not less than 100×10 ⁹ /L, a hemoglobin of not less than 9 g/dL, a serum bilirubin of not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, a CrCl of not less than 30 mL/min, and an ALT and AST of not more than 3 times ULN. In some embodiments, the human subjects to whom the methods provided herein can be used also have any two of the following conditions: an absolute neutrophil count of not less than 1.0×10 ⁹ /L, a platelet count of not less than 100×10 ⁹ /L, a hemoglobin of not less than 9 g/dL, a serum bilirubin of not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, a CrCl of not less than 30 mL/min, and an ALT and AST of not more than 3 times ULN, in any combination or arrangement. In some embodiments, the human subjects to whom the methods provided herein can be used also have any three of the following conditions: an absolute neutrophil count of not less than 1.0×10 ⁹ /L, a platelet count of not less than 100×10 ⁹ /L, a hemoglobin of not less than 9 g/dL, a serum bilirubin of not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, a CrCl of not less than 30 mL/min, and an ALT and AST of not more than 3 times ULN, in any combination or arrangement. In some embodiments, the human subjects to whom the methods provided herein can be used also have any four of the following conditions: an absolute neutrophil count of not less than 1.0×10 ⁹ /L, a platelet count of not less than 100×10 ⁹ /L, a hemoglobin of not less than 9 g/dL, a serum bilirubin of not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, a CrCl of not less than 30 mL/min, and an ALT and AST of not more than 3 times ULN, in any combination or arrangement. In some embodiments, the human subjects to whom the methods provided herein can be used also have any five of the following conditions: an absolute neutrophil count of not less than 1.0×10 ⁹ /L, a platelet count of not less than 100×10 ⁹ /L, a hemoglobin of not less than 9 g/dL, a serum bilirubin of not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, a CrCl of not less than 30 mL/min, and an ALT and AST of not more than 3 times ULN, in any combination or arrangement. In some embodiments, the human subjects to whom the methods provided herein can be used also have all six of the following conditions: an absolute neutrophil count of not less than 1.0×10 ⁹ /L, a platelet count of not less than 100×10 ⁹ /L, a hemoglobin of not less than 9 g/dL, a serum bilirubin of not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, a CrCl of not less than 30 mL/min, and an ALT and AST of not more than 3 times ULN.

In other embodiments of the methods provided herein, including the methods of the preceding paragraphs, human subjects to which the methods provided herein may be applied are human subjects free of certain conditions. In one embodiment, human subjects to which the methods provided herein may be applied may have no more than Grade 2 sensory or motor neuropathy. In some embodiments, human subjects to which the methods provided herein may be applied may not have active central nervous system metastasis. In certain embodiments, human subjects to which the methods provided herein may be applied may not have uncontrolled diabetes. In one embodiment, human subjects to which the methods provided herein may be applied may have no more than Grade 2 sensory or motor neuropathy and no active central nervous system metastasis. In some embodiments, human subjects to which the methods provided herein may be applied may have no more than Grade 2 sensory or motor neuropathy and no uncontrolled diabetes. In other embodiments, human subjects to which the methods provided herein may be applied may not have active central nervous system metastasis and uncontrolled diabetes. In other embodiments, human subjects to which the methods provided herein may be applied may have no more than Grade 2 sensory or motor neuropathy, no active central nervous system metastasis, and uncontrolled diabetes. In some embodiments, the human subjects to whom the methods provided herein can be used may have any one of the following: no more than grade 2 sensory or motor neuropathy, no active central nervous system metastasis, and uncontrolled diabetes. In some embodiments, the human subjects to whom the methods provided herein can be used may have any two of the following: no more than grade 2 sensory or motor neuropathy, no active central nervous system metastasis, and uncontrolled diabetes, in any combination or arrangement. In some embodiments, the human subjects to whom the methods provided herein can be used may have all three of the following: no more than grade 2 sensory or motor neuropathy, no active central nervous system metastasis, and uncontrolled diabetes. In one embodiment of the method provided in this paragraph, uncontrolled diabetes is determined by hemoglobin A1c (HbA1c) being no less than 8%. In some embodiments of the method provided in this paragraph, uncontrolled diabetes is determined by HbA1c being between 7% and 8% and accompanied by related diabetes symptoms that are not otherwise explained. In other embodiments of the method provided in this paragraph, related diabetes symptoms include or consist of polyuria. In some other embodiments of the method provided in this paragraph, related diabetes symptoms include or consist of polydipsia. In other embodiments of the methods provided in this paragraph, the relevant diabetic symptoms comprise or consist of both polyuria and polydipsia.

In some embodiments of the methods provided herein, CrCl is measured by 24-hour urine collection. In other embodiments of the methods provided herein, CrCl is estimated by the Cockcroft-Gault criteria.

In some embodiments of the methods provided herein, the subject has been treated with one or more other cancer therapies. In certain embodiments of the methods provided herein, urothelial carcinoma (including locally advanced or metastatic urothelial carcinoma) has been treated with one or more other cancer therapies.

In some embodiments, the CPI provided for use in the methods may comprise or consist of any CPI as described in this section (Section 5.2.1.1).

5.2.1.2 Treatment results of the methods provided herein

The methods provided herein, including the methods described in this section (Section 5.2) and Sections 3 and 6, can provide beneficial treatment results for these human subjects with cancer. In one embodiment, the human subject has a complete response after treatment by the methods provided herein. In another embodiment, the human subject has a partial response after treatment by the methods provided herein.

In some embodiments, the response (complete or partial response) is determined by assessing the tumor or cancer site (lesion). The criteria for determining complete response (CR), partial response (PR), progressive disease (PD) and stable disease (SD) are described in Section 6 (e.g., in Section 6.1.6.3).

Therefore, the treatment results of the methods provided herein can be evaluated based on any one or more of the response criteria described above. In one embodiment, a human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 30%, with the baseline sum diameter as a reference. In another embodiment, a human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 35%, with the baseline sum diameter as a reference. In another embodiment, a human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 40%, with the baseline sum diameter as a reference. In another embodiment, a human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 45%, with the baseline sum diameter as a reference. In one embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 50%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 55%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 60%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 65%, using the baseline sum diameter as a reference. In one embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 70%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 75%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 80%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 85%, using the baseline sum diameter as a reference. In one embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 90%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of the diameters of the target lesions by at least or about 95%, using the baseline sum diameter as a reference. In some embodiments, the diameter is determined according to the longest diameter of the lesion. In certain embodiments, the diameter is determined based on the longest diameter of the lesion in the measurement plane. In some embodiments, the diameter is determined based on the longest diameter of the lesion in the measurement plane, wherein the minimum dimension is 10 mm by CT scanning. In certain embodiments, the diameter is determined based on the longest diameter of the lesion in the measurement plane, wherein the minimum dimension is 10 mm by CT scanning, and the CT slice thickness does not exceed 5 mm.

The therapeutic outcome of the method provided herein can also be assessed based on whether the disease is stable after treatment. In one embodiment, the human subject has stable disease after treatment by the method provided herein. In another embodiment, the human subject does not have progressive disease after treatment by the method provided herein.

Alternatively, the treatment results based on complete response, partial response or stable disease can be evaluated with respect to the human subject population treated by the methods provided herein, by evaluating the percentage of subjects with complete response, partial response or stable disease in the treatment population. Therefore, in some embodiments, the treatment results or efficacy measurements are applicable to the results achieved by actually treating the subject population. In other embodiments, the treatment results or efficacy measurements refer to the results or effects that can be achieved when treating the human subject population as disclosed herein. Although the following chapters discuss the treatment of actual human subject populations, it should be understood that the corresponding methods of the results or efficacy measurements that can be achieved in the patient population are also covered herein. In short, the two situations described above are applicable to the following chapters; for simplicity and to avoid redundancy, only one situation is described below.

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

In one embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 2%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 3%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 4%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 5%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 6%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 7%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 8%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 9%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 10%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 15%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 20%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 20.2%. In one embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 22%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 22.5%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with complete response in the treatment population is at least or about 23%. In one embodiment, the human subject population is treated by the method provided herein, wherein the percentage of the subject with a complete response in the treatment population is at least or about 25%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of the subject with a complete response in the treatment population is at least or about 30%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of the subject with a complete response in the treatment population is at least or about 35%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of the subject with a complete response in the treatment population is at least or about 40%.

Similarly, using the percentage of partial response as a standard, in one embodiment, a human subject population is treated by the method provided herein, wherein the percentage of subjects with partial response in the treatment population is at least or about 20%. In another embodiment, a human subject population is treated by the method provided herein, wherein the percentage of subjects with partial response in the treatment population is at least or about 25%. In one embodiment, a human subject population is treated by the method provided herein, wherein the percentage of subjects with partial response in the treatment population is at least or about 28%. In one embodiment, a human subject population is treated by the method provided herein, wherein the percentage of subjects with partial response in the treatment population is at least or about 30%. In one embodiment, a human subject population is treated by the method provided herein, wherein the percentage of subjects with partial response in the treatment population is at least or about 31%. In another embodiment, a human subject population is treated by the method provided herein, wherein the percentage of subjects with partial response in the treatment population is at least or about 32%. In another embodiment, a human subject population is treated by the method provided herein, wherein the percentage of subjects with partial response in the treatment population is at least or about 33%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with partial responses in the treatment population is at least or about 34%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with partial responses in the treatment population is at least or about 35%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with partial responses in the treatment population is at least or about 36%. In one embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with partial responses in the treatment population is at least or about 37%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with partial responses in the treatment population is at least or about 38%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with partial responses in the treatment population is at least or about 39%. In another embodiment, the human subject population is treated by the method provided herein, wherein the percentage of subjects with partial responses in the treatment population is at least or about 40%. In another embodiment, a human subject population is treated by the methods provided herein, wherein the percentage of subjects with a partial response in the treatment population is at least or about 45%. In another embodiment, a human subject population is treated by the methods provided herein, wherein the percentage of subjects with a partial response in the treatment population is at least or about 50%.

Similarly, the total response rate, which is the sum of the percentage of subjects with complete response and the percentage of subjects with partial response, can be used as an assessment standard for the treatment results in human subjects treated by the methods provided herein. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is at least or about 20%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is at least or about 25%. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is at least or about 30%. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is at least or about 35%. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is at least or about 36%. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is at least or about 37%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is at least or about 38%. In another embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is at least or about 39%. In one embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is at least or about 40%. In another embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is at least or about 41%. In another embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is at least or about 42%. In one embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is at least or about 43%. In another embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is at least or about 44%. In another embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is at least or about 45%. In another embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is at least or about 50%. In one embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is at least or about 55%. In another embodiment, human subject colony is treated by the method provided herein, wherein the total response rate in the treatment colony is at least or about 60%. In another embodiment, human subject colony is treated by the method provided herein, wherein the total response rate in the treatment colony is at least or about 65%. In another embodiment, human subject colony is treated by the method provided herein, wherein the total response rate in the treatment colony is at least or about 70%. In another embodiment, human subject colony is treated by the method provided herein, wherein the total response rate in the treatment colony is at least or about 75%. In another embodiment, human subject colony is treated by the method provided herein, wherein the total response rate in the treatment colony is at least or about 80%.

In one embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 40% to 65%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 40% to 65%. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 40% to 60%. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 40% to 55%. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 40% to 50%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 40% to 45%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 35% to 65%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 35% to 65%. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 35% to 60%. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 35% to 55%. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 35% to 50%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 35% to 45%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 35% to 40%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 30% to 65%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 30% to 65%. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total response rate in the treatment population is in the range of 30% to 60%. In another embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is in the range of 30% to 55%. In another embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is in the range of 30% to 50%. In one embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is in the range of 30% to 45%. In one embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is in the range of 30% to 40%. In one embodiment, the human subject population is treated by the method provided herein, wherein the total response rate in the treatment population is in the range of 30% to 35%.

In addition, the percentage of subjects with stable disease can be used as an assessment standard for the treatment results in human subjects treated by the methods provided herein. In one embodiment, the human subject population is treated by the methods provided herein, wherein the percentage of subjects with stable disease in the treatment population is at least or about 10%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the percentage of subjects with stable disease in the treatment population is at least or about 15%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the percentage of subjects with stable disease in the treatment population is at least or about 20%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the percentage of subjects with stable disease in the treatment population is at least or about 25%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the percentage of subjects with stable disease in the treatment population is at least or about 26%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the percentage of subjects with stable disease in the treatment population is at least or about 27%. In one embodiment, the human subject population is treated by the methods provided herein, wherein the percentage of subjects with stable disease in the treatment population is at least or about 28%. In one embodiment, the human subject population is treated by the method provided herein, wherein the percentage of the subject with stable disease in the treatment group is at least or about 29%. In one embodiment, the human subject population is treated by the method provided herein, wherein the percentage of the subject with stable disease in the treatment group is at least or about 30%. In one embodiment, the human subject population is treated by the method provided herein, wherein the percentage of the subject with stable disease in the treatment group is at least or about 31%. In one embodiment, the human subject population is treated by the method provided herein, wherein the percentage of the subject with stable disease in the treatment group is at least or about 32%. In one embodiment, the human subject population is treated by the method provided herein, wherein the percentage of the subject with stable disease in the treatment group is at least or about 33%. In one embodiment, the human subject population is treated by the method provided herein, wherein the percentage of the subject with stable disease in the treatment group is at least or about 34%. In one embodiment, the human subject population is treated by the method provided herein, wherein the percentage of the subject with stable disease in the treatment group is at least or about 35%. In one embodiment, the human subject population is treated by the method provided herein, wherein the percentage of the subject with stable disease in the treatment group is at least or about 40%. In one embodiment, human subject colony is treated by the method provided herein, wherein the percentage of the subject with stable disease in the treatment colony is at least or about 45%. In one embodiment, human subject colony is treated by the method provided herein, wherein the percentage of the subject with stable disease in the treatment colony is at least or about 50%. In one embodiment, human subject colony is treated by the method provided herein, wherein the percentage of the subject with stable disease in the treatment colony is at least or about 55%. In one embodiment, human subject colony is treated by the method provided herein, wherein the percentage of the subject with stable disease in the treatment colony is at least or about 60%.

In addition, the treatment results of the methods provided herein can be evaluated based on the duration of response as described in Section 6.1.8.4. In one embodiment, the human subject has a duration of response of at least or about 4 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 5 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 6 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 7 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 8 months after treatment. In one embodiment, the human subject has a duration of response of at least or about 9 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 10 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 11 months after treatment. In one embodiment, the human subject has a duration of response of at least or about 12 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 13 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 14 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 15 months after treatment. In one embodiment, the human subject has a response duration of at least or about 16 months after treatment. In another embodiment, the human subject has a response duration of at least or about 17 months after treatment. In another embodiment, the human subject has a response duration of at least or about 18 months after treatment. In another embodiment, the human subject has a response duration of at least or about 19 months after treatment. In another embodiment, the human subject has a response duration of at least or about 20 months after treatment.

In certain embodiments, the human subject has a duration of response in the range of 4 to 22 months after treatment. In certain embodiments, the human subject has a duration of response in the range of 5 to more than 22 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 21 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 20 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 19 months after treatment. In one embodiment, the human subject has a duration of response in the range of 5 to 18 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 17 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 16 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 15 months after treatment. In one embodiment, the human subject has a duration of response in the range of 5 to 14 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 13 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 12 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 11 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 10 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 9 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 8 months after treatment. In another embodiment, the human subject has a duration of response in the range of 5 to 7 months after treatment. In another embodiment, the human subject has a duration of response in the range of 6 to 22 months after treatment. In another embodiment, the human subject has a duration of response in the range of 6 to 21 months after treatment. In another embodiment, the human subject has a duration of response in the range of 6 to 20 months after treatment. In another embodiment, the human subject has a duration of response in the range of 6 to 19 months after treatment. In one embodiment, the human subject has a duration of response in the range of 6 to 18 months after treatment. In another embodiment, the human subject has a duration of response in the range of 6 to 17 months after treatment. In another embodiment, the human subject has a duration of response in the range of 6 to 16 months after treatment. In another embodiment, the human subject has a duration of response within the range of 6 to 15 months after treatment. In one embodiment, the human subject has a duration of response within the range of 6 to 14 months after treatment. In another embodiment, the human subject has a duration of response within the range of 6 to 13 months after treatment. In another embodiment, the human subject has a duration of response within the range of 6 to 12 months after treatment. In another embodiment, the human subject has a duration of response within the range of 6 to 11 months after treatment. In another embodiment, the human subject has a duration of response within the range of 6 to 10 months after treatment. In another embodiment, the human subject has a duration of response within the range of 6 to 9 months after treatment. In another embodiment, the human subject has a duration of response within the range of 6 to 8 months after treatment. In one embodiment, the human subject has a duration of response within the range of 7 to 22 months after treatment. In another embodiment, the human subject has a duration of response within the range of 7 to 21 months after treatment. In another embodiment, the human subject has a duration of response within the range of 7 to 20 months after treatment. In another embodiment, the human subject has a duration of response within the range of 7 to 19 months after treatment. In one embodiment, the human subject has a duration of response within the range of 7 to 18 months after treatment. In another embodiment, the human subject has a duration of response within the range of 7 to 17 months after treatment. In another embodiment, the human subject has a duration of response within the range of 7 to 16 months after treatment. In another embodiment, the human subject has a duration of response within the range of 7 to 15 months after treatment. In one embodiment, the human subject has a duration of response within the range of 7 to 14 months after treatment. In another embodiment, the human subject has a duration of response within the range of 7 to 13 months after treatment. In another embodiment, the human subject has a duration of response within the range of 7 to 12 months after treatment. In another embodiment, the human subject has a duration of response within the range of 8 to 22 months after treatment. In another embodiment, the human subject has a duration of response within the range of 9 to 22 months after treatment. In another embodiment, the human subject has a duration of response within the range of 10 to 22 months after treatment. In another embodiment, the human subject has a duration of response within the range of 11 to 22 months after treatment. In one embodiment, the human subject has a duration of response within the range of 12 to 22 months after treatment. In another embodiment, the human subject has a duration of response within the range of 13 to 22 months after treatment. In another embodiment, the human subject has a duration of response within the range of 14 to 22 months after treatment. In another embodiment, the human subject has a duration of response within the range of 15 to 22 months after treatment. In another embodiment, the human subject has a duration of response within the range of 16 to 22 months after treatment. In one embodiment, the human subject has a duration of response within the range of 17 to 22 months after treatment. In another embodiment, the human subject has a duration of response within the range of 18 to 22 months after treatment. In another embodiment, the human subject has a duration of response within the range of 6 to 21 months after treatment. In another embodiment, the human subject has a duration of response within the range of 7 to 20 months after treatment. In another embodiment, the human subject has a duration of response within the range of 8 to 19 months after treatment. In one embodiment, the human subject has a duration of response within the range of 9 to 18 months after treatment. In another embodiment, the human subject has a duration of response within the range of 10 to 17 months after treatment. In another embodiment, the human subject has a duration of response within the range of 11 to 16 months after treatment. In another embodiment, the human subject has a duration of response in the range of 12 to 15 months following treatment. In another embodiment, the human subject has a duration of response in the range of 13 to 14 months following treatment.

In some embodiments, the duration of response of a human subject population treated by the methods provided herein is assessed by assessing the median or average duration of response in the treatment population. In one embodiment, a human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 5 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 6 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 7 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 8 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 9 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 10 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 11 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 12 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 13 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 14 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 15 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 16 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 17 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 18 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average duration of response in the treatment population is at least or about 19 months. In one embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or mean duration of response in the treated population is at least or about 20 months.

In certain embodiments, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 5 to 22 months. In some embodiments, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 5 to 21 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 5 to 20 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 5 to 19 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 5 to 18 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 5 to 17 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 5 to 16 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 5 to 15 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 5 to 14 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 5 to 13 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 5 to 12 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 5 to 11 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 5 to 10 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 5 to 9 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 5 to 8 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 5 to 7 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 6 to 22 months. In some embodiments, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 6 to 21 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 6 to 20 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 6 to 19 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 6 to 18 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 6 to 17 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 6 to 16 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 6 to 15 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 6 to 14 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 6 to 13 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 6 to 12 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 6 to 11 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 6 to 10 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 6 to 9 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 6 to 8 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 7 to 22 months. In some embodiments, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 7 to 21 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 7 to 20 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 7 to 19 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 7 to 18 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 7 to 17 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 7 to 16 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 7 to 15 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 7 to 14 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 7 to 13 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 7 to 12 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 7 to 11 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 7 to 10 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is within the range of 7 to 9 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 8 to 22 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 9 to 22 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 10 to 22 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 11 to 22 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 12 to 12 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 13 to 22 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 14 to 22 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 15 to 22 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the duration of response in the treatment population is within the range of 6 to 21 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is in the range of 7 to 20 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is in the range of 8 to 19 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is in the range of 9 to 18 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is in the range of 10 to 17 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is in the range of 11 to 16 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the duration of response in the treatment population is in the range of 12 to 15 months.

Alternatively, the treatment results of the methods provided herein can be evaluated based on the progression-free survival as described in Section 6.1.8.4. In one embodiment, the human subject has a progression-free survival of at least or about 2 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 3 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 4 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 5 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 6 months after treatment. In one embodiment, the human subject has a progression-free survival of at least or about 6.7 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 7 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 8 months after treatment. In one embodiment, the human subject has a progression-free survival of at least or about 9 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 10 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 11 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 12 months after treatment. In one embodiment, the human subject has a progression-free survival of at least or about 13 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 14 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 15 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 16 months after treatment. In one embodiment, the human subject has a progression-free survival of at least or about 17 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 18 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 19 months after treatment. In another embodiment, the human subject has a progression-free survival of at least or about 20 months after treatment.

In one embodiment, the human subject has a progression-free survival in the range of 5 to 10 months after treatment. In some embodiments, the human subject has a progression-free survival in the range of 5 to 9 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 5 to 8 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 5 to 7 months after treatment. In one embodiment, the human subject has a progression-free survival in the range of 5 to 6 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 6 to 10 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 7 to 10 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 8 to 10 months after treatment. In one embodiment, the human subject has a progression-free survival in the range of 9 to 10 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 4 to 11 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 4 to 10 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 4 to 9 months after treatment. In one embodiment, the human subject has a progression-free survival in the range of 4 to 8 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 4 to 7 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 5 to 11 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 6 to 11 months after treatment. In one embodiment, the human subject has a progression-free survival in the range of 7 to 11 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 8 to 11 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 9 to 11 months after treatment. In another embodiment, the human subject has a progression-free survival in the range of 10 to 11 months after treatment.

In addition, in some embodiments, the progression-free survival of the human subject population treated by the methods provided herein is assessed by assessing the median or average progression-free survival in the treatment population. In one embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 2 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 3 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 4 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 5 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 6 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 7 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 8 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 9 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 10 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 11 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 12 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 13 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 14 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 15 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 16 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 17 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 18 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 19 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the median or average progression-free survival in the treatment population is at least or about 20 months.

In one embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 5 to 9 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 5 to 8 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 5 to 7 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 5 to 6 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 6 to 9 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 7 to 9 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 8 to 9 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 4 to 10 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 5 to 10 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 6 to 10 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 7 to 10 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 8 to 10 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 9 to 10 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 4 to 10 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 4 to 9 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 4 to 8 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 4 to 7 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 4 to 6 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 4 to 5 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 4 to 11 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 5 to 11 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 6 to 11 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 7 to 11 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is within the range of 8 to 11 months. In another embodiment, human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is in the range of 9 to 11 months. In another embodiment, human subject population is treated by the method provided herein, wherein the progression-free survival in the treatment population is in the range of 10 to 11 months.

Alternatively, the therapeutic outcome of the methods provided herein can be assessed based on total survival. In one embodiment, the human subject has a total survival of at least or about 5 months after treatment. In another embodiment, the human subject has a total survival of at least or about 6 months after treatment. In another embodiment, the human subject has a total survival of at least or about 7 months after treatment. In another embodiment, the human subject has a total survival of at least or about 8 months after treatment. In one embodiment, the human subject has a total survival of at least or about 9 months after treatment. In another embodiment, the human subject has a total survival of at least or about 10 months after treatment. In another embodiment, the human subject has a total survival of at least or about 11 months after treatment. In another embodiment, the human subject has a total survival of at least or about 12 months after treatment. In one embodiment, the human subject has a total survival of at least or about 13 months after treatment. In another embodiment, the human subject has a total survival of at least or about 14 months after treatment. In another embodiment, the human subject has a total survival of at least or about 15 months after treatment. In one embodiment, the human subject has a total survival of at least or about 16 months after treatment. In another embodiment, the human subject has a total survival of at least or about 17 months after treatment. In another embodiment, the human subject has a total survival of at least or about 18 months after treatment. In another embodiment, the human subject has a total survival of at least or about 19 months after treatment. In one embodiment, the human subject has a total survival of at least or about 20 months after treatment. In another embodiment, the human subject has a total survival of at least or about 21 months after treatment. In another embodiment, the human subject has a total survival of at least or about 22 months after treatment. In another embodiment, the human subject has a total survival of at least or about 23 months after treatment. In another embodiment, the human subject has a total survival of at least or about 24 months after treatment. In one embodiment, the human subject has a total survival of at least or about 25 months after treatment. In another embodiment, the human subject has a total survival of at least or about 26 months after treatment. In another embodiment, the human subject has a total survival of at least or about 27 months after treatment. In one embodiment, the human subjects have an overall survival of at least or about 28 months after treatment. In another embodiment, the human subjects have an overall survival of at least or about 29 months after treatment. In another embodiment, the human subjects have an overall survival of at least or about 30 months after treatment.

In one embodiment, the human subject has a total survival in the range of 10 to 19 months after treatment. In another embodiment, the human subject has a total survival in the range of 10 to 18 months after treatment. In another embodiment, the human subject has a total survival in the range of 10 to 17 months after treatment. In another embodiment, the human subject has a total survival in the range of 10 to 16 months after treatment. In one embodiment, the human subject has a total survival in the range of 10 to 15 months after treatment. In another embodiment, the human subject has a total survival in the range of 10 to 14 months after treatment. In another embodiment, the human subject has a total survival in the range of 10 to 13 months after treatment. In another embodiment, the human subject has a total survival in the range of 10 to 12 months after treatment. In one embodiment, the human subject has a total survival in the range of 10 to 11 months after treatment. In another embodiment, the human subject has a total survival in the range of 11 to 19 months after treatment. In another embodiment, the human subject has a total survival in the range of 12 to 19 months after treatment. In another embodiment, the human subject has a total survival in the range of 13 to 19 months after treatment. In one embodiment, the human subject has a total survival in the range of 14 to 18 months after treatment. In one embodiment, the human subject has a total survival in the range of 14 to 19 months after treatment. In one embodiment, the human subject has a total survival in the range of 15 to 18 months after treatment. In another embodiment, the human subject has a total survival in the range of 15 to 19 months after treatment. In another embodiment, the human subject has a total survival in the range of 16 to 19 months after treatment. In another embodiment, the human subject has a total survival in the range of 17 to 19 months after treatment. In one embodiment, the human subject has a total survival in the range of 18 to 19 months after treatment. In another embodiment, the human subject has a total survival in the range of 11 to 18 months after treatment. In another embodiment, the human subject has a total survival in the range of 12 to 17 months after treatment. In another embodiment, the human subject has a total survival in the range of 13 to 16 months after treatment. In another embodiment, the human subject has a total survival in the range of 14 to 15 months after treatment. In one embodiment, the human subject has a total survival in the range of 10 to 20 months after treatment. In another embodiment, the human subject has a total survival in the range of 11 to 20 months after treatment. In one embodiment, the human subject has a total survival in the range of 11 to 24 months after treatment. In one embodiment, the human subject has a total survival in the range of 11 to 25 months after treatment. In one embodiment, the human subject has a total survival in the range of 12 to 24 months after treatment. In one embodiment, the human subject has a total survival in the range of 12 to 25 months after treatment. In another embodiment, the human subject has a total survival in the range of 12 to 20 months after treatment. In one embodiment, the human subject has a total survival in the range of 13 to 20 months after treatment. In another embodiment, the human subject has a total survival in the range of 14 to 20 months after treatment. In another embodiment, the human subject has a total survival in the range of 15 to 20 months after treatment. In another embodiment, the human subject has a total survival in the range of 16 to 20 months after treatment. In one embodiment, the human subject has a total survival in the range of 17 to 20 months after treatment. In another embodiment, the human subject has a total survival in the range of 18 to 20 months after treatment. In another embodiment, the human subject has a total survival in the range of 19 to 20 months after treatment. In one embodiment, the human subject has a total survival in the range of 9 to 20 months after treatment. In another embodiment, the human subject has a total survival in the range of 9 to 19 months after treatment. In another embodiment, the human subject has a total survival in the range of 9 to 18 months after treatment. In one embodiment, the human subject has a total survival in the range of 9 to 17 months after treatment. In another embodiment, the human subject has a total survival in the range of 9 to 16 months after treatment. In another embodiment, the human subject has a total survival in the range of 9 to 15 months after treatment. In one embodiment, the human subject has a total survival in the range of 9 to 14 months after treatment. In another embodiment, the human subjects have an overall survival in the range of 9 to 13 months after treatment. In another embodiment, the human subjects have an overall survival in the range of 9 to 12 months after treatment. In one embodiment, the human subjects have an overall survival in the range of 9 to 11 months after treatment. In another embodiment, the human subjects have an overall survival in the range of 9 to 10 months after treatment.

In addition, in some embodiments, the total survival of the human subject population treated by the methods provided herein is assessed by assessing the median or average total survival in the treatment population. In one embodiment, the human subject population is treated by the methods provided herein, wherein the median or average total survival in the treatment population is at least or about 5 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average total survival in the treatment population is at least or about 6 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average total survival in the treatment population is at least or about 7 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average total survival in the treatment population is at least or about 8 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the median or average total survival in the treatment population is at least or about 9 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average total survival in the treatment population is at least or about 10 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 11 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 12 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 13 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 14 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 15 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 16 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 17 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 18 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 19 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 20 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 21 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 22 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 23 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 24 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 25 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 26 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 27 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 28 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 29 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the median or average overall survival in the treatment population is at least or about 30 months.

In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 10 to 19 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 10 to 18 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 10 to 17 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 10 to 16 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 10 to 15 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 10 to 14 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 10 to 13 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 10 to 12 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 10 to 11 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 10 to 19 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 11 to 19 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 11 to 24 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 11 to 25 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 12 to 24 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 12 to 25 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 12 to 19 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 13 to 19 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 14 to 19 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 15 to 19 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 16 to 19 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 17 to 19 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 18 to 19 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 11 to 18 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 12 to 17 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 13 to 16 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 14 to 15 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 10 to 20 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 11 to 20 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 12 to 20 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 13 to 20 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 14 to 20 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 15 to 20 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 16 to 20 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 17 to 20 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 18 to 20 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 19 to 20 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 9 to 20 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 9 to 19 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 9 to 18 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 9 to 17 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 9 to 16 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 9 to 15 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 9 to 14 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 9 to 13 months. In one embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 9 to 12 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 9 to 11 months. In another embodiment, the human subject population is treated by the method provided herein, wherein the total survival in the treatment population is within the range of 9 to 10 months.

5.2.2 Methods of treating cancer in selected patient populations

Provided herein are methods for treating various cancers in a subject, wherein the cancer has any of the suitable markers and/or features provided in Section 6. Also provided herein are methods for treating various cancers in a subject, wherein the subject has any of the suitable features provided in Section 6.

In one aspect, provided herein is a method for preventing or treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 conjugated to one or more monomethyl auristatin E (MMAE) units, 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 heavy chain variable region shown 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 shown in SEQ ID NO: 23; and wherein the subject has any one of the suitable characteristics provided in Section 6.

In some aspects, provided herein is a method for preventing or treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 conjugated to one or more monomethyl auristatin E (MMAE) units, 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 heavy chain variable region shown 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 shown in SEQ ID NO: 23; and wherein the cancer has any one of the suitable markers and/or characteristics provided in Section 6.

In some aspects, provided herein is a method for treating urothelial carcinoma or bladder cancer in a human subject having liver metastases, the method comprising administering to the subject having liver metastases an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 conjugated to one or more monomethyl auristatin E (MMAE) units, wherein the subject has received immune checkpoint inhibitor (CPI) therapy, and wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 conjugated to one or more monomethyl auristatin E (MMAE) units, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a complementarity determining region (CDR) comprising an amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 22; and a light chain variable region comprising a CDR comprising an amino acid sequence of a CDR of a light chain variable region shown in SEQ ID NO: 23; and wherein the subject has any one of the suitable characteristics provided in Section 6.

In some aspects, provided herein is a method for treating urothelial carcinoma or bladder cancer in a human subject having liver metastases, the method comprising administering an effective amount of an antibody drug conjugate to the subject having liver metastases, wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 conjugated to one or more monomethyl auristatin E (MMAE) units, wherein the subject has received immune checkpoint inhibitor (CPI) therapy, and wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 conjugated to one or more monomethyl auristatin E (MMAE) units, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a complementarity determining region (CDR) comprising an amino acid sequence of a heavy chain variable region shown in SEQ ID NO: 22; and a light chain variable region comprising a CDR comprising an amino acid sequence of a CDR of a light chain variable region shown in SEQ ID NO: 23; and wherein the urothelial carcinoma or bladder cancer has any one of the suitable markers and/or characteristics provided in Section 6.

In some aspects, provided herein is a method for treating urothelial carcinoma or bladder cancer in a human subject having a primary tumor site in the upper urinary tract, the method comprising administering to the subject having a primary tumor site in the upper urinary tract an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or more monomethyl auristatin E (MMAE) units, wherein the subject has received immune checkpoint inhibitor (CPI) therapy, and wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or more monomethyl auristatin E (MMAE) units, 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 heavy chain variable region shown 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 shown in SEQ ID NO: 23; and wherein the subject has any one of the suitable characteristics provided in Section 6.

In some aspects, provided herein is a method for treating urothelial carcinoma or bladder cancer in a human subject having a primary tumor site in the upper urinary tract, the method comprising administering to the subject having a primary tumor site in the upper urinary tract an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or more monomethyl auristatin E (MMAE) units, wherein the subject has received immune checkpoint inhibitor (CPI) therapy, and wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or more monomethyl auristatin E (MMAE) units, 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 heavy chain variable region shown in SEQ ID NO: 22; and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the heavy chain variable region shown in SEQ ID NO: 22; CDRs of the amino acid sequence of the CDRs of the light chain variable region shown in NO:23; and wherein the urothelial carcinoma or bladder cancer has any one of the suitable markers and/or characteristics provided in Section 6.

In some aspects, provided herein is a method for treating urothelial carcinoma or bladder cancer in a human subject, the method comprising administering to the subject an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 conjugated to one or more monomethyl auristatin E (MMAE) units, wherein the subject has received immune checkpoint inhibitor (CPI) therapy, wherein the subject has progression or recurrence of the cancer during or after the CPI therapy, and wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 conjugated to one or more monomethyl auristatin E (MMAE) units, 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 heavy chain variable region shown 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 shown in SEQ ID NO: 23; and wherein the subject has any one of the suitable characteristics provided in Section 6.

In some aspects, provided herein is a method for treating urothelial carcinoma or bladder cancer in a human subject, the method comprising administering to the subject an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 that has been conjugated to one or more monomethyl auristatin E (MMAE) units, wherein the subject has received immune checkpoint inhibitor (CPI) therapy, wherein the subject has progression or recurrence of the cancer during or after the CPI therapy, and wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 that has been conjugated to one or more monomethyl auristatin E (MMAE) units, 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 heavy chain variable region shown in SEQ ID NO: 22; and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the heavy chain variable region shown in SEQ ID NO: 22; CDRs of the amino acid sequence of the CDRs of the light chain variable region shown in NO:23; and wherein the urothelial carcinoma or bladder cancer has any one of the suitable markers and/or characteristics provided in Section 6.

In all methods provided herein, and in particular the methods described in Sections 5.2.1 and 5.2.2: therapeutic agents that can be used are described in Sections 5.3 and 6; selection of patients for treatment is described herein and exemplified in Sections 5.2 (including Section 5.2.2) and 6; dosing regimens and pharmaceutical compositions for administering therapeutic agents are described in Sections 5.4, 5.6, 5.7, and 6 below; biomarkers that can be used to identify therapeutic agents, select patients, determine the results of these methods, and/or serve as criteria for these methods in any way are described herein and exemplified in Sections 5.2 (including Section 5.2.2) and 6; the treatment outcomes of the methods provided herein can be improvements of the biomarkers described herein, such as those described and exemplified in Sections 5.2 (including Section 5.2.2) and 6. Therefore, it should be understood by those skilled in the art that the methods provided herein include all permutations and combinations of patients, therapeutic agents, dosing regimens, biomarkers, and treatment outcomes as described above and below.

5.3 Antibody Drug Conjugates for Use in Methods

In various embodiments of the methods provided herein (including the methods provided in Section 5.2), the ADC used in the methods comprises or is an anti-191P4D12 ADC described herein and/or in U.S. Pat. No. 8,637,642, which is incorporated herein by reference in its entirety. In some embodiments, the anti-191P4D12 antibody drug conjugate provided in the methods herein comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 as provided herein (including subsection 5.3.1), the 191P4D12 being coupled to one or more cytotoxic agents (drug units or D) units as provided herein (including this section (Section 5.3), further disclosed in subsection 5.3.2). In certain embodiments, the cytotoxic agent (drug unit or D) may be covalently linked directly or via a linker unit (linker unit; LU).

In some embodiments, the antibody drug conjugate compound has the formula:

L-(LU-D) _p (I)

or a pharmaceutically acceptable salt or solvate thereof; wherein:

L is an antibody unit, such as an anti-Connexin-4 antibody or antigen-binding fragment thereof as provided in Subsection 5.3.1 below, and

(LU-D) is a Linker Unit-Drug Unit moiety, wherein:

LU- is the joint unit, and

D is a drug unit having cytostatic or cytotoxic activity against target cells; and

p is an integer of 1 to 20.

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, the antibody drug conjugate compound has the formula:

L-( _Aa - _Ww - _Yy -D) _p (II)

or a pharmaceutically acceptable salt or solvate thereof, wherein:

L is an antibody unit, such as an anti-Connexin-4 antibody or antigen-binding fragment thereof as provided in Subsection 5.3.1 below; and

_-Aa - _Ww - _Yy- is a linker unit (LU), wherein:

-A- is the extension subunit,

a is 0 or 1,

Each -W- is independently an amino acid unit,

w is an integer in the range of 0 to 12,

-Y- is a self-degradable spacer unit,

y is 0, 1, or 2;

D is a drug unit having cytostatic or cytotoxic activity against target cells; and

p is an integer of 1 to 20.

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 to 12, y is 1 or 2. In some embodiments, w is 2 to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

In some specific embodiments of the methods provided herein, including the methods provided in Section 5.2, the cytotoxic agent that is part of any one of the ADCs provided herein for the methods comprises, consists of, or is MMAE.

For compositions comprising a variety of antibodies or their antigen-binding fragments, drug loading is represented by p (average number of drug molecules/antibody unit). Drug loading can be in the range of 1 to 20 drugs (D)/antibody. The average number of drugs/antibody in the preparation of the coupling reaction can be characterized by conventional means such as mass spectrometry, ELISA determination and HPLC. The quantitative distribution of antibody drug conjugates is also determined according to p. In some cases, the separation, purification and characterization of homogeneous antibody drug conjugates with a specific value of antibody drug conjugates with other drug loads, can be achieved by means such as reversed-phase HPLC or electrophoresis. In an exemplary embodiment, p is 2 to 8.

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

5.3.1 Anti-191P4D12 Antibodies or Antigen-Binding Fragments

In one embodiment, the antibody or antigen-binding fragment thereof that binds to a connexin-4-related protein is an antibody or antigen-binding fragment that specifically binds to a connexin-4 protein comprising an amino acid sequence of SEQ ID NO: 2 (see FIG. 1A ). The corresponding cDNA encoding the 191P4D12 protein has a sequence of SEQ ID NO: 1 (see FIG. 1A ).

Antibodies that specifically bind to a connexin-4 protein comprising the amino acid sequence of SEQ ID NO: 2 include antibodies that can bind to other connexin-4-related proteins. For example, antibodies that bind to a connexin-4 protein comprising the amino acid sequence of SEQ ID NO: 2 can bind to connexin-4-related proteins, such as connexin-4 variants and homologs or analogs thereof.

In some embodiments, the anti-Connexin-4 antibodies provided herein are monoclonal antibodies.

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.

In some embodiments, the anti-Connexin-4 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 heavy chain variable region shown in SEQ ID NO:22 (which is an 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 comprising the amino acid sequence of the CDR of the light chain variable region shown in SEQ ID NO:23 (which is an 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, an anti-connexin-4 antibody or an antigen-binding fragment thereof comprises: a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 comprising the amino acid sequences of corresponding complementarity determining region 1 (CDR-H1), CDR-H2 and CDR-H3 in the heavy chain variable region sequence shown in SEQ ID NO:22 (which is an 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 comprising the amino acid sequences of corresponding CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region sequence shown in SEQ ID NO:23 (which is an 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-Connexin-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 shown in SEQ ID NO:22 (which is an 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 shown in SEQ ID NO:23 (which is an 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, an anti-connexin-4 antibody or an antigen-binding fragment thereof comprises: a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 consisting of the amino acid sequences of corresponding complementarity determining region 1 (CDR-H1), CDR-H2 and CDR-H3 in the heavy chain variable region sequence shown in SEQ ID NO:22 (which is an 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 consisting of the amino acid sequences of corresponding CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region sequence shown in SEQ ID NO:23 (which is an amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 130th amino acid (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:

SEO ID NO: 22

SEQ ID NO:23

SEQ ID NO7

SEQ ID NO: 8

The CDR sequence can be determined according to a well-known numbering system. As described above, the CDR region is well known to those skilled in the art and is defined by a well-known numbering system. For example, the Kabat complementary determining region (CDR) is based on sequence variability and is most commonly used (see, for example, Kabat et al., supra). Chothia actually refers to the position of the structural loop (see, for example, Chothia and Lesk, 1987, J.Mol.Biol.196:901-17). When numbering using the Kabat numbering convention, the end of the Chothia CDR-H1 loop changes between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B exists, the loop end is at 32; if only 35A exists, the loop end is at 33; if both 35A and 35B exist, the loop end is at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Volume 2 (Kontermann and Dübel, ed., 2nd ed., 2010)). The "contact" hypervariable regions are based on 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 dedicated to immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complexes (MHC) of humans and other vertebrates. Herein, CDRs are referred to with respect to amino acid sequences and positions within light or heavy chains. Since the "position" of CDRs within the immunoglobulin variable domain structure is conserved between species and is present in structures called loops, CDRs and framework residues are easily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to transplant and replace CDR residues from immunoglobulins of one species into a receptor framework typically from human antibodies. Honegger and Plückthun, 2001, J. Mol. Biol. 309:657-70 have developed another numbering system (AHon). The correspondence between numbering systems (including, for example, Kabat numbering and the IMGT unique numbering system) is well known to those skilled in the art (see, for example, Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from each of these hypervariable regions or CDRs are indicated in Table 1 above.

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

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

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

In other embodiments, the anti-connexin-4 antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) comprising the amino acid sequence of the CDRs of the heavy chain variable region shown in SEQ ID NO:22 according to Contact numbering; and a light chain variable region comprising CDRs comprising the amino acid sequence of the CDRs of the light chain variable region shown in SEQ ID NO:23 according to Contact numbering.

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

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

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

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

In other embodiments, the anti-connexin-4 antibody or its antigen-binding fragment comprises: a heavy chain variable region comprising CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) consisting of the amino acid sequence of the CDR of the heavy chain variable region shown in SEQ ID NO:22 according to Contact numbering; and a light chain variable region comprising CDRs consisting of the amino acid sequence of the CDR of the light chain variable region shown in SEQ ID NO:23 according to Contact numbering.

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

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

Table 4

For another example, as determined by ANARCI, the heavy chain CDR sequence within SEQ ID NO: 22 and the light chain CDR sequence within SEQ ID NO: 23 according to IMGT numbering are listed in Table 5 below.

Table 5

VH of SEQ ID NO:22 VL of SEQ ID NO:23 CDR1 GFTFSSYN (SEQ ID NO: 16) QGISGW (SEQ ID NO: 19) CDR2 ISSSSSTI (SEQ ID NO: 17) AAS (SEQ ID NO: 20) CDR3 ARAYYYGMDV (SEQ ID NO: 18) QQANSFPPT (SEQ ID NO: 21)

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

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

In some embodiments, the antibody or its antigen-binding fragment comprises: CDR-H1, which consists of the amino acid sequence of SEQ ID NO:9; CDR-H2, which consists of the amino acid sequence of SEQ ID NO:10; CDR-H3, which consists of the amino acid sequence of SEQ ID NO:11; CDR-L1, which consists of the amino acid sequence of SEQ ID NO:12; CDR-L2, which consists of the amino acid sequence of SEQ ID NO:13; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO:14.

In some embodiments, the antibody or its antigen-binding fragment comprises: CDR-H1, which consists of the amino acid sequence of SEQ ID NO: 16; CDR-H2, which consists of the amino acid sequence of SEQ ID NO: 17; CDR-H3, which consists of the amino acid sequence of SEQ ID NO: 18; CDR-L1, which consists of the amino acid sequence of SEQ ID NO: 19; CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 20; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 21.

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.

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.

In some embodiments, the antibody comprises: a heavy chain comprising an amino acid sequence within the range of amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7; and a light chain comprising an amino acid sequence within the range of amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8.

In some embodiments, the antibody comprises: a heavy chain consisting of 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 consisting of an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8.

In some embodiments, amino acid sequence modifications 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. Therefore, in addition to the antibodies described herein, it is contemplated that antibody variants may be prepared. For example, antibody variants may be prepared by introducing appropriate nucleotide changes in the encoding DNA and/or by synthesizing the desired antibody or polypeptide. Those skilled in the art who understand amino acid changes may alter the post-translational processes of the antibody, such as changing the number or position of glycosylation sites or changing membrane anchoring characteristics.

In some embodiments, the antibodies provided herein are chemically modified, for example, by covalently linking any type of molecule to the antibody. Antibody derivatives may include chemically modified antibodies, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, by known protective groups/capping group derivatization, protein cleavage, connection to cell ligands or other proteins, etc. Chemically modified antibodies. Any of a variety of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In addition, the antibody may contain one or more non-classical amino acids.

The variation may be a substitution, deletion or insertion of one or more codons encoding a single domain antibody or polypeptide that results in an amino acid sequence change compared to the original antibody or polypeptide. An amino acid substitution may be the result of an amino acid being replaced by another amino acid comprising similar structure and/or chemical properties, such as leucine being replaced by serine, such as a conservative amino acid substitution. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis that cause amino acid substitutions. Insertions or deletions may optionally be within the range of about 1 to 5 amino acids. In certain embodiments, substitutions, deletions or insertions include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions relative to the original molecule. In a specific embodiment, substitutions are conservative amino acid substitutions produced at one or more predicted non-essential amino acid residues. Allowed variations can be determined by systematically generating amino acid insertions, deletions or substitutions in the sequence and testing the activity exhibited by the parent antibody of the resulting variant.

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. Examples of terminal insertions include antibodies with an N-terminal methionyl residue.

The antibodies produced by conservative amino acid substitution are included in the present disclosure. In conservative amino acid substitution, amino acid residues are replaced by amino acid residues comprising side chains with similar charges. As described above, the families of amino acid residues comprising 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-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, e.g., by saturation mutation induction, and the resulting mutants can be screened according to biological activity to identify the mutants that maintain activity. Following mutagenesis, the encoded protein can be expressed and the activity of the protein can be assayed, and conservative (eg, within a group of amino acids with similar properties and/or side chains) substitutions can be made to maintain or not significantly change the properties.

Amino acids can be grouped according to the similarity of their side chain properties (see, e.g., Lehninger, Biochemistry 73-75 (2nd 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 grouped 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 affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

For example, any cysteine residue not involved in maintaining the proper conformation of the antibody may also be substituted with another amino acid (eg, alanine or serine) to improve the oxidative stability of the molecule and prevent aberrant cross-linking.

Variants can be generated using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. The cloned DNA can be subjected to 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 to generate anti-MSLN antibody variant DNA.

Covalent modification of antibodies is included within the scope of the present disclosure. Covalent modification includes reacting the target amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or N-terminal or C-terminal residues of the antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; hydroxyl phosphorylation 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.

Other types of covalent modifications of antibodies included 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) in a manner such as 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.

In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having a certain homology or identity with the heavy chain as shown in SEQ ID NO: 7 and a light chain having a certain homology or identity with the light chain as shown in SEQ ID NO: 8. Such embodiments of heavy chain/light chain having homology or identity are further provided as follows. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having more than 70% homology or identity with the heavy chain as shown in SEQ ID NO: 7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having more than 75% homology or identity with the heavy chain as shown in SEQ ID NO: 7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having more than 80% homology or identity with the heavy chain as shown in SEQ ID NO: 7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having more than 85% homology or identity with the heavy chain as shown 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 one of the homology or identity provided to the heavy chain set forth in SEQ ID NO: 7, wherein the 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 a light chain as 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 a light chain as 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 a light chain as 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 a light chain as 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 homology or identity provided to a light chain as set forth in SEQ ID NO: 8, wherein the CDRs (CDR-L1, CDR-L2, and CDR-L3) are identical to the CDRs in the light chain as set forth in SEQ ID NO: 8. In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise any combination or arrangement of any cognate light chain and any cognate heavy chain as provided in this paragraph.

In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having a certain homology or identity with the heavy chain variable region as shown in SEQ ID NO: 22 and a light chain variable region having a certain homology or identity with the light chain variable region as shown in SEQ ID NO: 23. Such embodiments of heavy chain variable regions and light chain variable regions having homology or identity are further provided as follows. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having more than 70% homology or identity with the heavy chain variable region as shown in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having more than 75% homology or identity with the heavy chain variable region as shown in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having more than 80% homology or identity with the heavy chain variable region as shown 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 homology or identity provided to the heavy chain variable region set forth in SEQ ID NO: 22, wherein the 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 provided homologies or identities with the light chain variable region set forth in SEQ ID NO: 23, wherein 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 combination or arrangement of any cognate light chain variable region and any cognate heavy chain variable region as provided in this paragraph.

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

In some embodiments, the anti-connexin-4 antibodies provided herein comprise: the heavy chain 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 a hybridoma (Accession No.: PTA-11267) deposited with the American Type Culture Collection (ATCC)); or heavy chain and light chain CDR regions consisting of amino acid sequences homologous to the amino acid sequences of the heavy chain and light chain CDR regions of Ha22-2(2,4)6.1, and wherein the antibody retains the desired functional properties of the anti-connexin-4 antibody designated Ha22-2(2,4)6.1 (produced by a hybridoma (Accession No.: PTA-11267) deposited with the American Type Culture Collection (ATCC)).

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, wherein:

(a) the heavy chain variable region comprises CDRs (CDR-H1, CDR-H2 and CDR-H3) comprising the amino acid sequence of the heavy chain variable region CDRs set forth in the antibody produced by the hybridoma deposited with the American Type Culture Collection (ATCC) under Accession No.: PTA-11267;

(b) The light chain variable region comprises CDRs (CDR-L1, CDR-L2 and CDR-L3) comprising the amino acid sequence of the light chain variable region CDRs set forth in the antibody produced by the hybridoma deposited with the American Type Culture Collection (ATCC) under Accession No.: PTA-11267.

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, wherein:

(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 set forth in the antibody produced by the hybridoma deposited with the American Type Culture Collection (ATCC) under Accession No.: PTA-11267;

(b) The light chain variable region comprises CDRs (CDR-L1, CDR-L2 and CDR-L3) consisting of the amino acid sequences of the light chain variable region CDRs set forth in the antibody produced by the hybridoma deposited with the American Type Culture Collection (ATCC) under Accession No.: PTA-11267.

In some embodiments, the anti-Connexin-4 antibodies provided herein comprise: the heavy chain and light chain variable regions of the antibody designated Ha22-2(2,4)6.1 (produced by a hybridoma deposited under the American Type Culture Collection (ATCC) Accession No.: PTA-11267), or heavy chain and light chain variable regions comprising amino acid sequences homologous to the amino acid sequences of the heavy chain and light chain variable regions of Ha22-2(2,4)6.1, and wherein the antibodies retain the desired functional properties of the anti-Connexin-4 antibodies provided herein. In some embodiments, the connexin-4 antibodies provided herein comprise: the heavy and light chain variable regions of the antibody designated Ha22-2(2,4)6.1 (produced by a hybridoma deposited under the 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 wherein the antibodies retain the desired functional properties of the anti-connexin-4 antibodies provided herein. Any subclass of constant regions may be selected as the constant region of the antibodies of the present disclosure. In one embodiment, the human IgG1 constant region may be used as the heavy chain constant region and the human Igκ constant region may be used as the light chain constant region.

In some embodiments, the anti-connexin-4 antibodies provided herein comprise the heavy and light chains of the antibody designated Ha22-2(2,4)6.1 (produced by a hybridoma deposited under the American Type Culture Collection (ATCC) Accession No.: PTA-11267), or heavy and light chains comprising amino acid sequences homologous to the amino acid sequences of the heavy and light chains of Ha22-2(2,4)6.1, and wherein the antibodies retain the desired functional properties of the anti-connexin-4 antibodies provided herein. In some embodiments, the anti-connexin-4 antibodies provided herein comprise the heavy and light chains of the antibody designated Ha22-2(2,4)6.1 (produced by a hybridoma deposited under the American Type Culture Collection (ATCC) Accession No.: PTA-11267), or heavy and light chains consisting of amino acid sequences homologous to the amino acid sequences of the heavy and light chains of Ha22-2(2,4)6.1, and wherein the antibodies retain the desired functional properties of the anti-connexin-4 antibodies provided herein.

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 amino acid sequence of the heavy chain variable region of an antibody produced by a hybridoma deposited with the American Type Culture Collection (ATCC) under 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 amino acid sequence of the light chain variable region of an antibody produced by a hybridoma deposited with the American Type Culture Collection (ATCC) under Accession No.: PTA-11267.

In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having a certain homology or identity to the heavy chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under the American Type Culture Collection (ATCC) Accession No.: PTA-11267, and a light chain variable region having a certain homology or identity to the light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under the American Type Culture Collection (ATCC) Accession No.: PTA-11267. Such embodiments of heavy chain variable regions and light chain variable regions having homology or identity are further provided as follows. 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 the 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 amino acid sequence of the heavy chain variable region of an antibody produced by a hybridoma deposited under the 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 95% homologous or identical to the amino acid sequence of the heavy chain variable region of an antibody produced by a hybridoma deposited under the 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 amino acid sequence of the heavy chain variable region of an antibody produced by a hybridoma deposited under the 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 amino acid sequence of the light chain variable region of an antibody produced by a hybridoma deposited under the 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 amino acid sequence of the light chain variable region of an antibody produced by a hybridoma deposited under the 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 95% homologous or identical to the amino acid sequence of the light chain variable region of an antibody produced by a hybridoma deposited under the American Type Culture Collection (ATCC) Accession No.: PTA-11267. In other embodiments, the light 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 light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited with the American Type Culture Collection (ATCC) under Accession No. PTA-11267. In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise any combination or arrangement of any homologous light chain variable region and any homologous heavy chain variable region provided in such paragraphs.

In other embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain and a light chain, wherein:

(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 an antibody produced by a hybridoma deposited with the American Type Culture Collection (ATCC) under 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 a hybridoma deposited with the American Type Culture Collection (ATCC) under Accession No.: PTA-11267.

In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having a certain homology or identity to the heavy chain amino acid sequence of an antibody produced by a hybridoma deposited under the American Type Culture Collection (ATCC) Accession No.: PTA-11267, and a light chain having a certain homology or identity to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under the American Type Culture Collection (ATCC) Accession No.: PTA-11267. Such embodiments of heavy and light chains having homology or identity are further provided as follows. 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 an antibody produced by a hybridoma deposited under the 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 an antibody produced by a hybridoma deposited under the American Type Culture Collection (ATCC) Accession No.: PTA-11267. In 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 an antibody produced by a hybridoma deposited under the 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 an antibody produced by a hybridoma deposited under the 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 the 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 the American Type Culture Collection (ATCC) Accession No.: PTA-11267. In 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 the American Type Culture Collection (ATCC) Accession No.: PTA-11267. In other embodiments, the light chain may 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 an antibody produced by a hybridoma deposited under the American Type Culture Collection (ATCC) Accession No.: PTA-11267. In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise any combination or arrangement of any cognate light chain and any cognate heavy chain as provided in this paragraph.

In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to a specific epitope in 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the VC1 domain of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the VC1 domain of 191P4D12 but do not bind to the C1C2 domain. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 1st to 147th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to epitopes located in the 1st to 147th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 1st to 10th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 11th to 20th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 21st to 30th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 31st to 40th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 41st to 50th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 51st to 60th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 61st to 70th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 71st to 80th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 81st to 90th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 91st to 100th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 101st to 110th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 111th to 120th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 121st to 130th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 131st to 140th amino acid residues of 191P4D12. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to the 141st to 147th amino acid residues of 191P4D12. The binding epitopes of certain embodiments of the antibodies or antigen-binding fragments thereof provided herein have been determined and described in WO 2012/047724, which is incorporated herein by reference in its entirety.

In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to epitopes in 191P4D12 that are common between 191P4D12 variants observed in humans. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to epitopes in 191P4D12 that are common between 191P4D12 polymorphisms observed in humans. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to epitopes in 191P4D12 that are common between 191P4D12 polymorphisms observed in human cancers. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to epitopes in 191P4D12 that will bind, internalize, destroy or modulate the biological functions of 191P4D12 or 191P4D12 variants. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to epitopes in 191P4D12 that will destroy the interactions between 191P4D12 and ligands, substrates, and binding partners.

The engineered antibodies provided herein include those in which the framework residues in VH and/or VL have been modified (e.g., to improve the properties of the antibody). Typically, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one method is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that are different from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. In order to restore the framework region sequence to its germline configuration, somatic mutations can be "backmutated" to the germline sequence (e.g., leucine "backmutates" to methionine) by, for example, site-directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed within the present disclosure.

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

In addition to or alternatively to modifications made in the framework region or CDR region, the antibodies of the present disclosure may be engineered to include modifications in the Fc region, typically to change one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular toxicity. In addition, the anti-191P4D12 antibodies provided herein may be chemically modified (e.g., one or more chemical moieties may be connected to the antibody) or modified to change its glycosylation, to again change one or more functional properties of the antibody. Each of these embodiments is further described in detail below.

In one embodiment, the hinge region of CH1 is modified so that the number of cysteine residues in the hinge region is changed, for example, increased or decreased. This method is further described in U.S. Pat. No. 5,677,425 to Bodmer et al. The number of cysteine residues in the hinge region of CH1 is changed, for example, to facilitate the assembly of light and heavy chains or to increase or decrease the stability of anti-191P4D12 antibodies.

In another embodiment, the Fc hinge region of the antibody is mutated to reduce 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 to reduce the binding of the antibody to Staphylococcus protein A (Staphylococcyl protein A; SpA) relative to the native Fc hinge domain SpA binding. This method is further described in detail in U.S. Pat. No. 6,165,745 to Ward et al.

In another embodiment, the anti-191P4D12 antibody is modified to increase its biological half-life. A variety of methods are possible. For example, mutations can be introduced as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively, in order to increase the biological half-life, the antibody can be changed in the CH1 or CL region to contain salvage receptor binding epitopes of two loops of the CH2 domain obtained from the Fc region of IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 to Presta et al.

In other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids selected from a particular amino acid residue can be replaced with a different amino acid residue so that the affinity of the antibody for the effector ligand is changed but the antigen binding ability of the parent antibody is retained. The effector ligand with changed affinity can be, for example, an Fc receptor or the C1 component of complement. This method is further described in detail, for example, in U.S. Pat. Nos. 5,624,821 and 5,648,260 to Winter et al.

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

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

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

Additional embodiments of anti-Connexin-4 antibodies have been described in U.S. Pat. No. 8,637,642 and International Application No. PCT/US2019/056214 (Publication No. WO2020/117373), both of which are hereby incorporated by reference in their entirety.

5.3.2 Cytotoxic Agents (Drug Units)

Since the ADC used in the method provided herein comprises an antibody or antigen-binding fragment thereof coupled to a cytotoxic agent, the present disclosure further provides various embodiments of a cytotoxic agent as a part of the ADC used in the method. In various embodiments of the method provided herein (including the method provided in section 5.2), the cytotoxic agent as a part of any ADC provided herein to the method comprises a tubulin rupture agent, is composed of a tubulin rupture agent, or is a tubulin rupture agent. In one embodiment, the cytotoxic agent is a tubulin rupture agent. In some embodiments, the tubulin rupture agent is selected from the group consisting of: dolastatin, auristatin, hemiasterlin, vinca alkaloids, maytansinoids, eribulin, colchicine, plocabulin, phomopsin, epothilone, cryptophycin, and taxane. In a specific embodiment, the tubulin rupture agent is auristatin. In another specific embodiment, the auristatin is monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), AFP or auristatin T. In another specific embodiment, the auristatin is monomethyl auristatin E (MMAE).

In various embodiments of the methods provided herein, including the methods provided in Section 5.2, the cytotoxic agent as part of any ADC 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 hereby incorporated by reference in their entirety.

In some embodiments, the auristatin is MMAE (wherein the wavy line indicates the covalent attachment to the linker of the antibody drug conjugate).

In some embodiments, an exemplary embodiment comprising MMAE and a linker component (described further herein) has the following structure (wherein L represents an antibody (eg, an anti-connexin-4 antibody or antigen-binding fragment thereof) and p ranges from 1 to 12):

In some embodiments of the formula described in the preceding paragraph, 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 of the formula described in the preceding paragraph, 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 of the formula described 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 formula described in the preceding paragraph, p is about 1. In some embodiments of the formula described in the preceding paragraph, p is about 2. In some embodiments of the formula described in the preceding paragraph, p is about 3. In some embodiments of the formula described in the preceding paragraph, p is about 4. In some embodiments of the formula described in the preceding paragraph, p is about 3.8. In some embodiments of the formula described in the preceding paragraph, p is about 5. In some embodiments of the formula described in the preceding paragraph, p is about 6. In some embodiments of the formula described in the preceding paragraph, p is about 7. In some embodiments of the formula described in the preceding paragraph, p is about 8. In some embodiments of the formula described in the preceding paragraph, p is about 9. In some embodiments of the formula described in the preceding paragraph, p is about 10. In some embodiments of the formula described in the preceding paragraph, p is about 11. In some embodiments of the formula described in the preceding paragraph, p is about 12. In some embodiments of the formula described in the preceding paragraph, p is about 13. In some embodiments of the formula described in the preceding paragraph, p is about 14. In some embodiments of the formula described in the preceding paragraph, p is about 15. In some embodiments of the formula described in the preceding paragraph, p is about 16. In some embodiments of the formula described in the preceding paragraph, p is about 17. In some embodiments of the formula described in the preceding paragraph, p is about 18. In some embodiments of the formula described in the preceding paragraph, p is about 19. In some embodiments of the formula described in the preceding paragraph, p is about 20.

Typically, a peptide-based drug unit 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 liquid phase synthesis methods well known in the field of peptide chemistry (see E. and K. Lübke, "The Peptides", Vol. 1, pp. 76-136, 1965, Academic Press). The auristatin/dolastatin drug unit can be prepared according to the methods of the following documents: US 5635483; US 5780588; 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.

Additional embodiments of cytotoxic agents have been described in U.S. Pat. No. 8,637,642 and International Application No. PCT/US2019/056214 (Publication No. WO2020/117373), both of which are hereby incorporated by reference in their entirety.

5.3.3 Connectors

Typically, the antibody drug conjugate comprises a linker unit between a drug unit (e.g., MMAE) and an antibody unit (e.g., an anti-191P4D12 antibody or an antigen-binding fragment thereof). In some embodiments, the linker is cleavable under intracellular conditions, so that the linker is cleaved in the intracellular environment and releases the drug unit from the antibody. In other embodiments, the linker unit is not cleavable and releases the drug, for example, by antibody degradation. In some embodiments, the linker is cleavable by a cleavage agent present in the intracellular environment (e.g., lysosomes or endosomes or caveolae). The linker may be, for example, a peptidyl linker that can be cleaved by an intracellular peptidase or protease (including but not limited to lysosomal or endosomal proteases). For example, a peptidyl linker (e.g., Phe-Leu or Gly-Phe-Leu-Gly linker (SEQ ID NO: 15)) that can be cleaved by a thiol-dependent protease cathepsin B can be used, and the cathepsin B is highly expressed in cancer tissues. In some embodiments, the length of the peptidyl linker is at least two amino acids or at least three amino acids. In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Generally, pH sensitive joints can be hydrolyzed under acidic conditions. For example, acid labile joints (for example, hydrazone, semicarbazone, sulfur semicarbazone, cis-aconitamide, orthoester, acetal, ketal, etc.) that can be hydrolyzed in lysosome can be used. In other embodiments, joints can be cleaved (for example, disulfide joints) under reducing conditions. Various disulfide joints are known in the art, including, for example, those joints that can be formed using N-succinimidyl-S-acetylthioacetate (SATA), N-succinimidyl-3-(2-pyridyldisulfide) propionate (SPDP), N-succinimidyl-3-(2-pyridyldisulfide) butyrate (SPDB) and N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-disulfide) toluene (SMPT), SPDB and SMPT.

A "Linker Unit" (LU) is a bifunctional compound that can be used to link a Drug unit to an Antibody unit to form an Antibody Drug Conjugate. In some embodiments, the Linker Unit has the following formula:

-A _a -W _w -Y _y -

Wherein: -A- is an extension subunit,

a is 0 or 1,

Each -W- is independently an amino acid unit,

w is an integer in the range of 0 to 12,

-Y- is a self-immolative spacer unit, and

y is 0, 1, or 2.

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 to 12, y is 1 or 2. In some embodiments, w is 2 to 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 have been described in U.S. Pat. No. 8,637,642 and International Application No. PCT/US2019/056214 (Publication No. WO2020/117373), both of which are hereby incorporated by reference in their entirety.

Embodiments of antibody-drug conjugates may include:

where w and y are each 0, 1, or 2, and

Where w and y are each 0,

5.3.4 Drug loading

Drug loading is represented by p, and is the average number of drug units/antibody in the molecule. Drug loading can be in the range of 1 to 20 drug units (D)/antibody. ADC provided herein includes a collection of antibodies or antigen-binding fragments coupled to a series of drug units (e.g., 1 to 20). When ADC is prepared by coupling reaction, the average number of drug units/antibody can be characterized by conventional means such as mass spectrometry and ELISA determination. The quantitative distribution of ADC can also be determined according to p. In some cases, the separation, purification and characterization of homogeneous ADCs with specific values from ADCs with other drug loads can be achieved by means such as electrophoresis.

In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 20. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 18. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 15. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 12. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 10. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 9. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 8. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 7. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 6. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 5. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 4. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 3. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2 to 12. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2 to 10. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2 to 9. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2 to 8. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2 to 7. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2 to 6. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2 to 5. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2 to 4. In certain embodiments, the drug loading of the ADC provided herein is in the range of 3 to 12. In certain embodiments, the drug loading of the ADC provided herein is in the range of 3 to 10. In certain embodiments, the drug loading of the ADC provided herein is in the range of 3 to 9. In certain embodiments, the drug loading of the ADC provided herein is in the range of 3 to 8. In certain embodiments, the drug loading of the ADC provided herein is in the range of 3 to 7. In certain embodiments, the drug loading of the ADC provided herein is in the range of 3 to 6. In certain embodiments, the drug loading of the ADC provided herein is in the range of 3 to 5. In certain embodiments, the drug loading of the ADC provided herein is in the range of 3 to 4.

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.

In certain embodiments, the drug loading of the ADC 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 some embodiments, the drug loading of the ADC 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.

In some embodiments, the drug loading of the ADC 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 ADC 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 ADC provided herein is about 1. In some embodiments, the drug loading of the ADC provided herein is about 2. In some embodiments, the drug loading of the ADC provided herein is about 3. In some embodiments, the drug loading of the ADC provided herein is about 4. In some embodiments, the drug loading of the ADC provided herein is about 3.8. In some embodiments, the drug loading of the ADC provided herein is about 5. In some embodiments, the drug loading of the ADC provided herein is about 6. In some embodiments, the drug loading of the ADC provided herein is about 7. In some embodiments, the drug loading of the ADC provided herein is about 8. In some embodiments, the drug loading of the ADC provided herein is about 9. In some embodiments, the drug loading of the ADC provided herein is about 10. In some embodiments, the drug loading of the ADC provided herein is about 11. In some embodiments, the drug loading of the ADC provided herein is about 12. In some embodiments, the drug loading of the ADC provided herein is about 13. In some embodiments, the drug loading of the ADC provided herein is about 14. In some embodiments, the drug loading of the ADC provided herein is about 15. In some embodiments, the drug loading of the ADC provided herein is about 16. In some embodiments, the drug loading of the ADC provided herein is about 17. In some embodiments, the drug loading of the ADC provided herein is about 18. In some embodiments, the drug loading of the ADC provided herein is about 19. In some embodiments, the drug loading of the ADC provided herein is about 20.

In certain embodiments, during the coupling reaction, less than the theoretical maximum number of drug units are coupled to the antibody. The antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent. In general, antibodies do not contain many free and reactive cysteine thiol groups that can be connected to the drug unit; in fact, most of the cysteine thiol residues in the antibody exist in the form of disulfide bridges. In certain embodiments, the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or complete reducing conditions to produce reactive cysteine thiol groups. In certain embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine. In some embodiments, the linker unit or drug unit is coupled via a lysine residue on the antibody unit. In some embodiments, the linker unit or drug unit is coupled via a cysteine residue on the antibody unit.

In some embodiments, the amino acid connected to the joint unit or drug unit is in the heavy chain of an antibody or its antigen-binding fragment. In some embodiments, the amino acid connected to the joint unit or drug unit is in the light chain of an antibody or its antigen-binding fragment. In some embodiments, the amino acid connected to the joint unit or drug unit is in the hinge region of an antibody or its antigen-binding fragment. In some embodiments, the amino acid connected to the joint unit or drug unit is in the Fc region of an antibody or its antigen-binding fragment. In other embodiments, the amino acid connected to the joint unit or drug unit is in the constant region (e.g., CH1, CH2 or CH3 of the heavy chain, or CH1 of the light chain) of an antibody or its antigen-binding fragment. In other embodiments, the amino acid connected to the joint unit or drug unit is in the VH framework region of an antibody or its antigen-binding fragment. In other embodiments, the amino acid connected to the joint unit or drug unit is in the VL framework region of an antibody or its antigen-binding fragment.

The loading (drug/antibody ratio) of the ADC can be controlled in various ways, for example by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the coupling reaction time or temperature, (iii) partial or limiting reducing conditions for cysteine thiol modification, (iv) engineering the amino acid sequence of the antibody by recombinant technology so that the number and position of cysteine residues are modified to control the number and/or position of linker-drug attachments (e.g., thioMab or thioFab prepared as disclosed herein and in WO2006/034488, which is incorporated herein by reference in its entirety).

It is understood that where more than one nucleophilic group reacts with a drug-linker intermediate or linker reagent, and subsequently reacts with a drug unit reagent, then the resulting product is a distributed mixture of ADC compounds and one or more drug units linked to an antibody unit. The average number of drugs per antibody can be calculated from the mixture by a dual ELISA antibody assay specific for the antibody and specific for the drug. Individual ADC molecules can be identified in the mixture by mass spectrometry and separated by HPLC (e.g., hydrophobic interaction chromatography) (see, e.g., Hamblett, K.J. 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, AACR Conference Proceedings, Vol. 45, March 2004; Alley, S.C. 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, AACR Conference Proceedings, Vol. 45, March 2004). In certain embodiments, homogeneous ADCs having a single loading value can be separated from the conjugated mixture by electrophoresis or chromatography.

Methods for making, screening, and characterizing antibody drug conjugates are known to those of ordinary skill in the art, for example, as described in U.S. Pat. No. 8,637,642, which is incorporated herein by reference in its entirety.

In some embodiments, the antibody drug conjugate used in the methods provided herein is AGS-22M6E, which is prepared according to the method described in U.S. Pat. No. 8,637,642 and has the following formula:

wherein L is Ha22-2(2,4)6.1, and p is 1 to 20.

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.

In some embodiments, the ADC used in the methods provided herein is Ennomonab Vedotin. Ennomonab Vedotin is an ADC comprising a fully human immunoglobulin G1κ (IgG1 _K ) antibody coupled to a microtubule disruptor (MMAE) via a protease-cleavable linker (Challita-Eid PM et al., Cancer Res. 2016; 76 (10): 3003-13). Ennomonab Vedotin binds to the 191P4D12 protein on the cell surface, causing the ADC-191P4D12 complex to internalize, then transport to the lysosomal compartment and release MMAE via proteolytic cleavage of the linker in the compartment to induce anti-tumor activity. Intracellular release of MMAE subsequently interferes with tubulin polymerization, leading to G2/M phase cell cycle arrest and apoptotic cell death (Francisco JA et al., Blood. August 15, 2003; 102 (4): 1458-65).

As described above and in U.S. Pat. No. 8,637,642, AGS-22M6E is an ADC derived from a murine hybridoma cell line. Ennomonab Vedotin is an equivalent of AGS-22M6E ADC derived from a Chinese hamster ovary (CHO) cell line and is an exemplary product for human treatment. Ennomonab Vedotin has the same amino acid sequence, linker and cytotoxic drug as AGS-22M6E. The comparability between Ennomonab Vedotin and AGS-22M6E is determined through in-depth analysis and biocharacterization studies, such as binding affinity to 191P4D12, in vitro cytotoxicity and in vivo anti-tumor activity.

In one embodiment, the ADC provided herein is ennobotin, also known as EV, PADCEV, AGS-22M6E, AGS-22C3E, ASG-22C3E. Ennobotin includes anti-191P4D12 antibodies, 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.

Ennosumab Vedotin is an antibody-drug conjugate (ADC) against connexin-4, which consists of a fully human anti-connexin-4 IgG1κ monoclonal antibody (AGS-22C3) coupled to a small molecule microtubule disruptor monomethyl auristatin E (MMAE) via a protease-cleavable maleimidohexanoyl valine-citrulline (vc) linker (SGD-1006). The coupling occurs on a cysteine residue containing an interchain disulfide bond of the antibody, resulting in a product with a drug: antibody ratio of approximately 3.8:1. The molecular weight is approximately 152 kDa.

Ennosumab vedotin has the following structural formula:

Approximately 4 molecules of MMAE are attached to each antibody molecule. Ennosumab vedotin is produced by chemically coupling the antibody and small molecule components. The antibody is produced by mammalian (Chinese hamster ovary) cells, and the small molecule components are produced by chemical synthesis.

Ennomonab Vedotin injection is provided in single-dose vials as a sterile, preservative-free white to off-white lyophilized powder for intravenous use. Ennomonab Vedotin is supplied in the form of 20mg/vial and 30mg/vial, and needs to be reconstituted with USP sterile water for injection (2.3mL and 3.3mL, respectively), producing a transparent to slightly milky, colorless to slightly yellow solution with a final concentration of 10mg/mL. After reconstruction, each vial allows 2mL (20mg) and 3mL (30mg) to be extracted. Each mL of the reconstituted solution contains 10mg Ennomonab Vedotin, histidine (1.4mg), monohydrated histidine hydrochloride (2.31mg), polysorbate 20 (0.2mg) and dihydrated trehalose (55mg), with a pH of 6.0.

5.4 Pharmaceutical Compositions

In certain embodiments of the methods provided herein, the ADC used in the methods is provided in the form of a "pharmaceutical composition". Such pharmaceutical compositions include the antibody drug conjugates provided herein, and one or more pharmaceutically acceptable or physiologically acceptable excipients. In certain embodiments, the antibody drug conjugates are provided in combination with one or more additional agents or in a separate form therefrom. A composition is also provided, which comprises such one or more additional agents and one or more pharmaceutically acceptable or physiologically acceptable excipients. In a specific embodiment, the antibody drug conjugate and the additional agent are present in a therapeutically acceptable amount. The pharmaceutical composition can be used according to the methods and uses provided herein. Thus, for example, the pharmaceutical composition can be administered to a subject ex vivo or in vivo to practice the methods and uses of treatment 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.

In some embodiments, pharmaceutical compositions having antibody drug conjugates are provided that modulate cancer or tumors.

In certain embodiments of the methods provided herein, the pharmaceutical compositions comprising ADCs may also comprise other therapeutic agents or compounds disclosed herein or known to those skilled in the art, which may be used to treat or prevent various diseases and disorders (e.g., cancer) as described herein. As described above, additional therapeutic agents or compounds may be present in separate pharmaceutical compositions.

The pharmaceutical composition generally comprises 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.

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

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.

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

In some embodiments, the pharmaceutical compositions provided herein comprise 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.

In some embodiments, the concentration of L-histidine suitable for use in the pharmaceutical compositions provided herein is in the range of 5 to 50 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is in the range of 10 to 40 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is in the range of 15 to 35 mM.

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

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

In one embodiment, the concentration of dihydrated trehalose suitable for use in the pharmaceutical composition provided herein is in the range of 1% to 20% (w/v). In another embodiment, the concentration of dihydrated trehalose is in the range of 2% to 15% (w/v). In one embodiment, the concentration of dihydrated trehalose is in the range of 3% to 10% (w/v). In another embodiment, the concentration of dihydrated trehalose is in the range of 4% to 9% (w/v). In another embodiment, the concentration of dihydrated trehalose is in the range of 4% to 8% (w/v). In another embodiment, the concentration of dihydrated trehalose is in the range of 4% to 7% (w/v). In another embodiment, the concentration of dihydrated trehalose is in the range of 4% to 6% (w/v). In another embodiment, the concentration of dihydrated trehalose is in the range of 4.5% to 6% (w/v). In another embodiment, the concentration of dihydrated trehalose 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). In another embodiment, the concentration of trehalose dihydrate is about 6.5% (w/v).

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

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

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

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

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.

In some embodiments, the pH is measured at room temperature. In other embodiments, the pH is measured at 15°C to 27°C. In other embodiments, the pH is measured at 4°C. In other embodiments, the pH is measured at 25°C.

In some embodiments, the pH is adjusted by 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.

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

In some embodiments, pH is adjusted by 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.

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

In some specific embodiments, the pharmaceutical compositions provided herein comprise at least one of about 20 mM L-histidine, about 0.02% (w/v) TWEEN-20 and 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.

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.

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.

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.

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.

In a specific embodiment, provided herein is a

(a) an antibody-drug conjugate comprising the following structure:

wherein L- represents an antibody or an antigen-binding fragment thereof (eg, an anti-connexin-4 antibody or an 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 concentration of the antibody drug conjugate is about 10 mg/mL, and wherein the pH is about 6.0 at 25°C.

In another specific embodiment, the pharmaceutical composition provided herein comprises:

(a) an antibody-drug conjugate comprising the following structure:

wherein L- represents an antibody or an antigen-binding fragment thereof (eg, an anti-connexin-4 antibody or an 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,

wherein the concentration of the antibody drug conjugate is about 10 mg/mL, and wherein the pH is about 6.0 at 25°C.

In another specific embodiment, the pharmaceutical composition provided herein comprises:

(a) an antibody-drug conjugate comprising the following structure:

wherein L- represents an antibody or an antigen-binding fragment thereof (eg, an anti-connexin-4 antibody or an 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,

wherein the concentration of the antibody drug conjugate is about 10 mg/mL, and wherein the pH is about 6.0 at 25°C.

While certain numbers (and numerical ranges thereof) are provided, it is understood that in certain embodiments, values within, for example, 2%, 5%, 10%, 15%, or 20% of the stated number (or numerical range) are also contemplated.

The main solvent in the vehicle can be aqueous or non-aqueous in nature. In addition, the vehicle may contain other pharmaceutically acceptable excipients for modifying or maintaining the pH, molar osmotic pressure concentration, viscosity, sterility or stability of the pharmaceutical composition. In certain embodiments, the pharmaceutically acceptable vehicle is an aqueous buffer. In other embodiments, the vehicle comprises, for example, sodium chloride and/or sodium citrate.

The pharmaceutical compositions provided herein may also contain other pharmaceutically acceptable formulations for modifying or maintaining the release rate of the antibody drug conjugates and/or additional agents as described herein. Such formulations include those substances known to skilled artisans for preparing sustained release formulations. For other references on pharmaceutically and physiologically acceptable formulations, see, for example, Remington's Pharmaceutical Sciences, 18th edition (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, The Merck Index, 12th edition (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 can be applied to the methods and compositions provided herein.

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

The pharmaceutical composition can be formulated to suit its intended route of administration. Thus, the pharmaceutical composition includes excipients suitable for administration by routes including parenteral (e.g., subcutaneous (s.c.), intravenous, intramuscular or intraperitoneal), intradermal, oral (e.g., ingestion), inhalation, intracavity, intracranial and transdermal (topical). Other exemplary routes of administration are described herein.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension. Such suspensions may be prepared using suitable dispersants or wetting agents and suspending agents disclosed herein or known to those skilled in the art. Sterile injectable preparations may also be sterile injectable solutions or suspensions in nontoxic parenterally acceptable diluents or solvents, such as in the form of solutions 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. In addition, sterile non-volatile oils are conventionally used as solvents or suspension media. For this purpose, any mild non-volatile oil may be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid may be used to prepare injectables. Prolonged absorption of certain injectable formulations can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate or gelatin.

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

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

In one embodiment, pharmaceutical compositions intended for parenteral administration may include one or more pharmaceutically acceptable excipients including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives to combat the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, chelating agents or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusters, and inert gases.

In one embodiment, suitable aqueous vehicles include, but are not limited to, water, saline, physiological 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 plant origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, medium chain triglycerides of hydrogenated soybean oil and coconut oil, and palm kernel oil. Water-miscible vehicles include, but are not limited to, ethanol, 1,3-butylene glycol, liquid polyethylene glycols (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerol, N-methyl-2-pyrrolidone, N,N-dimethylacetamide and dimethyl sulfoxide.

In one embodiment, suitable antimicrobial agents or preservatives include, but are not limited to, phenol, cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl and propyl parabens, and sorbic acid. Suitable isotonic agents include, but are not limited to, sodium chloride, glycerol, and dextrose. Suitable buffers include, but are not limited to, phosphates and citrates. Suitable antioxidants are those described herein, including bisulfites and sodium metabisulfite. Suitable local anesthetics include, but are not limited to, procaine hydrochloride. Suitable suspending and dispersing agents are suspending and dispersing agents as described herein, including sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Suitable emulsifiers include those described herein, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate. Suitable chelating agents 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, including α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, and sulfobutyl ether 7-β-cyclodextrin ( CyDex, Lenexa, KS).

In one embodiment, the pharmaceutical composition provided herein can be formulated for single dose or multiple dose administration. Single dose preparations are packaged in ampoules, vials or syringes. Multiple dose parenteral preparations can contain antimicrobial agents at antibacterial or antifungal concentrations. As known and practiced in the art, all parenteral preparations must be sterile.

In one embodiment, the pharmaceutical composition is provided in the form of a ready-to-use sterile solution. In another embodiment, the pharmaceutical composition is provided as a sterile dry soluble product to be reconstituted with a vehicle before use, including lyophilized powders and subcutaneous tablets. In another embodiment, the pharmaceutical composition is provided in the form of a ready-to-use sterile suspension. In another embodiment, the pharmaceutical composition is provided in the form of a sterile dry insoluble product to be reconstituted with a vehicle before use. In another embodiment, the pharmaceutical composition is provided in the form of a ready-to-use sterile emulsion.

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

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, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.

The pharmaceutical composition may also include excipients to protect the composition from rapid degradation or discharge from the body, such as controlled release formulations, including implants, liposomes, hydrogels, prodrugs and microencapsulation delivery systems. For example, time delay materials, such as glyceryl monostearate or glyceryl stearate, may be used alone or in combination with wax. The extended absorption of injectable pharmaceutical compositions may be achieved by including agents (such as aluminum monostearate or gelatin) that delay absorption. The prevention of microbial action may be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.

The pharmaceutical compositions provided herein can be stored at -80°C, 4°C, 25°C, or 37°C.

Lyophilized compositions can be prepared by freeze drying the liquid pharmaceutical compositions provided herein. In a specific embodiment, the pharmaceutical compositions provided herein are lyophilized pharmaceutical compositions. In some embodiments, the pharmaceutical preparations are lyophilized powders that can be reconstituted for administration in the form of solutions, emulsions, and other mixtures. They can also be reconstituted and formulated as solids or gels.

In some embodiments, preparation of the lyophilized formulations provided herein involves batching the formulated bulk solution for lyophilization, sterile filtering, filling into vials, freezing the vials in a freeze dryer chamber, followed by lyophilization, stoppering, and capping.

A freeze dryer can be used to prepare freeze-dried formulations. For example, a VirTis Genesis Model EL test unit can be used. The unit has a chamber with three working shelves (total usable shelf area of about 0.4 square meters), an external condenser, and a mechanical vacuum pump system. Cascaded mechanical refrigeration allows the shelves to be cooled to -70°C or lower, and allows the external condenser to be cooled to -90°C or lower. The shelf temperature and chamber pressure are automatically controlled to +/-0.5°C and +/-2 microns (mitorr), respectively. The unit is equipped with a vacuum capacitance manometer, a Pirani vacuum gauge, a pressure converter (measuring range: 0 to 1 atmosphere), and a relative humidity sensor.

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 sterile. The solution is then sterile filtered and then lyophilized under standard conditions known to those skilled in the art to obtain the desired formulation. In one embodiment, the resulting solution will be distributed into vials for lyophilization. Each vial will contain 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.

Reconstitute this lyophilized powder with water for injection to obtain a formulation for parenteral administration. For reconstitution, add the lyophilized powder to sterile water or other suitable excipients. This amount can be determined empirically and adjusted according to specific needs.

An exemplary reconstitution procedure is described as follows: (1) equip a 5 mL or 3 mL syringe with an 18 or 20 gauge needle and fill the syringe with water for injection (WFI) grade water; (2) measure the appropriate amount of WFI using the syringe scale, ensuring that the syringe contains no bubbles; (3) insert the needle through the rubber stopper; (4) dispense the entire contents of the syringe down the wall of the vial into the container, remove the syringe and needle and place in the pointed container; (4) continue to vortex the vial to carefully dissolve the entire vial contents until completely reconstituted (e.g., about 20-40 seconds) and to minimize excessive agitation of the protein solution which can cause foaming.

In some embodiments, the pharmaceutical composition provided herein is supplied in a hermetic sealed container in the form of a dry sterile lyophilized powder or anhydrous concentrate, and can be reconstituted to an appropriate concentration with, for example, water or saline to be administered to a subject. In certain embodiments, the antibody drug conjugate is supplied in a hermetic sealed container in the form of a dry sterile lyophilized powder with the following unit doses: 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 between 2 and 8 ° C, and the antibody drug conjugate can be administered within 12 hours after reconstitution, for example, within 6 hours, within 5 hours, within 3 hours, or within 1 hour. In an alternative embodiment, the pharmaceutical composition comprising the antibody drug conjugate provided herein is supplied in a gas-tight sealed container in liquid form, and the container indicates the quantity and concentration of the antibody drug conjugate. In certain embodiments, the antibody drug conjugate in liquid form is supplied in a gas-tight sealed container with the following dosage: 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.

Additional embodiments of pharmaceutical compositions have been described in U.S. Pat. No. 8,637,642 and International Application No. PCT/US2019/056214 (Publication No. WO2020/117373), both of which are hereby incorporated by reference in their entirety.

5.5 Combination treatment approaches

Methods for inhibiting tumor cell growth using the pharmaceutical composition provided herein and chemotherapy or radiation or a combination of the two, including before, during or after starting chemotherapy or radiotherapy and any combination thereof (i.e., before and during the start of chemotherapy and/or radiotherapy, before and after, during and after, or before, during and after), administering the pharmaceutical composition of the present invention. Depending on the treatment regimen and specific patient needs, the method is performed in a manner that will provide the most effective treatment and ultimately extend the patient's life. Additional embodiments of such combination therapies have been 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 entirety.

5.6 Dosage of immune checkpoint inhibitors

In some embodiments, the amount of a checkpoint inhibitor used in the various methods provided herein is determined by standard clinical techniques.

The dose of the checkpoint inhibitor produces a serum titer of about 0.1 μg/ml to about 450 μg/ml, and in some embodiments, the following amounts can be administered to humans to prevent and/or treat cancer: at least 0.1 μg/ml, at least 0.2 μg/ml, at least 0.4 μg/ml, at least 0.5 μg/ml, at least 0.6 μg/ml, at least 0.8 μg/ml, at least 1 μg/ml, at least 1.5 μg/ml, for example, at least 2 μg/ml, at least 5 μg/ml, at least 10 μg/ml. l, at least 15μg/ml, at least 20μg/ml, at least 25μg/ml, at least 30μg/ml, at least 35μg/ml, at least 40μg/ml, at least 50μg/ml, at least 75μg/ml, at least 100μg/ml, at least 125μg/ml, at least 150μg/ml, at least 200μg/ml, at least 250μg/ml, at least 300μg/ml, at least 350μg/ml, at least 400μg/ml or at least 450μg/ml. It should be understood that the precise dose of the checkpoint inhibitor to be employed will also depend on the route of administration and the severity of the subject's cancer, and should be determined according to the judgment of the practitioner and the circumstances of each patient.

In some embodiments, the dosage of the checkpoint inhibitor (e.g., PD-1 inhibitor or PD-L1 inhibitor) administered to the patient is generally 0.1 mg/kg to 100 mg/kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 1 mg/kg to about 75 mg/kg of the subject's body weight. In some embodiments, the dosage administered to the patient is between 1 mg/kg and 20 mg/kg of the subject's body weight, for example, 1 mg/kg to 5 mg/kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 1 mg/kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 1.5 mg/kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 2 mg/kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 2.5 mg/kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 3 mg/kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 3.5 mg/kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 4 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 4.5 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 5 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 5.5 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 6 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 6.5 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 7 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 7.5 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 8 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 8.5 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 9.0 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 10.0 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 15.0 mg/kg of the subject's body weight. In some embodiments, the dose administered to a patient is about 20.0 mg/kg of subject body weight.

5.7 Dosage of ADC for Methods

In some embodiments, the amount of a prophylactic or therapeutic agent (eg, an antibody drug conjugate provided herein) or a pharmaceutical composition provided herein that will be effective in preventing and/or treating cancer can be determined by standard clinical techniques.

In some embodiments, the ADC of the methods describing various doses in this section (Section 5.7) is enroku vedotin (EV).

Thus, the dose of the antibody drug conjugate in the pharmaceutical composition produces a serum titer of about 0.1 μg/ml to about 450 μg/ml, and in some embodiments, the following doses can be administered to humans to prevent and/or treat cancer: at least 0.1 μg/ml, at least 0.2 μg/ml, at least 0.4 μg/ml, at least 0.5 μg/ml, at least 0.6 μg/ml, at least 0.8 μg/ml, at least 1 μg/ml, at least 1.5 μg/ml, for example, at least 2 μg/ml, at least 5 ... 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 30 μg/ml, at least 35 μg/ml, at least 40 μg/ml, at least 50 μg/ml, at least 75 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 200 μg/ml, at least 250 μg/ml, at least 300 μg/ml, at least 350 μg/ml, at least 400 μg/ml or at least 450 μg/ml. It will be understood that the precise dose to be used in the formulation will also depend on the route of administration and the severity of the subject's cancer, and should be decided according to the judgment of the practitioner and each patient's circumstances.

Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For pharmaceutical compositions comprising antibody drug conjugates provided herein, the dosage of the antibody drug conjugates administered to the patient is generally 0.1 mg/kg to 100 mg/kg of subject weight. In some embodiments, the dosage administered to the patient is about 1 mg/kg to about 75 mg/kg of subject weight. In some embodiments, the dosage administered to the patient is between 1 mg/kg and 20 mg/kg of subject weight, for example, 1 mg/kg to 5 mg/kg of subject weight. In some embodiments, the dosage administered to the patient is about 0.5 mg/kg of subject weight. In some embodiments, the dosage administered to the patient is about 0.75 mg/kg of subject weight. In some embodiments, the dosage administered to the patient is about 1 mg/kg of subject weight. In some embodiments, the dosage administered to the patient is about 1.25 mg/kg of subject weight. In some embodiments, the dosage administered to the patient is about 1.5 mg/kg of subject weight. In some embodiments, the dosage administered to the patient is about 2 mg/kg of subject weight. In some embodiments, the dosage administered to the patient is about 2.5 mg/kg of subject weight. In some embodiments, the dose administered to the patient is about 3 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 3.5 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 4 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 4.5 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 5 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 5.5 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 6 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 6.5 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 7 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 7.5 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 8 mg/kg of the subject's body weight. In some embodiments, the dose administered to the patient is about 8.5 mg/kg of the subject's body weight.

In some embodiments, the antibody drug conjugate formulated in the pharmaceutical composition provided herein is administered based on the patient's actual body weight at baseline, and unless the patient's body weight changes ≥10% from the baseline of the previous cycle or meets the dose adjustment criteria, the dose will remain unchanged. In some embodiments, the actual weight will be used unless the patient weighs more than 100 kg, in which case the dose will be calculated based on a weight of 100 kg. In some embodiments, for patients receiving a 1.00 mg/kg dose level, the maximum dose is 100 mg; and for patients receiving a 1.25 mg/kg dose level, the maximum dose is 125 mg.

In one embodiment, about 100 mg/kg or less, about 75 mg/kg or less, about 50 mg/kg or less, about 25 mg/kg or less, about 10 mg/kg or less, about 5 mg/kg or less, about 1.5 mg/kg or less, about 1.25 mg/kg or less, about 1 mg/kg or less, about 0.75 mg/kg or less, about 0.5 mg/kg or less, or about 0.1 mg/kg or less dose of the antibody drug conjugate formulated into the pharmaceutical composition of the present invention is administered 5 times, 4 times, 3 times, 2 times or 1 time to treat cancer. In some embodiments, the pharmaceutical composition comprising the antibody drug conjugate provided herein is administered about 1-12 times, wherein the dose can be administered as needed, for example, once a week, once every two weeks, once a month, once every two months, once every three months, etc., as determined by a physician. In some embodiments, lower doses (e.g., 0.1-15 mg/kg) can be administered more frequently (e.g., 3-6 times). In other embodiments, higher doses (eg, 25-100 mg/kg) may be administered less frequently (eg, 1-3 times).

In some embodiments, for each two-week cycle (e.g., about 14 days) for a certain period of time (e.g., one year), a single dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 times of the antibody drug conjugate formulated in the pharmaceutical composition provided herein is administered to the patient to prevent and/or treat cancer, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, about 2 mg/kg g, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or a combination thereof (i.e., each monthly dose may be the same or may be different).

In some embodiments, for each three-week cycle (e.g., about 21 days) for a certain period of time (e.g., one year), a single dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 times of the antibody drug conjugate formulated in the pharmaceutical composition provided herein is administered to the patient to prevent and/or treat cancer, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, about 2 mg/kg g, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or a combination thereof (i.e., each monthly dose may be the same or may be different).

In some embodiments, for each four-week cycle (e.g., about 28 days) for a certain period of time (e.g., one year), a single dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 times of the antibody drug conjugate formulated in the pharmaceutical composition provided herein is administered to the patient to prevent and/or treat cancer, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, about 2 mg/kg g, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or a combination thereof (i.e., each monthly dose may be the same or may be different).

In another embodiment, a single dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 times of an antibody drug conjugate formulated in a pharmaceutical composition provided herein is administered to a patient at about monthly intervals (e.g., about 30 days) for a certain period of time (e.g., one year) to prevent and/or treat cancer, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, About 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or a combination thereof (i.e., each monthly dose may or may not be the same).

In another embodiment, a single dose of an antibody drug conjugate formulated in a pharmaceutical composition provided herein is administered to a patient 1, 2, 3, 4, 5 or 6 times at intervals of about two months (e.g., about 60 days) for a certain period of time (e.g., one year) to prevent and/or treat cancer, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg , about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or a combination thereof (i.e., each monthly dose may or may not be the same).

In another embodiment, a single dose of an antibody drug conjugate formulated in a pharmaceutical composition provided herein is administered to a patient 1, 2, 3 or 4 times at intervals of about three months (e.g., about 120 days) for a certain period of time (e.g., one year) to prevent and/or treat cancer, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about In some embodiments, the dosage may be about 50 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or a combination thereof (i.e., each monthly dosage may or may not be the same).

In certain embodiments, the route of administration for administering to a patient a dosage of an antibody drug conjugate formulated in a pharmaceutical composition provided herein is intranasal, intramuscular, intravenous, or a combination thereof, but other routes described herein are also acceptable. Each dose may or may not be administered by the same route of administration. In some embodiments, the antibody drug conjugate formulated in a pharmaceutical composition provided herein may be administered simultaneously or thereafter with one or more additional therapeutic agents of other doses via a variety of routes of administration.

In some more specific embodiments, the antibody drug conjugate formulated in the pharmaceutical compositions provided herein is administered by intravenous (IV) injection or infusion at a dose of about 0.5 mg/kg, about 0.75 mg/kg, about 1 mg/kg, about 1.25 mg/kg, or about 1.5 mg/kg of the subject's body weight.

In some more specific embodiments, the antibody drug conjugate formulated in the pharmaceutical composition provided herein is a dose of about 0.5 mg/kg, about 0.75 mg/kg, about 1 mg/kg, about 1.25 mg/kg or about 1.5 mg/kg of the subject's body weight, and is administered by intravenous (IV) injection or infusion twice for about 30 minutes per three-week cycle. In some embodiments, the antibody drug conjugate formulated in the pharmaceutical composition is administered by intravenous (IV) injection or infusion for about 30 minutes on the 1st and 8th day of each three-week cycle. In some embodiments, the method also includes administering immune checkpoint inhibitors by intravenous (IV) injection or infusion once or more in each three-week cycle. In some embodiments, the method also includes administering immune checkpoint inhibitors by intravenous (IV) injection or infusion on the 1st day of each three-week cycle. In some embodiments, the immune checkpoint inhibitor is pembrolizumab, and wherein pembrolizumab is administered over about 30 minutes in an amount of about 200 mg. In other embodiments, the immune checkpoint inhibitor is atezolizumab, and atezolizumab is administered in an amount of about 1200 mg over about 60 minutes or 30 minutes. In some embodiments, an antibody-drug conjugate is administered to patients with urothelial carcinoma or bladder cancer who have shown disease progression or recurrence during or after treatment with another cancer treatment. In some embodiments, an antibody-drug conjugate is administered to patients with metastatic urothelial carcinoma or bladder cancer who have shown disease progression or recurrence during or after treatment with another cancer treatment. In some embodiments, an antibody-drug conjugate is administered to patients with locally advanced urothelial carcinoma or bladder cancer who have shown disease progression or recurrence during or after treatment with another cancer treatment. In some embodiments, the ADC of the method describing various doses in this paragraph is ennomonab vedotin (EV).

In other more specific embodiments, the antibody drug conjugate formulated in the pharmaceutical composition provided herein is a dose of about 0.5 mg/kg, about 0.75 mg/kg, 1 mg/kg, about 1.25 mg/kg or about 1.5 mg/kg of the subject's body weight, administered by three about 30-minute intravenous (IV) injections or infusions every four weeks. In some embodiments, the antibody drug conjugate formulated in the pharmaceutical composition is administered on the 1st day, 8th day and 15th day of every 28-day (four weeks) cycle. In some embodiments, the antibody drug conjugate formulated in the pharmaceutical composition is administered by about 30-minute intravenous (IV) injections or infusions on the 1st day, 8th day and 15th day of every 28-day (four weeks) cycle. In some embodiments, the method also includes administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion once or more in each four-week cycle. In some embodiments, the immune checkpoint inhibitor is pembrolizumab. In other embodiments, the immune checkpoint inhibitor is atezolizumab. In some embodiments, an antibody drug conjugate is administered to a patient with urothelial carcinoma or bladder cancer who has shown disease progression or recurrence during or after treatment with another cancer therapy. In some embodiments, an antibody drug conjugate is administered to a patient with metastatic urothelial carcinoma or bladder cancer who has shown disease progression or recurrence during or after treatment with another cancer therapy. In some embodiments, an antibody drug conjugate is administered to a patient with locally advanced urothelial carcinoma or bladder cancer who has shown disease progression or recurrence during or after treatment with another cancer therapy. In some embodiments, the ADC of the method describing various doses in this paragraph is ennomonab vedotin (EV).

In some embodiments of the various methods provided herein, ADC is administered at a dosage of about 0.25 to about 10 mg/kg subject weight, about 0.25 to about 5 mg/kg subject weight, about 0.25 to about 2.5 mg/kg subject weight, about 0.25 to about 1.25 mg/kg subject weight, about 0.5 to about 10 mg/kg subject weight, about 0.5 to about 5 mg/kg subject weight, about 0.5 to about 2.5 mg/kg subject weight, about 0.5 to about 1.25 mg/kg subject weight, about 0.75 to about 10 mg/kg subject weight, about 0.75 to about 5 mg/kg subject weight, about 0.75 to about 2.5 mg/kg subject weight, or about 0.75 to about 1.25 mg/kg subject weight. In some embodiments, ADC is administered at a dosage of about 1 to about 10 mg/kg subject weight. In certain embodiments, ADC is administered at a dosage of about 1 to about 5 mg/kg subject weight. In other embodiments, ADC is administered at a dosage of about 1 to about 2.5 mg/kg subject weight. In other embodiments, the ADC is administered at a dose of about 1 to about 1.25 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of about 0.25 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of about 0.5 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of about 0.75 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of about 1.0 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of about 1.25 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of about 1.5 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of about 1.75 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of about 2.0 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of about 2.25 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of about 2.5 mg/kg of the subject's body weight.

In certain embodiments of the various methods provided herein, ADC is administered at a dosage of 0.25 to 10 mg/kg subject weight, 0.25 to 5 mg/kg subject weight, 0.25 to 2.5 mg/kg subject weight, 0.25 to 1.25 mg/kg subject weight, 0.5 to 10 mg/kg subject weight, 0.5 to 5 mg/kg subject weight, 0.5 to 2.5 mg/kg subject weight, 0.5 to 1.25 mg/kg subject weight, 0.75 to 10 mg/kg subject weight, 0.75 to 5 mg/kg subject weight, 0.75 to 2.5 mg/kg subject weight, or 0.75 to 1.25 mg/kg subject weight. In some embodiments, ADC is administered at a dosage of 1 to 10 mg/kg subject weight. In certain embodiments, ADC is administered at a dosage of 1 to 5 mg/kg subject weight. In other embodiments, ADC is administered at a dosage of 1 to 2.5 mg/kg subject weight. In other embodiments, the ADC is administered at a dose of 1 to 1.25 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of 0.25 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of 0.5 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of 0.75 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of 1.0 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of 1.25 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of 1.5 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of 1.75 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of 2.0 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of 2.25 mg/kg of the subject's body weight. In some embodiments, the ADC is administered at a dose of 2.5 mg/kg of the subject's body weight.

In some embodiments of the various methods provided herein (including those requiring a first and second doses), the first ADC dose is a dose of about 0.25 to about 10 mg/kg subject body weight, about 0.25 to about 5 mg/kg subject body weight, about 0.25 to about 2.5 mg/kg subject body weight, about 0.25 to about 1.25 mg/kg subject body weight, about 0.5 to about 10 mg/kg subject body weight, about 0.5 to about 5 mg/kg subject body weight, about 0.5 to about 2.5 mg/kg subject body weight, about 0.5 to about 1.25 mg/kg subject body weight, about 0.75 to about 10 mg/kg subject body weight, about 0.75 to about 5 mg/kg subject body weight, about 0.75 to about 2.5 mg/kg subject body weight, or about 0.75 to about 1.25 mg/kg subject body weight. In some embodiments of the various methods provided herein (including those requiring a first and second doses), the first ADC dose is a dose of about 1 to about 10 mg/kg subject body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the first ADC dose is a dose of about 1 to about 5 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the first ADC dose is a dose of about 1 to about 2.5 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the first ADC dose is a dose of about 1 to about 1.25 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the first ADC dose is a dose of about 0.25 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the first ADC dose is a dose of about 0.5 mg/kg of the subject's body weight. In some embodiments, the first ADC dose is a dose of about 0.75 mg/kg of the subject's body weight. In some embodiments, the first ADC dose is a dose of about 1.0 mg/kg of the subject's body weight. In some embodiments, the first ADC dose is a dose of about 1.25 mg/kg of the subject's body weight. In some embodiments, the first ADC dose is a dose of about 1.5 mg/kg of subject body weight. In some embodiments, the first ADC dose is a dose of about 1.75 mg/kg of subject body weight. In some embodiments, the first ADC dose is a dose of about 2.0 mg/kg of subject body weight. In some embodiments, the first ADC dose is a dose of about 2.25 mg/kg of subject body weight. In some embodiments, the first ADC dose is a dose of about 2.5 mg/kg of subject body weight.

In certain embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the first ADC dose is a dose of 0.25 to 10 mg/kg subject body weight, 0.25 to 5 mg/kg subject body weight, 0.25 to 2.5 mg/kg subject body weight, 0.25 to 1.25 mg/kg subject body weight, 0.5 to 10 mg/kg subject body weight, 0.5 to 5 mg/kg subject body weight, 0.5 to 2.5 mg/kg subject body weight, 0.5 to 1.25 mg/kg subject body weight, 0.75 to 10 mg/kg subject body weight, 0.75 to 5 mg/kg subject body weight, 0.75 to 2.5 mg/kg subject body weight, or 0.75 to 1.25 mg/kg subject body weight. In certain embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the first ADC dose is a dose of 1 to 10 mg/kg subject body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the first ADC dose is a dose of 1 to 5 mg/kg of subject body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the first ADC dose is a dose of 1 to 2.5 mg/kg of subject body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the first ADC dose is a dose of 1 to 1.25 mg/kg of subject body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the first ADC dose is a dose of 0.25 mg/kg of subject body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the first ADC dose is a dose of 0.5 mg/kg of subject body weight. In some embodiments, the first ADC dose is a dose of 0.75 mg/kg of subject body weight. In some embodiments, the first ADC dose is a dose of 1.0 mg/kg of subject body weight. In some embodiments, the first ADC dose is a dose of 1.25 mg/kg of subject body weight. In some embodiments, the first ADC dose is a dose of 1.5 mg/kg of subject body weight. In some embodiments, the first ADC dose is a dose of 1.75 mg/kg of subject body weight. In some embodiments, the first ADC dose is a dose of 2.0 mg/kg of subject body weight. In some embodiments, the first ADC dose is a dose of 2.25 mg/kg of subject body weight. In some embodiments, the first ADC dose is a dose of 2.5 mg/kg of subject body weight.

In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.1 mg/kg to about 2 mg/kg of subject weight lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.1 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.2 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.25 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.3 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.4 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.5 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.6 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.7 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.75 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.8 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.9 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 1 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 1.1 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 1.2 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 1.25 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 1.3 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 1.4 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 1.5 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 1.6 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 1.7 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 1.75 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 1.8 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 1.9 mg/kg lower than the first dose. In some embodiments of the various methods provided herein, including those requiring a first dose and a second dose, the second ADC dose is about 2 mg/kg body weight of the subject lower than the first dose.

In certain embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 0.1 mg/kg to 2 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is about 0.1 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 0.2 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 0.25 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 0.3 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 0.4 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 0.5 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 0.6 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 0.7 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 0.75 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 0.8 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 0.9 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 1 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 1.1 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 1.2 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 1.25 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 1.3 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 1.4 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 1.5 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 1.6 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 1.7 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 1.75 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 1.8 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 1.9 mg/kg lower than the first dose. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is 2 mg/kg lower than the first dose.

In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 0.25 to about 10 mg/kg subject body weight, about 0.25 to about 5 mg/kg subject body weight, about 0.25 to about 2.5 mg/kg subject body weight, about 0.25 to about 1.25 mg/kg subject body weight, about 0.5 to about 10 mg/kg subject body weight, about 0.5 to about 5 mg/kg subject body weight, about 0.5 to about 2.5 mg/kg subject body weight, about 0.5 to about 1.25 mg/kg subject body weight, about 0.75 to about 10 mg/kg subject body weight, about 0.75 to about 5 mg/kg subject body weight, about 0.75 to about 2.5 mg/kg subject body weight, or about 0.75 to about 1.25 mg/kg subject body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 1 to about 10 mg/kg subject body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 1 to about 5 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 1 to about 2.5 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 1 to about 1.25 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 0.25 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 0.5 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 0.75 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 1.0 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 1.25 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 1.5 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 1.75 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 2.0 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of about 2.25 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein, including those requiring a first dose and a second dose, the second ADC dose is a dose of about 2.5 mg/kg body weight of the subject.

In certain embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 0.25 to 10 mg/kg subject body weight, 0.25 to 5 mg/kg subject body weight, 0.25 to 2.5 mg/kg subject body weight, 0.25 to 1.25 mg/kg subject body weight, 0.5 to 10 mg/kg subject body weight, 0.5 to 5 mg/kg subject body weight, 0.5 to 2.5 mg/kg subject body weight, 0.5 to 1.25 mg/kg subject body weight, 0.75 to 10 mg/kg subject body weight, 0.75 to 5 mg/kg subject body weight, 0.75 to 2.5 mg/kg subject body weight, or 0.75 to 1.25 mg/kg subject body weight. In certain embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 1 to 10 mg/kg subject body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 1 to 5 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 1 to 2.5 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 1 to 1.25 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 0.25 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 0.5 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 0.75 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 1.0 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 1.25 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 1.5 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 1.75 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 2.0 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 2.25 mg/kg of the subject's body weight. In some embodiments of the various methods provided herein (including those requiring a first dose and a second dose), the second ADC dose is a dose of 2.5 mg/kg of the subject's body weight.

In certain embodiments of the various methods provided herein, including those methods requiring a first dose and a second dose, the second ADC dose is the same as the first ADC dose.

In some embodiments of the methods provided herein, ADC is administered by intravenous (IV) injection or infusion. In one embodiment, the first ADC dose is administered by IV injection. In another embodiment, the first ADC dose is administered by IV infusion. In another embodiment, the second ADC dose is administered by IV injection. In another embodiment, the second ADC dose is administered by IV infusion. In one embodiment, the first ADC dose is administered by IV injection and the second ADC dose is administered by IV injection. In another embodiment, the first ADC dose is administered by IV infusion and the second ADC dose is administered by IV injection. In another embodiment, the first ADC dose is administered by IV injection and the second ADC dose is administered by IV infusion. In another embodiment, the first ADC dose is administered by IV injection and the second ADC dose is administered by IV infusion. In another embodiment, the first ADC dose is administered by IV infusion and the second ADC dose is administered by IV infusion. In another embodiment, the first ADC dose is administered by IV infusion and the second ADC dose is administered by IV infusion. In some embodiments, the ADC of the method describing various doses in this paragraph is Ennomonab Vedotin (EV).

In certain embodiments of the methods provided herein, the ADC is administered by three IV injections or infusions every four weeks. In some embodiments of the methods provided herein, the first ADC dose is administered by three IV injections or infusions every four weeks. In some embodiments of the methods provided herein, the second ADC dose is administered by three IV injections or infusions every four weeks. In some embodiments of the methods provided herein, the first ADC dose is administered by three IV injections or infusions every four weeks, and the second ADC dose is administered by three IV injections or infusions every four weeks. In some embodiments of the methods provided herein, the first ADC dose is administered by three IV injections or infusions every four weeks, and the second ADC dose is administered by three IV injections or infusions every four weeks. In some embodiments, the ADC of the method describing various doses in this paragraph is Ennomonab Vedotin (EV).

In some embodiments of the methods provided herein, ADC is administered by IV injection or infusion on the 1st day, 8th day and 15th day of every four-week cycle. In some embodiments, the first ADC dose is administered by IV injection or infusion on the 1st day, 8th day and 15th day of every four-week cycle. In some embodiments, the second ADC dose is administered by IV injection or infusion on the 1st day, 8th day and 15th day of every four-week cycle. In some embodiments, the first ADC dose is administered by IV injection or infusion on the 1st day, 8th day and 15th day of every four-week cycle, and the second ADC dose is administered by IV injection or infusion on the 1st day, 8th day and 15th day of every four-week cycle. In some embodiments, the ADC of the method for describing various doses in this paragraph is Ennomonab Vedotin (EV).

In certain embodiments of the methods provided herein, ADC is administered by three about 30-minute IV injections or infusions every four weeks. In some embodiments, the first ADC dose is administered by three about 30-minute IV injections or infusions every four weeks. In some embodiments, the second ADC dose is administered by three about 30-minute IV injections or infusions every four weeks. In some embodiments, the first ADC dose is administered by three about 30-minute IV injections or infusions every four weeks, and the second ADC dose is administered by three about 30-minute IV injections or infusions every four weeks. In some embodiments, the ADC of the method for describing various doses in this paragraph is Ennomonab Vedotin (EV).

In some embodiments of the methods provided herein, the ADC is administered by about 30 minutes of IV injection or infusion on the 1st day, 8th day and 15th day of every four-week cycle. In some embodiments of the methods provided herein, the first ADC dose is administered by about 30 minutes of IV injection or infusion on the 1st day, 8th day and 15th day of every four-week cycle. In some embodiments of the methods provided herein, the second ADC dose is administered by about 30 minutes of IV injection or infusion on the 1st day, 8th day and 15th day of every four-week cycle. In some embodiments of the methods provided herein, the first ADC dose is administered by about 30 minutes of IV injection or infusion on the 1st day, 8th day and 15th day of every four-week cycle, and the second ADC dose is administered by about 30 minutes of IV injection or infusion on the 1st day, 8th day and 15th day of every four-week cycle. In some embodiments, the ADC of the method describing various doses in this paragraph is Ennomonab Vedotin (EV).

In other more specific embodiments, the antibody drug conjugate formulated in the pharmaceutical composition provided herein is at a dose of about 1 mg/kg, 1.25 mg/kg or about 1.5 mg/kg of the subject's body weight, administered by three about 30-minute intravenous (IV) injections or infusions per 28-day cycle. In some embodiments, the antibody drug conjugate formulated in the pharmaceutical composition is administered by about 30-minute intravenous (IV) injections or infusions on the 1st, 8th and 15th days of each 28-day cycle. In some embodiments, the method further comprises administering immune checkpoint inhibitors by intravenous (IV) injection or infusion once or more in each four-week cycle. In some embodiments of the method provided herein, ADC is administered three times within a 28-day cycle. In some embodiments of the method provided herein, ADC is administered on the 1st, 8th and 15th days of a 28-day cycle. In some embodiments, the ADC of the method describing various doses in this paragraph is Ennomonab Vedotin (EV).

5.8 Methods for Determining Biomarkers

The disclosure provides that the expression of any marker provided herein can be measured by various methods known in the art. In some embodiments, the expression of the marker can be measured according to the amount or relative amount of mRNA transcribed from the marker gene. In one embodiment, the expression of the marker gene can be measured according to the amount or relative amount of the protein product encoded by the marker gene. In another embodiment, the expression of the marker gene can be measured according to the biological or chemical reaction level induced by the protein product encoded by the marker gene. In addition, in certain embodiments, the expression of the marker gene can be measured according to the expression of one or more genes related to the expression of the marker gene.

As described above, the level or amount of the gene transcript (e.g., mRNA) of the marker gene can be used as an indication of the expression level of the marker gene. A variety of different PCR or qPCR protocols are known in the art, including those illustrated herein. In some embodiments, a variety of PCR or qPCR methods are applied to or adapted to measure the mRNA levels of various marker genes. Quantitative PCR (qPCR) (also referred to as real-time PCR) is applied to and adapted to be used in some embodiments, because it not only provides quantitative measurement results, but also reduces time and pollution. As used herein, "quantitative PCR (or "qPCR")" refers to direct monitoring of the PCR amplification process as it is occurring, without the need for repeated sampling of the reaction products. In quantitative PCR, the reaction product can be monitored via a signal transduction mechanism (e.g., fluorescence) when it is produced and tracked when the signal rises above the background level, but before the reaction reaches a stable stage. The number of cycles required to reach a detectable or "threshold" fluorescence level varies directly with the concentration of the amplifiable target at the beginning of the PCR process, thereby allowing the signal intensity to be measured so as to measure the target nucleic acid content in the sample in real time. When qPCR is applied to determine mRNA expression levels, an additional step of reverse transcription of mRNA into DNA is performed prior to qPCR analysis. Examples of PCR methods can be found in the 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. Fluorescent in situ PCR methods are described in U.S. Patent No. 7,186,507, which is incorporated herein by reference in its entirety.

In a specific embodiment, qPCR can be performed as follows to determine or measure the mRNA level of a marker gene. In short, the average Ct (cycle threshold) value (or interchangeably referred to herein as Cq (quantitative cycle)) of replicate qPCR reactions of a 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 example formula: marker gene ΔCt=(average Ct of the marker gene-average Ct of housekeeping gene A). The relative level of the marker gene mRNA can then be determined using the relative marker gene ΔCt, for example, by using the mRNA expression formula=2 ^-ΔCt . 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)).

To quantify RNA transcripts of marker genes in a sample as an indication of marker gene expression, other commonly used methods known in the art can also be used, including northern blotting and in situ hybridization (Parker and Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNase protection assay (Hod, Biotechniques 13:852-854 (1992)); microarray method (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 molecular biology technique that is widely used to measure and locate specific RNA sequences in cells (e.g., circulating tumor cells (CTCs)) or tissue sections, such as messenger RNA (mRNA), long non-coding RNA (lncRNA), and microRNA (miRNA), while preserving the cell and tissue environment. ISH is a type of hybridization that uses a directly or indirectly labeled complementary DNA or RNA chain (e.g., a probe) to bind and locate a specific nucleic acid, such as DNA or RNA, in a sample (part of a tissue or cell (in situ) or a section). The probe type 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 an ISH type using fluorescent labels. The term "chromogenic in situ hybridization" or "CISH" refers to an ISH type using chromogenic labels. ISH, FISH and CISH methods are well known to those skilled in the art (see, e.g., 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)). RNA ISH thus provides spatial-temporal visualization and quantification of gene expression within cells and tissues. It has wide applications in research and diagnosis (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 uses fluorescent dyes and fluorescent microscopy for RNA labeling and detection, respectively. Fluorescent RNA ISH can provide multitasking of four to five target sequences.

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

In some embodiments, the expression of the marker gene can be determined according to the relative abundance of the RNA transcripts (including, for example, mRNA) of the marker gene in the total transcribed RNA pool. Such relative abundance of the RNA transcripts of the marker gene can be determined by the next generation sequencing referred to as RNA-seq. In one example of the RNA-seq program, RNA from different sources (blood, tissue, cells) is purified, optionally enriched (e.g., using oligonucleotide (dT) primers), converted into cDNA and fragmented. Millions or even billions of short sequence reads are generated using random fragmentation cDNA libraries. See Zhao et al., BMC genomics 16: 97 (2015); Zhao et al., Scientific Reports 8: 4781 (2018); Shanrong Zhao et al., RNA, published in advance on April 13, 2020, doi: 10.1261 / rna.074922.120, which are all incorporated herein by reference as a whole. The expression level of each mRNA transcript of the marker gene is determined according to the total number of fragments located after normalization, which is proportional to its abundance level. Several normalization schemes are known and used to facilitate the use of RNA transcript abundance as a parameter for determining gene expression, including the number of reads per kilobase per million (RPKM), the number of fragments per kilobase per million (FPKM), and/or the number of transcripts per kilobase per million (TPM). In short, RPKM can be calculated as follows: the total reads in the sample are counted and the number is divided by 1,000,000 (which is the "per million" scale factor); the read count is divided by the "per million" scale factor, which is normalized for sequencing depth to obtain the number of reads per million (RPM); and the RPM value is divided by the gene length (in kilobases) to obtain RPKM. In addition to replacing reads with fragments, FPKM is closely related to RPKM. RPKM is generated from single-end RNA-seq, where each read corresponds to a single fragment sequenced. FPKM is generated from paired-end RNA-seq, where two reads can correspond to a single fragment, or if one read in a pair is not located, one read can correspond to a single fragment. TPM is very similar to RPKM and FPKM and is calculated as follows: divide the read count by the length of each gene in kilobases to get the number of reads per kilobase (RPK); count all RPK values for a sample and divide this number by 1,000,000 to get the "per million" scaling factor; divide the RPK value by the "per million" scaling factor to get TPM. See Zhao et al., BMC genomics 16:97 (2015); Zhao et al., Scientific Reports 8:4781 (2018); Shanrong Zhao et al., RNA, published in advance on April 13, 2020, doi: 10.1261/rna.074922.120, all of which are incorporated herein by reference in their entirety.

In one embodiment, the expression of the marker gene is determined by RNA-seq (e.g., TPM, RPKM and/or FPKM). In some embodiments, the expression of the marker gene is determined by TPM. In some embodiments, the expression of the marker gene is determined by RPKM. In some embodiments, the expression of the marker gene is determined by FPKM.

As described earlier, the expression of marker genes in a sample from a subject can be determined. 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 tissue around cancer). In some embodiments, the sample is a tissue sample. In some embodiments, a tissue sample is a tissue portion separated or extracted from a mammal, particularly a human being. In some embodiments, a tissue sample is a cell colony separated or extracted from a mammal, particularly a human being. In some embodiments, a tissue sample is a sample obtained from a biopsy. In certain embodiments, the sample may be obtained from a variety of organs of a subject (including a human subject). In some embodiments, the sample is obtained from an organ of a subject suffering from cancer. In some embodiments, the sample is obtained from an organ suffering from cancer in a subject suffering from cancer. In other embodiments, a sample (e.g., a reference sample) is obtained from a normal organ of a patient or a second human subject.

In certain embodiments of the methods provided herein, the tissue comprises 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, tonsil, thymus, heart, stomach, small intestine, liver, skeletal muscle, peripheral nerve, mesothelium, or eye.

In other embodiments of the methods provided herein, the expression of various marker genes can be detected by a variety of immunoassays known in the art, including immunohistochemistry (IHC) assays, immunoblotting assays, FACS assays, and ELISA.

In a variety of IHC assays, the expression of various marker genes can be detected by antibodies against protein products encoded by marker genes. IHC staining of tissue sections has been shown to be a reliable method for assessing or detecting the presence of proteins in samples. IHC techniques utilize antibodies to detect and visualize in situ cell antigens, usually by color development or fluorescence methods. Primary antibodies or antisera, such as polyclonal antisera and monoclonal antibodies that specifically target protein products encoded by marker genes, can be used to detect the expression of marker genes in IHC assays. In some embodiments, tissue samples are contacted with primary antibodies for specific targets for a time sufficient for antibody-target binding to occur. As discussed in more detail earlier, the antibodies can be detected by directly labeling the antibodies themselves with, for example, radioactive labels, fluorescent labels, hapten labels (e.g., biotin) or enzymes (e.g., horseradish peroxidase or alkaline phosphatase). Alternatively, unlabeled primary antibodies are used in combination with labeled secondary antibodies (including antisera, polyclonal antisera or monoclonal antibodies) that are specific to the primary antibodies. IHC protocols and kits are well known in the art and commercially available. Automated systems for slide preparation and IHC processing are commercially available. The Leica BOND automated stainer and the Leica Bond Refine detection system are examples of such automated systems.

In some embodiments, an IHC assay is performed using an unlabeled primary antibody in combination with a labeled secondary antibody in an indirect assay. An indirect assay uses two antibodies to detect protein products encoded by marker genes in a tissue sample. First, an uncoupled primary antibody is applied to the tissue (first layer) to react with the target antigen in the tissue sample. Next, an enzyme-labeled secondary antibody is applied that specifically recognizes the antibody isotype of the primary antibody (second layer). The secondary antibody reacts with the primary antibody, followed by application of a substrate-chromogen. The second layer of antibodies can be labeled with an enzyme (e.g., peroxidase) that reacts with the chromogen 3,3'-diaminobenzidine (DAB) to produce a brown precipitate at the reaction site. This method is sensitive and versatile because the potential signal is amplified via a signal amplification system.

In certain embodiments, in order to increase the detection sensitivity, a signal amplification system can be used. As used herein, a "signal amplification system" means a system of reagents and methods that can be used to enhance the signal from the primary or secondary antibody bound to the detection. The signal amplification system enhances the detection sensitivity of the target protein, enhances the detected signal, and reduces the lower boundary of the detection limit. There are several types of signal amplification systems, including enzyme labeling systems and macro labeling systems. These systems/methods are not mutually exclusive and can be used in combination to obtain an additive effect.

Macrolabels/macrolabeling systems are collections of labels that are connected to or incorporated into a common scaffold, ranging in number from tens (e.g., phycobiliproteins) to millions (e.g., fluorescent microspheres). The scaffold can be coupled to a target-specific affinity reagent (e.g., an antibody), and the incorporated labels are thus associated with the target together after binding. The label in the macrolabel can be any label described herein, such as a fluorophore, a hapten, an enzyme, and/or a radioisotope. In one embodiment of the signal amplification system, a secondary antibody coupled to a labeled chain polymer is used. Polymer technology utilizes inert "spike" glucan molecules labeled with HRP enzymes that can be linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50 or more secondary antibody molecules, making the system even more sensitive.

The signal amplification system based on the enzyme labeling system utilizes the catalytic activity of enzyme (for example horseradish peroxidase (HRP) or alkaline phosphatase) to produce the high density labeling of target protein or nucleic acid sequence in situ. In one embodiment, tyramide can be used to enhance HRP signal. In this system, HRP enzymatically converts the labeled tyramide derivative into a tyramide group with high reactivity and short life. The labeled active tyramide group is then covalently coupled with the residue (mainly the phenol part of protein tyrosine residue) near the HRP antibody-target interaction site, causing the number of labels at the site to amplify and the signal localization loss associated with diffusion to be minimum. Therefore, the signal can be amplified by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 75 or 100 times. As known to those skilled in the art, the label on tyramide can be any label described herein, including fluorophore, enzyme, hapten, radioisotope and/or luminophore. Other reactions based on enzyme can also be used to generate signal amplification. For example, enzyme-labeled fluorescence (ELF) signal amplification can be used for alkaline phosphatase, wherein alkaline phosphatase enzymatically cleaves a weak blue fluorescent substrate (ELF 97 phosphate) and converts it into a bright yellow-green fluorescent precipitate, which exhibits an unusually large Stokes shift and excellent light stability. Signal amplification systems based on tyramide and ELF signal amplification can be purchased from, for example, ThermoFisher Scientific (Waltham, MA USA 02451).

Thus, in some embodiments of the methods provided herein, the expression level of the marker gene is detected using IHC using a signal amplification system. In some embodiments, the sample is then subjected to counterstaining to identify cells and subcellular elements.

In some embodiments, the expression level of the protein product encoded by the marker gene can also be detected by using an antibody to the protein product encoded by the marker gene and an immunoblot assay. In some embodiments of the immunoblot assay, the protein is usually (but not necessarily) separated by electrophoresis and transferred to a membrane (usually a nitrocellulose or PVDF membrane). Similar to IHC assays, primary antibodies or antisera, such as polyclonal antisera and monoclonal antibodies specifically targeting protein products encoded by marker genes, can be used to detect the expression of marker genes. In some embodiments, the membrane is contacted with a primary antibody for a specific target for a time sufficient for antibody-antigen binding to occur, and the bound antibody can be detected by directly labeling the primary antibody itself with, for example, a radioactive label, a fluorescent label, a hapten label (e.g., biotin) or an enzyme (e.g., horseradish peroxidase or alkaline phosphatase). In other embodiments, in an indirect assay as described above, an unlabeled primary antibody is used in combination with a labeled secondary antibody specific for the primary antibody. As described herein, the secondary antibody can be labeled with, for example, an enzyme or other detectable label (e.g., a fluorescent label, a luminescent label, a colorimetric label, or a radioisotope). Immunoblotting protocols and kits are well known and commercially available in the art. Automated systems for immunoblotting, such as the iBind Western system (ThermoFisher, Waltham, MA USA 02451) for western blotting are commercially available. Immunoblotting includes but is not limited to western blotting, intracellular western blotting and dot blotting. Dot blotting is a simplified procedure in which protein samples are not separated by electrophoresis, but are directly spotted on the membrane. Intracellular western blotting includes inoculating cells in microtiter plates, fixing/permeating cells, and subsequently detecting with primary labeled primary antibodies or unlabeled primary antibodies, followed by detection with labeled secondary antibodies, as described herein.

In other embodiments, the expression level of the protein product encoded by the marker gene can also be detected using antibodies described herein in flow cytometry assays (including fluorescence activated cell sorting (FACS) assays).Similar to IHC or immunoblotting assays, primary antibodies or antisera (e.g., polyclonal antisera and monoclonal antibodies specifically targeting protein products encoded by marker genes) can be used in FACS assays to detect protein expression.In some embodiments, cells are stained with primary antibodies for a specific target protein for a time sufficient for antibody-antigen binding to occur and the bound antibodies can be detected by direct labeling (e.g., fluorescent labeling or hapten labeling, such as biotin on primary antibodies) on the primary antibodies.In other embodiments, in an indirect assay as described above, unlabeled primary antibodies are used in combination with fluorescently labeled secondary antibodies specific to the primary antibodies.FACS provides a method (one cell at a time) for sorting or analyzing a mixture of fluorescently labeled biological cells based on the specific light scattering and fluorescence characteristics of each cell.Therefore, flow cytometers detect and report the intensity of fluorescently labeled antibodies, which indicates the expression level of the target protein. Therefore, the expression level of the protein product encoded by the marker gene can be detected using antibodies against such protein products. Non-fluorescent cytoplasmic proteins can also be observed by staining permeabilized cells. Methods for performing FACS staining and analysis are well known to those skilled in the art and are described in Teresa S. Hawley and Robert G. Hawley in Flow Cytometry Protocols, Humana Press, 2011 (ISBN 1617379506, 9781617379505).

In other embodiments, the expression level of the protein product encoded by the marker gene can also be detected using an immunoassay such as an enzyme immunoassay (EIA) or ELISA. EIA and ELISA assays are known in the art, for example, for assaying a variety of tissues and samples, including blood, plasma, serum or bone marrow. There are a variety of ELISA assay formats available, see, for example, U.S. Patents Nos. 4,016,043, 4,424,279 and 4,018,653, which are incorporated herein by reference in their entirety. These include non-competitive single-point and two-point or "sandwich" assays, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to the target protein. Sandwich assays are commonly used assay formats. There are a variety of variations in sandwich assay technology. For example, in a typical forward assay, an unlabeled antibody is fixed to a solid substrate, and the sample to be tested is contacted with the bound molecule. After incubation for a suitable period of time, i.e., a period sufficient to allow the formation of an antibody-antigen complex, a second antibody specific for the antigen labeled with a reporter molecule capable of producing a detectable signal is then added and incubated to give sufficient time to form another complex of antibody-antigen-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observing the signal produced by the reporter molecule. The result can be qualitatively determined by simply observing the visible signal, or can be quantitatively determined by comparison with a control sample containing a known amount of the target protein.

In some embodiments of EIA or ELISA assays, an enzyme is coupled to a second antibody. In other embodiments, a fluorescently labeled secondary antibody can be used in an ELISA assay format to replace an enzyme-labeled secondary antibody to produce a detectable signal. When activated by irradiation with light of a specific wavelength, the fluorescent dye-labeled antibody absorbs light energy, induces an excitable state of the molecule, and then emits light of a characteristic color that can be visually detected with an optical microscope. Like EIA and ELISA, a fluorescently labeled antibody is allowed to bind to the first antibody-target protein complex. After washing off the unbound reagent, the residual ternary complex is then exposed to light of an appropriate wavelength, and the observed fluorescence indicates the presence of the target protein of interest. Immunofluorescence and EIA techniques have been used for a long time in the art and are disclosed herein.

For the immunoassay described herein, any of a variety of enzymes or non-enzyme labels can be used, as long as the enzyme activity or non-enzyme label can be detected respectively. The enzyme thus produces a detectable signal, which can be used to detect the target protein. Particularly suitable detectable signals are colorimetric or fluorescent signals. Therefore, particularly suitable enzymes used as labels include enzymes that can utilize colorimetric or fluorescent substrates. Such colorimetric or fluorescent substrates can be converted into easily detectable colorimetric or fluorescent products by enzyme reactions, and the products can be easily detected and/or quantified using microscopy or spectroscopy. Such enzymes are well known to those skilled in the art, including but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, etc. (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Other enzymes using well-known colorimetric or fluorescent substrates include various peptidases, in which colorimetric or fluorescent peptide substrates can be used to detect protein decomposition cleavage reactions. The use of chromogenic and fluorogenic substrates in bacterial diagnostics is also well known, including but not limited to the use of α-galactosidases and β-galactosidases, β-glucuronidases, 6-phospho-β-D-galactoside 6-phosphogalactohydrolases, β-glucosidases, α-glucosidases, amylases, neuraminidase, esterases, lipases, etc. (Manafi et al., Microbiol. Rev. 55:335-348 (1991)), and such enzymes for which chromogenic or fluorogenic substrates are known can be readily adapted for use in the disclosed methods.

Various chromogenic or fluorogenic substrates that produce detectable signals are well known to those skilled in the art and are commercially available. Exemplary substrates that can be used to produce detectable signals include, but are not limited to, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), chloronaphthol (Chloronaphthol) (4-CN) (4-chloro-1-naphthol), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine dihydrochloride (OPD), and 3-amino-9-ethylcarbazole (AEC) for horseradish peroxidase; alkaline phosphoric acid 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP), nitro blue tetrazolium (NBT), fast red (fast red TR/AS-MX) and p-nitrophenyl phosphate (PNPP) for enzymes; 1-methyl-3-indolyl-β-D-galactopyranoside and 2-methoxy-4-(2-nitrovinyl)phenyl β-D-galactopyranoside for β-galactosidase; 2-methoxy-4-(2-nitrovinyl)phenyl β-D-glucopyranoside for β-glucosidase; and the like. Exemplary fluorogenic substrates include, but are not limited to, 4-(trifluoromethyl)umbelliferyl phosphate for alkaline phosphatase; 4-methylumbelliferyl phosphate bis(2-amino-2-methyl-1,3-propanediol), 4-methylumbelliferyl phosphate bis(cyclohexylammonium), and 4-methylumbelliferyl phosphate for phosphatase; QuantaBlu ^™ and QuantaRed ^™ for horseradish peroxidase; 4-methylumbelliferyl β-D-galactopyranoside, fluorescein di(β-D-galactopyranoside), and naphthofluorescein di-(β-D-galactopyranoside) for β-galactosidase; 3-acetylumbelliferyl β-D-glucopyranoside and 4-methylumbelliferyl-β-D-glucopyranoside for β-glucosidase; and 4-methylumbelliferyl-α-D-galactopyranoside for α-galactosidase. Exemplary enzymes and substrates for producing detectable signals are also described in, for example, U.S. Publication 2012/0100540. Various detectable enzyme substrates (including chromogenic or fluorescent substrates) are well known and commercially available (Pierce, Rockford IL; Santa Cruz Biotechnology, Dallas TX; Invitrogen, Carlsbad CA; 42 Life Science; Biocare). In general, the substrate is converted to a product to form a precipitate that is deposited at the target nucleic acid site. Other exemplary substrates include, but are not limited to, HRP-green (42 Life Science), Betazoid DAB, Cardassian DAB, Romulin AEC, Bajoran purple, Vina green, Deep Space Black ^TM , Warp Red ^TM , Biocare's Vulcan Fast Red and Ferangi Blue (Concord CA; biocare.net/products/detection/chromogens).

In some embodiments of immunoassays, a detectable label may be directly coupled to a primary antibody that may be present or a secondary antibody that detects an unlabeled primary antibody. Exemplary detectable labels are well known to those skilled in the art and include, but are not limited to, chromogenic or fluorescent labels (see Hermanson, Bioconjugate Techniques, Academic Press, San Diego (1996)). Exemplary fluorophores suitable for use as labels include, but are not limited to, rhodamine derivatives, such as tetramethylrhodamine, rhodamine B, rhodamine 6G, sulforhodamine B, Texas Red (sulforhodamine 101), rhodamine 110 and derivatives thereof, such as tetramethylrhodamine-5-(or 6), lissaminerhodamine B, etc.; 7-nitrobenz-2-oxa-1,3-diazole (NBD); fluorescein and derivatives thereof; naphthalene, such as dansyl (5-dimethylaminonaphthalene-1-sulfonyl); coumarin derivatives, such as 7-amino-4-methylcoumarin-3-acetic acid (AMCA), 7-diethylamino-3-[(4'-(iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA), Alexa fluor dyes (Molecular Probes), etc.; 4,4-difluoro-4-bora-3a,4a-diaza-s-dicyclopentadienyl acene (BODIPY ^™ ) and its derivatives (Molecular Probes; Eugene Oreg.); pyrene and sulfonated pyrene, such as Cascade Blue ^™ and its derivatives, including 8-methoxypyrene-1,3,6-trisulfonic acid, etc.; pyridyl oxazole derivatives and dapoxyl derivatives (Molecular Probes); fluorescein (3,6-disulfonate-4-amino-naphthalimide) and its derivatives; CyDye ^™ fluorescent dyes (Amersham/GE Healthcare Life Sciences; Piscataway NJ), etc. Exemplary chromophores include, but are not limited to, phenolphthalein, malachite green, nitroaromatic hydrocarbons (e.g., nitrophenyl), diazo dyes, dabsyl (4-dimethylaminoazobenzene-4'-sulfonyl), etc.

The chromogenic or fluorescent detectable signal associated with the bound primary or secondary antibody can be visualized using methods well known to those skilled in the art, such as microscopy or spectroscopy.

The method provided in this section (section 5.8) can be used in conjunction with various cancer models known in the art. In one embodiment, a mouse xenograft cancer model is used. In brief, T-24 and UM-UC-3 cells are purchased from ATCC and cultured using recommended culture medium conditions. Using the pRCDCMEP-CMV-hNectin-4EF1-Puro construct, T-24hNectin-4 (human connexin-4) and UM-UC-3 connexin-4 cells are produced by transducing parental cells with a lentivirus containing human connexin-4, and puromycin is used to select. T-24 connexin-4 (clone 1A9) cells are implanted into nude mice and passaged via trocars, allowing to reach approximately 200mm ³ tumor volume, and subsequently treated with a single dose of ennomonab vedotin (3mg/kg) or non-binding ADC (3mg/kg) intraperitoneally (IP), 7 animals per treatment group. The follow-up ICD study using this model involved collecting tumors on the 5th day after treatment for downstream analysis by RNA-seq, flow, immunohistochemistry (IHC) and Luminex. The tumors were fixed in formalin and prepared into FFPE tissue blocks. 4 μm blocks were cut and immunohistochemistry was performed using F4/80 and CD11c. Immunohistochemically stained slide sections were scanned with a Leica AT2 digital complete slide scanner, and images were analyzed using an algorithm customized for connexin 4, CD11c and F4/80 staining, using Visiopharm software. Based on staining intensity and background staining, the algorithm was optimized. The percentage of positive staining for connexin 4 was calculated, and the number of positive cells per mm ² for F480 and CD11c was calculated.

Tumor sections were lysed in cell lysis buffer 2 (R&D Cytokines and chemokines in tumor samples were measured using the MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (Millipore) and read on the LUMINEX MAGPIX System.

For RNA-seq analysis, RNA was isolated from snap-frozen tumors using the TRIZOL Plus RNA purification kit (Life Technologies) according to the manufacturer's protocol to generate high-quality RNA (average RNA integrity number >8). RNA selection was performed using Poly(A) selection and Illumina's mRNA library Prep kit and read on a single index Hi-Seq 2×150 bp (Illumina). Sequence reads were mapped relative to human and mouse transcriptomes and the total number of reads per million was determined.

The disclosure is generally provided using affirmative language to describe numerous embodiments. The disclosure also particularly includes embodiments in which specific subject matter is excluded in whole or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analyses. Thus, although the disclosure is not generally described herein in terms of what is not included in the disclosure, aspects that are not explicitly included in the disclosure are disclosed herein.

Specific embodiments of the present disclosure are described herein, including the best mode known to the inventors for implementing the present disclosure. After reading the foregoing description, variations of the disclosed embodiments may become apparent to individuals working in the art, and it is contemplated that such variations may be adopted by those skilled in the art when appropriate. Therefore, it is intended that the present disclosure be practiced differently from as specifically described herein, and the present disclosure includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. In addition, unless otherwise indicated herein or otherwise clearly contradictory to the context, the present disclosure encompasses any combination of the elements described above in all possible variations thereof.

All publications, patent applications, accession numbers, and other references cited in this specification are hereby incorporated by reference in their entirety into the specification to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Several embodiments of the present disclosure have been described. However, it will be appreciated that various modifications can be made without departing from the spirit and scope of the present disclosure.

6. Examples

The following is a description of various methods and materials used in the studies, and is presented to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and is not intended to limit the scope of what the inventors regard as their invention, nor is it intended to represent that the following experiments have been performed and are all that can be performed. It should be understood that the exemplary descriptions written in the present tense are not necessarily performed, but rather that the descriptions may be performed to generate data, etc., relevant to the teachings of the present invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered.

6.1 Example 1 - An Open-Label, Randomized Phase 3 Study (EV-301) to Evaluate Enrotumab Vedotin Versus Chemotherapy in Subjects with Previously Treated Locally Advanced or Metastatic Urothelial Carcinoma.

6.1.1 Drugs used in clinical studies

In one embodiment, the ADC provided herein is ennomonab vedotin, also known as PADCEV. In a specific embodiment tested in this example (6.1), ennomonab vedotin-ejfv includes an anti-191P4D12 antibody, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising amino acid residues 20 to amino acid residues 466 of SEQ ID NO: 7 and a light chain comprising amino acid residues 23 to amino acid residues 236 of SEQ ID NO: 8.

Ennomonab Vedotin-ejfv is an antibody-drug conjugate (ADC) against connexin-4, which comprises a fully human anti-connexin-4 IgG1κ monoclonal antibody (AGS-22C3) coupled to a small molecule microtubule disruptor monomethyl auristatin E (MMAE) via a protease-cleavable maleimidocaproyl valine-citrulline (vc) linker (SGD-1006). The coupling occurs on a cysteine residue containing an interchain disulfide bond of the antibody, resulting in a product with a drug: antibody ratio of approximately 3.8:1. The molecular weight is approximately 152 kDa.

Ennosumab vedotin-ejfv has the following structural formula:

Approximately 4 molecules of MMAE are attached to each antibody molecule. Ennomonab Vedotin-ejfv is produced by chemically coupling the antibody and small molecule components. The antibody is produced by mammalian (Chinese hamster ovary) cells, and the small molecule components are produced by chemical synthesis.

PADCEV for injection (ennosumab vedotin-ejfv) is provided as a sterile, preservative-free white to off-white lyophilized powder in a single-dose vial for intravenous use. PADCEV is supplied in the form of 20 mg/vial and 30 mg/vial, and requires reconstitution with USP sterile water for injection (2.3 mL and 3.3 mL, respectively), resulting in a transparent to slightly opalescent, colorless to slightly yellow solution with a final concentration of 10 mg/mL (see Dosage and Administration (6.1.6.1 (i))). After reconstitution, each vial allows the extraction of 2 mL (20 mg) and 3 mL (30 mg). Each mL of the reconstituted solution contains 10 mg of ennosumab vedotin-ejfv, histidine (1.4 mg), monohydrated histidine hydrochloride (2.31 mg), polysorbate 20 (0.2 mg) and dihydrated trehalose (55 mg), with a pH of 6.0.

6.1.2 Research Overview

6.1.2.1 Outline

(i) Name of study drug

Ennosumab vedotin (ASG-22CE)

(ii) Development Phase

Phase 3

(iii) Research title

An Open-Label, Randomized, Phase 3 Study to Evaluate Enrotumab Vedotin Versus Chemotherapy in Subjects with Previously Treated Locally Advanced or Metastatic Urothelial Carcinoma (EV-301)

(iv) Planned research period

2Q2018 to 2Q2021. Planned study participation is approximately 24 months from the first subject enrollment, with an additional 12 months expected for overall survival (OS) tracking after the last subject enrollment. The total study duration will be approximately 36 months.

(v) Research objectives

(a) Main objectives

To compare the OS of subjects with locally advanced or metastatic urothelial carcinoma treated with EVs with that of patients treated with chemotherapy.

(b) Secondary Objectives

To compare the progression-free survival (PFS1) of subjects treated with EVs to those treated with chemotherapy according to Response Evaluation Criteria in Solid Tumors (RECIST) V1.1

To compare the overall response rate (ORR) of EV versus chemotherapy according to RECIST V1.1

To evaluate the duration of response (DOR) of EV and chemotherapy according to RECIST V1.1

To compare the disease control rate (DCR) of EV versus chemotherapy according to RECIST V1.1

To assess the safety and tolerability of EVs

To assess quality of life (QOL) and patient-reported outcome (PRO) parameters

(c) Research objectives

Exploratory genomic and/or other biomarkers in tumor tissue and peripheral blood that may correlate with treatment outcome, including connexin-4 expression

To evaluate the pharmacokinetics of EVs (total antibody (Tab), antibody-drug conjugate (ADC), and monomethyl auristatin E (MMAE))

To assess the incidence of anti-therapeutic antibodies (ATA)

To evaluate the PFS of EV compared with chemotherapy as the next line of treatment (PFS2)

Healthcare Resource Utilization (HRU)

(vi) Total number and locations of planned research centers

Approximately 185 research centers in North America, Europe, Asia Pacific and Latin America

(vii) Research Group

Subjects with locally advanced or metastatic urothelial carcinoma previously treated with platinum-based chemotherapy and an immune checkpoint inhibitor (CPI)

(viii) Number of subjects to be enrolled/randomized

Approximately 600 subjects

(ix) Overview of research design

This is a global open-label, randomized Phase 3 study in adult subjects with locally advanced or metastatic urothelial carcinoma who have received platinum-containing chemotherapy and have experienced disease progression or recurrence during or after treatment with immune checkpoint inhibitors. Approximately 600 subjects will be randomly assigned to EV group (Group A) or chemotherapy (Group B) at a 1:1 ratio. Subjects will be stratified according to the following: Eastern Cooperative Oncology Group Performance Status (ECOG PS), world region, and liver metastasis.

OS is the primary endpoint. OS is defined as the time from randomization to the date of death. Secondary endpoints include PFS1, ORR, DOR, DCR, safety, and QOL/PRO.

Subjects in Group A will receive EV on Days 1, 8, and 15 of each 28-day cycle. Subjects in Group B will receive docetaxel, paclitaxel, or vinflunine on Day 1 of each 21-day cycle (as determined by the investigator prior to randomization: vinflunine will be an option as a comparator only in countries where it is approved for urothelial carcinoma). Within the control group, the upper limit of the overall proportion of subjects receiving vinflunine will be approximately 35%. Subjects will continue to receive study treatment until they meet radiographic disease progression or other interruption criteria as determined by the investigator's assessment, or at the time of study termination or completion, whichever occurs first. Crossover in the study will not be allowed. This study will consist of 3 phases: screening, treatment, and follow-up.

Screening will be performed up to 28 days prior to randomization. Subjects will start in Cycle 1 and continue to subsequent 21-day or 28-day cycles until one of the discontinuation criteria is met. Treatment cycles are defined as 28 days for Group A and 21 days for Group B. Subjects randomized to Group A (EV) will be treated and evaluated on Days 1, 8, and 15 of all treatment cycles. Subjects randomized to Group B (docetaxel, paclitaxel, or vinflunine) will be treated and evaluated on Day 1 of all treatment cycles.

Subjects will be assessed for response according to RECIST V1.1. Imaging will be performed in both groups at baseline and every 56 days (±7 days) throughout the study from the first dose of study treatment until PFS1 is documented by radiographic disease progression or the subject is lost to follow-up, dies, withdraws study consent, or initiates subsequent anticancer therapy.

Baseline imaging performed as standard of care prior to informed consent may be used as long as it is performed within 28 days prior to randomization. All subjects will undergo a bone scan (scintigraphy) at screening/baseline. Subjects with a positive bone scan at baseline will undergo a bone scan every 56 days (± 7 days) or more frequently (if clinically indicated) throughout the study. If clinically indicated and not related to baseline status, the subject should undergo a follow-up bone scan. Brain scans (computed tomography with contrast/magnetic resonance imaging (MRI)) will only be performed at screening/baseline if clinically indicated and repeated as clinically indicated or according to standard of care during the entire study.

QOL assessments and PROs will be collected from all randomized subjects at protocol-specified time points. The following validated tools will be used: European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ-C30) and EuroQOL 5 Dimensions (EQ-5D-5L). Healthcare resource utilization (HRU) information will be collected at protocol-specified time points, with a particular focus on the number of subjects with no planned use of healthcare resources related to clinical or AEs from subjects assigned to treatment groups A and B.

Blood samples for pharmacokinetics and ATA will be collected throughout the study for subjects randomized to Group A. Confirmatory assays will be used to measure the concentrations of EV ADC and MMAE in serum or plasma and assess ATA. Pharmacokinetic samples will not be collected from subjects randomized to Group B. Samples for exploring biomarkers will be collected at protocol-specified time points. Biomarker assessments will not be used for subject selection.

After discontinuation of study drug, subjects will be followed up for 30 days (+7 days) after their last dose for safety assessment. If a subject discontinues study drug before radiographic disease progression (i.e., PFS1), the subject should enter the post-treatment follow-up period and continue to undergo imaging assessments every 56 days (±7 days) until PFS1 is documented or the subject starts another anticancer treatment, whichever occurs earlier.

After PFS1, subjects will enter the long-term follow-up period and be followed according to institutional guidelines, but no less than every 3 months from the date of the follow-up visit for survival status and progression status on subsequent therapy (ie, PFS2).

Subjects will be followed until PFS2 is documented, or the subject starts another anticancer therapy, whichever occurs first. All subsequent anticancer therapies, including the date of PFS2 and site of progression, will be recorded on the case report form.

After PFS2, subjects will enter the survival follow-up period and will be followed every 3 months until death, loss of follow-up, withdrawal of study consent, or termination of the study by the sponsor. This study is expected to end once the final survival analysis is completed.

An Independent Data Monitoring Committee (IDMC) will be chartered to oversee safety and planned interim efficacy analyses, which will occur after at least 285 OS events (about 65% of the total planned events) are observed. The primary analysis will be conducted at 439 OS events. The IDMC can recommend to the sponsor whether the trial should be terminated, modified, or continued unchanged based on the ongoing review of safety data and interim efficacy analysis. Other details will be outlined in the IDMC concession.

(x) Inclusion/Exclusion Criteria

Inclusion: Subjects were eligible for the study if all of the following applied:

1. Written informed consent and privacy language approved by the Institutional Review Board (IRB)/Independent Ethics Committee (IEC) in accordance with national regulations (e.g., Health Insurance Portability and Accountability Act (HIPAA) authorization for research sites in the United States) must be obtained from the subjects before any research-related procedures (including withdrawal of prohibited medications, if applicable).

2. Subjects were legal adults according to local regulations when signing the informed consent form.

3. The subject has histologically or cytologically confirmed urothelial carcinoma (ie, bladder, renal pelvis, ureter, or urethra). Subjects with urothelial carcinoma (transitional cell) with squamous differentiation or mixed cell types are eligible.

4. Subjects must have experienced radiographic progression or recurrence of locally advanced or metastatic disease during or after a CPI (anti-programmed cell death-1 (PD-1) or anti-programmed cell death-ligand 1 (PD-L1)). Subjects who discontinued CPI therapy due to toxicity were eligible if they had evidence of disease progression after the discontinuation. The CPI does not have to be the most recent therapy. Subjects are eligible if their most recent therapy has been a non-CPI-based regimen and they progressed/relapsed during or after their most recent therapy. Locally advanced disease must not be resectable with curative intent by the treating physician.

5. Subjects must have received a platinum-containing regimen (cisplatin or carboplatin) in the metastatic/locally advanced, neoadjuvant or adjuvant setting. If platinum was administered in the adjuvant/neoadjuvant setting, subjects must have progressed within 12 months of completion.

6. Subjects have radiographically documented metastatic or locally advanced disease at baseline.

7. Archived tumor tissue samples should be available for presentation to the central laboratory prior to study treatment. If archived tumor tissue samples are not available, fresh tissue samples should be provided. If fresh tissue samples cannot be provided due to safety concerns, study participants must discuss this with the medical monitor.

8. The subject's ECOG PS is 0 or 1

9. Subjects have the following baseline laboratory data:

Absolute neutrophil count (ANC) ≥1500/mm ³

Platelet count ≥100 × ¹⁰⁹ /L

Hemoglobin ≥9 g/dL

Serum total bilirubin ≤1.5× upper limit of normal (ULN)* or ≤3× ULN for subjects with Gilbert's disease

Creatinine clearance (CrCl) ≥30 mL/min as estimated by institutional criteria or as measured by a 24-hour urine collection (glomerular filtration rate (GFR) may also be used in lieu of CrCl)

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 2.5 × ULN, or

For subjects with liver metastases, ≤3×ULN*

*If total bilirubin > ULN, or if AST and/or ALT > 1.5× ULN with alkaline phosphatase > 2.5× ULN, docetaxel should not be selected as a comparator.

10. Female subjects must meet any of the following requirements:

No reproductive potential:

Postmenopausal (defined as the absence of any menstrual periods for at least one year without other obvious pathological or physiological etiology) prior to screening, or

●Documented surgical infertility (eg, hysterectomy, bilateral salpingectomy, bilateral oophorectomy).

NOTE: Those patients who are postmenopausal and attribute their menopause to alternative medical causes are not considered and must follow the criteria for subjects of childbearing potential.

●Or, if of childbearing potential:

Agree not to attempt to become pregnant during the study and for at least 6 months after the last dose of study drug,

● and had a negative urine or serum pregnancy test within 7 days prior to Day 1 (women with verification of a false-positive result and a documented negative pregnancy status were eligible to participate),

● and if heterosexual, agree to use condoms plus 1 form of highly effective birth control* according to locally accepted standards starting at Screening and continuously during the entire study period and for at least 6 months after the last study drug administration.

11. Female subjects must agree not to breastfeed or donate eggs starting at screening and throughout the study period and for at least 6 months after the last study drug administration.

12. Sexually active male subjects with female partners of reproductive potential are eligible if:

Agree to use male condoms starting at Screening and continuing throughout study treatment and for at least 6 months after the last study drug administration. If the male subject has not had a vasectomy or is not infertile as defined below, his female partner is to utilize 1 form of highly effective birth control* according to locally accepted standards starting at Screening and continuing throughout study treatment and for at least 6 months after the male subject receives his final study drug administration.

*Highly effective forms of birth control include:

■consistently and correctly use established hormonal contraceptives that suppress ovulation,

■ Established intrauterine device (IUD) or intrauterine hormone releasing system (IUS).

Bilateral fallopian tube occlusion

■ Vasectomy (Vasectomy is a highly effective method of contraception, provided that the absence of sperm has been confirmed. Otherwise, an additional highly effective method of contraception should be used)

■ Male infertility due to bilateral orchiectomy or radical cystoprostatectomy/removal of the seminal vesicles

■ Abstinence is considered highly effective only when it is defined as abstention from heterosexual intercourse throughout the risk period associated with study treatment. The reliability of abstinence needs to be assessed with respect to the duration of the study and the preferred and common lifestyle of the participants.

Note: Abstinence is not a sufficient method of contraception in Switzerland.

13. Starting at screening and throughout the study period, and for at least 6 months after the final study drug administration, male subjects are not allowed to donate sperm.

14. Male subjects with pregnant or breastfeeding partners must agree to abstain from sex or use condoms during pregnancy or during the entire study period and for at least 6 months after the final study drug administration.

15. The subjects agree not to participate in another intervention study while receiving the treatment in this study.

No waivers of the inclusion criteria were permitted.

Exclusions: Subjects will be excluded from participation if any of the following apply:

1. Subject has pre-existing sensory or motor neuropathy of ≥ Grade 2.

2. Subjects with active central nervous system (CNS) metastases. Subjects with treated CNS metastases are allowed into the study if all of the following are true:

●CNS metastases have been clinically stable for at least 6 weeks prior to screening

If steroids are required for CNS metastases, subjects are on a stable dose of ≤20 mg/day of prednisone or equivalent for at least 2 weeks

Baseline scans show no evidence of new or enlarging brain metastases

●The subject does not have leptomeningeal disease

3. Subjects are experiencing clinically significant toxicity (Grade 2 or higher, excluding alopecia) related to prior treatment (including systemic therapy, radiation therapy, or surgery). Subjects with ≤ Grade 2 immunotherapy-related hypothyroidism or panhypopituitarism may be enrolled if they are adequately maintained/controlled on stable doses of hormone replacement therapy (if indicated). Patients experiencing ≥ Grade 3 immunotherapy-related hypothyroidism or panhypopituitarism are excluded. Subjects experiencing immunotherapy-related colitis, uveitis, myocarditis, or pneumonitis or subjects with other immunotherapy-related AEs requiring high doses of steroids (>20 mg/day of prednisone or equivalent) are excluded.

4. Subjects previously treated with EV or other MMAE-based ADCs.

5. In the control group, subjects had received prior chemotherapy for urothelial carcinoma and all available study therapies (i.e., both prior paclitaxel and docetaxel in regions where vinflunine is not an approved therapy; or prior paclitaxel, docetaxel, and vinflunine in regions where vinflunine is an approved therapy).

NOTE: Subjects who have received both docetaxel and paclitaxel will be excluded after the vinflunine ceiling is reached.

6. Subject has received more than 1 prior chemotherapy regimen for locally advanced or metastatic urothelial carcinoma, including chemotherapy for adjuvant or neoadjuvant disease if relapse occurred within 12 months of completing therapy. Substitution of carboplatin for cisplatin does not constitute a new regimen, provided no new chemotherapeutic agent is added to the regimen.

7. Subjects with a history of another malignant disease within 3 years prior to the first dose of study drug, or any signs of residual disease from a previously diagnosed malignant disease. Subjects with non-melanoma skin cancer, localized prostate cancer that has undergone curative therapy and has no signs of progression, low-risk or very low-risk (according to standard guidelines) localized prostate cancer with no intention of treatment under active surveillance/watchful waiting, or any type of carcinoma in situ (if complete resection was performed) are permitted.

8. Subjects are currently receiving systemic antimicrobial therapy for viral, bacterial, or fungal infection at the time of the first dose of EV. Conventional antimicrobial prophylaxis is permitted.

9. The subject has known active hepatitis B (eg, HBsAg reactivity) or active hepatitis C (eg, HCV RNA detected (qualitative)).

10. Subjects are known to have a history of human immunodeficiency virus (HIV) infection (HIV 1 or 2).

11. Subjects have a documented history of cerebrovascular events (stroke or transient ischemic attack), unstable angina, myocardial infarction, or cardiac symptoms consistent with New York Heart Association Class III-IV (including congestive heart failure) within 6 months prior to the first dose of study drug.

12. The subject has received radiation therapy or major surgery within 4 weeks before the first dose of study drug.

13. Subjects receiving chemotherapy, biologics, investigational agents, and/or anti-tumor treatment with immunotherapy and not completing 2 weeks prior to the first dose of study drug.

14. The subject is known to be allergic to EV or to any excipients contained in EV drug formulations (including histidine, trehalose dihydrate, and polysorbate 20); or the subject is known to be allergic to biological agents prepared in Chinese hamster ovary (CHO) cells.

15. The subject is known to be allergic to the following:

Docetaxel or any other excipient listed in the product label, including polysorbate 80;

●paclitaxel or any other excipient listed in the product label, including polyethylene glycol glycerol ricinoleate 35 (Ph. Eur.); and

Vinflunine or any other excipient listed in the product label, including other vinca alkaloids (vinblastine, vincristine, vindesine, vinorelbine).

16. Subjects are known to have active keratitis or corneal ulcers. Subjects with superficial punctate keratitis are permitted if, in the opinion of the investigator, their condition is adequately treated.

17. The subject suffers from other underlying medical conditions which, in the opinion of the investigator, will impair the subject's ability to receive or tolerate the planned treatment and follow-up.

18. History of uncontrolled diabetes within 3 months of the first dose of study drug. Uncontrolled diabetes was defined as hemoglobin A1c (HbA1c) ≥ 8% or HbA1c between 7% and < 8% with associated unexplained diabetic symptoms (polyuria or polydipsia).

No waivers of the exclusion criteria are permitted.

(xi) Pharmaceutical product: Enrotumab vedotin: Dosage, mode of administration and dosage adjustment

EV 1.25 mg/kg will be administered on Days 1, 8, and 15 of each 28-day cycle. The drug will be administered intravenously over a 30-minute period.

EV will be administered on Day 1 of each cycle based on actual subject weight, except for subjects weighing more than 100 kg, in which case the dose will be calculated based on a maximum body weight of 100 kg. Group A does not need to recalculate the dose based on actual body weight on Days 8 and 15 of each cycle unless required by institutional standards.

Depending on the type and severity of toxicity, dose reductions to 1 mg/kg (dose level -1) and 0.75 mg/kg (dose level -2) will be allowed. Subjects who need to reduce the dose can be increased by 1 dose level (i.e., subjects who are reduced to 0.75 mg/kg can only be increased to 1 mg/kg), provided that the toxicity does not require interruption of the study drug and has returned to baseline or ≤ Grade 1. If toxicity recurs, no further increase will be allowed. Subjects with ≥ Grade 2 corneal AEs are not allowed to increase the dose. EV should not be administered to subjects with CrCl <30 mL/min. Dose adjustment recommendations for EV-related toxicity are presented in Tables 6 and 7.

Researchers at the study site may allow interruptions in dosing due to other EV-related toxicities at their discretion. Interruptions in dosing may last up to 8 weeks (2 cycles). If the subject's toxicity does not otherwise require permanent interruption of dosing, interruptions in dosing for subjects who have received clinical benefit from treatment may be extended beyond 8 weeks. If dosing interruptions occur, the time course of response assessments will not be adjusted.

Table 6 Recommended dose adjustments for enroku vedotin-related hematologic toxicities*

*Note: Hematologic toxicity refers to anemia, thrombocytopenia, neutropenia, and febrile neutropenia.

Table 7 Recommended dose adjustments for non-hematologic toxicities associated with enroku vedotin

AE: adverse event; EV: enrofloxacin

*Grade 3/4 electrolyte imbalances/laboratory abnormalities that are not associated with clinical sequelae and/or corrected with supplementation/appropriate management within 72 hours of their onset do not require interruption of treatment (e.g., Grade 4 hyperuricemia). Grade 3 rash that does not limit self-care activities of daily living or is associated with infection and requires systemic antibiotics does not require interruption of treatment, provided that symptoms are not severe and can be managed with supportive care.

(xii) Comparative drugs

In general, for drug-related grade 4 hematologic toxicity and non-hematologic toxicity ≥ grade 3, treatment with the chemotherapy comparator (docetaxel, paclitaxel, or vinflunine) should be discontinued, and subsequent doses should be adjusted according to Table 15. Dose adjustment guidelines specifically recommended for subjects receiving docetaxel, paclitaxel, or vinflunine are detailed below. Dose adjustment should also be considered based on the local product label or product characteristics (SmPC) and the overview of institutional guidelines. For docetaxel, paclitaxel, or vinflunine-related grade ≥ 3 hematologic toxicity, transfusions or growth factors may be used as indicated by institutional guidelines.

(a) Docetaxel: Dosage, mode of administration and dose adjustment

Docetaxel will be administered intravenously on Day 1 of each 21-day cycle. A starting dose of 75 mg/m ² of docetaxel will be administered over a 60-minute period or as required by local requirements. For additional guidance on docetaxel dosing, refer to local product labeling or institutional guidelines for SmPC and docetaxel.

Docetaxel should not be administered to subjects with total bilirubin > ULN, or to subjects with AST and/or ALT > 1.5×ULN and concomitant alkaline phosphatase > 2.5×ULN. Subjects with elevated bilirubin or abnormal transaminases and alkaline phosphatase are at increased risk for developing Grade 4 neutropenia, febrile neutropenia, infection, severe thrombocytopenia, severe stomatitis, severe skin toxicity, and toxic death. Docetaxel should also not be administered to subjects with neutrophil counts < 1500 cells/ ^mm3 . Severe fluid retention has been reported following docetaxel therapy.

Subjects should be pre-administered corticosteroids according to institutional guidelines before each docetaxel administration. Subjects with pre-existing effusions should be closely monitored for possible worsening of effusions from the first dose. Subjects who develop peripheral edema can be treated with standard measures, such as salt restriction and oral diuretics. Dosing interruption can last up to 6 weeks (2 cycles). If the subject's toxicity does not otherwise require permanent dosing interruption, the interruption of dosing for subjects who obtain clinical benefit from treatment can be extended for more than 6 weeks.

Dose adjustments not specified in Table 8 (e.g., severe or cumulative skin reactions) should also be considered based on local product labeling or the SmPC and institutional guidance.

Table 8 Suggested dose adjustments for subjects receiving docetaxel

ANC: absolute neutrophil count; N/A: not applicable; T: temperature

(b) Vinflunine: Dosage, mode of administration and dosage adjustment

Vinflunine will be administered intravenously on Day 1 of each 21-day cycle. Unless otherwise specified below, a starting dose of 320 mg/m ² of vinflunine will be administered over a 20-minute period (or as per local requirements). In cases of WHO/ECOG PS ≥ 1 or ECOGP 0 and prior pelvic irradiation, vinflunine treatment should be initiated at a dose of 280 mg/m ^2. In the absence of any hematologic toxicity causing treatment delay or dose reduction during the first cycle, the dose for subsequent cycles may be increased to 320 mg/m ² every 21 days.

In subjects with moderate renal impairment (40 mL/min ≤ CrCl ≤ 60 mL/min), the recommended dose is 280 mg/m ² given once every 21-day cycle. In subjects with renal impairment (30 mL/min ≤ CrCl < 40 mL/min), the recommended dose is 250 mg/m ² given once every 21-day cycle. In subjects with mild hepatic impairment (Child-Pugh Class A), the recommended dose of vinflunine is 250 mg/m ² given once every 21-day cycle.

The recommended doses for subjects ≥75 years of age are as follows:

- In subjects who are at least 75 years of age but less than 80 years of age, the dose of vinflunine to be administered is 280 mg/ ^m2 per 21 day cycle.

• In subjects 80 years of age and older, the dose of vinflunine to be administered is 250 mg/ ^m2 per 21 day cycle.

For additional guidance on the administration of vinflunine, refer to local product labeling or institutional guidelines for SmPC and vinflunine.

The interruption of dosing can last up to 6 weeks (2 cycles). If the toxicity of the subject does not otherwise require permanent interruption of dosing, the interruption of dosing for subjects who have obtained clinical benefit from treatment can be extended for more than 6 weeks. For specific dose adjustments for subjects receiving vinflunine, refer to the qualified product label.

(c) Paclitaxel: Dosage, Mode of Administration, and Dose Adjustment

Study treatment with paclitaxel should be administered intravenously on Day 1 of each 21-day cycle after all procedures/assessments have been completed. A starting dose of 175 mg/m ² of paclitaxel will be administered as an intravenous infusion over 3 hours or according to local requirements. See guidance on initial dose titration. For additional guidance on the administration of paclitaxel, refer to local product labeling or institutional guidelines for SmPC and paclitaxel.

All subjects should be premedicated according to institutional guidelines prior to paclitaxel administration to prevent severe allergic reactions. Such premedication may consist of: dexamethasone 20 mg orally approximately 12 and 6 hours before paclitaxel; diphenhydramine (or its equivalent) 50 mg IV 30 to 60 minutes before paclitaxel; and cimetidine (300 mg) or ranitidine (50 mg) IV 30 to 60 minutes before paclitaxel. Appropriate premedication regimens may be determined by the investigator.

Paclitaxel should not be administered to subjects with a baseline neutrophil count of less than 1500 cells/mm ^3. Subjects should not be retreated with paclitaxel in subsequent cycles until neutrophils recover to levels >1500 cells/mm ³ and platelets recover to levels >100,000/mm ^3. Severe conduction abnormalities have been documented in <1% of subjects during paclitaxel therapy and, in some cases, required placement of an atrial pulse generator. If a subject develops significant conduction abnormalities during paclitaxel infusion, appropriate therapy should be administered and continuous cardiac monitoring should be performed during subsequent paclitaxel therapy.

In cases of mild hepatic impairment (total bilirubin ≥1.25 ULN), paclitaxel should be initiated at a dose of 135 mg/ ^m2 .

Specific recommended dose adjustment guidelines for subjects receiving paclitaxel are detailed below in Table 9. Dose adjustments should also be considered based on local product labeling or SmPC and institutional guidelines.

The interruption of dosing may last up to 6 weeks (2 cycles). The interruption of dosing may be extended beyond 6 weeks for subjects who gain clinical benefit from treatment if their toxicity does not otherwise require permanent interruption of dosing.

Table 9 Suggested Dose Adjustments for Subjects Receiving Paclitaxel

N/A: Not applicable; T: Temperature

(xiii) Interruption criteria

Treatment interruption applies to subjects enrolled in the study and to subjects who permanently discontinue study treatment for any reason.

Subjects are free to withdraw from study treatment and/or study for any reason and at any time, without giving reasons for doing so and without penalty or prejudice. If the subject's clinical condition permits, the researchers are also free to interrupt the subject's study treatment or terminate the subject's participation in the study at any time. If a subject interrupts the study with persistent adverse events (AEs) or unresolved laboratory results that are significantly outside the reference range, the researchers will attempt to follow up until the condition is stable or no longer clinically significant.

The following are the treatment discontinuation criteria for individual subjects:

●The subject develops radiographic disease progression.

●The subject needs to receive another systemic anticancer treatment for the underlying or new cancer.

●Unacceptable toxicity to subjects.

●Female subjects are pregnant.

The investigator decides that interruption of treatment is in the best interest of the subject.

●The subject refuses further treatment.

- Subject is not complying with the protocol based on the assessment of the Investigator or Medical Monitor.

● Despite reasonable efforts by the investigators to locate the subject, the subject was lost to follow-up.

●Death.

Subjects who discontinue treatment prior to radiographic disease progression will enter a post-treatment follow-up period and continue to have imaging assessments every 56 days (±7 days) until PFS1 is documented by radiographic disease progression or the subject initiates another anticancer treatment, whichever occurs first.

After PFS1, subjects will enter the long-term follow-up period and will be followed according to institutional guidelines, but with survival status and progression to next-line therapy no less than every 3 months from the date of the follow-up visit until PFS2 is documented or the subject starts another anticancer treatment, whichever occurs first.

The subject will then enter the survival follow-up period. The subject's survival status will be tracked every 3 months until any of the OS discontinuation criteria are met. The subject will discontinue the OS post-treatment follow-up period if any of the following occurs:

●The subject refuses further study participation (ie, withdraws consent).

● Despite reasonable efforts by the investigators to locate the subject, the subject was lost to follow-up.

●Death.

●The study was terminated.

(xiv) Concomitant Medication Restrictions or Requirements:

Subjects must withdraw from further study treatment administration if the investigator determines that any of the following medications are required to provide adequate medical support to the subject:

Other research drugs

● Chemotherapy or other drugs intended to provide anti-tumor activity. This does not apply to subjects with a history of breast cancer in the setting of adjuvant endocrine therapy, or to subjects using agents intended for the treatment of bone metastases (e.g., bisphosphonates or RANK ligand inhibitors).

Radiation therapy

● NOTE: Radiation therapy to symptomatic solitary lesions or to bone may be considered on a case-by-case basis in special circumstances after consultation with the sponsor. The irradiated lesions must be non-target lesions and the subject must have clearly measurable disease outside the irradiated area according to RECIST V1.1.

Group A (ennolumab vedotin)

●Subjects receiving potent cytochrome P450 (CYP) 3A4 inhibitors or P-pg inhibitors concomitantly with EV should be closely monitored for adverse reactions.

Group B (docetaxel)

●Concomitant use of drugs that strongly inhibit or induce CYP3A4 may affect docetaxel exposure and should be avoided.

Group B (Vinflunine)

●The use of strong inhibitors or inducers of the CYP3A4 enzyme should be avoided in subjects receiving vinflunine.

●Medical products that prolong the QT/QTc interval should be avoided.

Group B (paclitaxel)

●Caution should be used when paclitaxel is administered with strong inhibitors or inducers of CYP3A4 and CYP2C8.

Refer to local drug inserts for concomitant medication restrictions or requirements for docetaxel, paclitaxel, and vinflunine.

(xv) Duration of treatment

Subjects will be allowed to receive EV or comparator until they meet study discontinuation criteria or upon study termination or study completion, whichever occurs first.

(xvi) Assessment endpoint

main

OS

secondary

●According to RECIST V1.1, PFS1

ORR (complete response (CR) + PR) according to RECIST V1.1

●DCR (CR+PR+stable disease (SD)) according to RECIST V1.1

●According to RECIST V1.1, DOR

Safety variables (eg, AEs, laboratory tests, vital sign measurements, 12-lead electrocardiogram, and ECOG PS)

QOL and PRO parameters (QLQ-C30 and EQ-5D-5L)

Exploratory

●Exploratory genomic and/or other biomarkers in tumor tissue and peripheral blood that may be associated with treatment outcome, including connexin-4 expression

Selected plasma or serum concentrations of TAb, ADC, and MMAE

●Incidence of ATA for EV

● According to RECIST V1.1, PFS2

HRU

(xvii) Statistical methods:

Approximately 600 subjects will be randomized in a 1:1 ratio to 2 treatment groups: Group A (EV) and Group B (docetaxel, paclitaxel, or vinflunine). Randomization will be stratified as follows:

Liver metastasis (yes/no)

ECOG PS (0 vs. 1)

World Region (United States, Western Europe, and Rest of the World)

(a) Sample size adjustment

Approximately 600 subjects (with a 10% dropout rate and 1 interim analysis) will be randomized in a 1:1 ratio to receive EV or chemotherapy.

Primary endpoint: OS

●2.5% I-type error on one side; 85% power

OS assumption: hazard ratio (HR) = 0.75 (median OS 10.7 m vs 8 m for EV vs chemotherapy)

A formal interim efficacy analysis will be performed when approximately 65% of deaths have occurred.

●Primary analysis will be performed at approximately 439 OS events

For the planned interim efficacy analysis, a group sequential design using O'Brien-Fleming boundaries implemented by the Lan-DeMets method will be employed to control for an overall one-sided 0.025 type I error. If the interim analysis confirms a statistically significant result for EV on efficacy, the study can be stopped for efficacy and concluded.

The full analysis set (FAS) will be used to perform efficacy analysis on OS and PFS1. All subjects who were randomized will be included in the FAS. For the time to the event endpoint, including the OS and PFS1 log-rank test stratified by randomization stratification factors (including liver metastases), the baseline ECOG PS and the world region at baseline will be used to compare the two treatment groups. The hazard ratio and corresponding 95% confidence interval from the stratified Cox proportional hazard regression model will also be presented. Median OS, PFS1 and DOR will be estimated using the Kaplan-Meier method and will be reported together with the corresponding 95% confidence interval by treatment group.

ORR and DCR will be compared between treatment groups using the Cochran-Mantel-Haenszel test and stratified by the same stratification factors used in the time-to-event analysis. The difference in response rate between treatment groups will be estimated with corresponding 95% confidence intervals.

(b) Security

The Safety Analysis Set (SAF) will be used for safety analysis. All subjects who underwent randomization and received study drug will be included in the SAF. The frequency of AEs and serious AEs will be summarized by MedDRA system organ class and preferred terms. In addition, summary statistics for the following safety parameters will be provided:

●Laboratory values

●Vital sign measurement

●ECOG PS

(c) Pharmacokinetics

Descriptive statistics (e.g., number, mean, standard deviation, minimum, median, maximum, coefficient of variation, and geometric mean) will be provided for plasma or serum concentrations of TAb, ADC, and MMAE. The incidence of ATA for EV will be summarized by cycle and overall and for possible relationships to the explored pharmacokinetics. Additional model-based analyses and exposure responses may be performed and reported separately.

6.1.3 Study objectives, design and endpoints

6.1.3.1 Research objectives

(i) Main objectives

To compare the OS of subjects with locally advanced or metastatic urothelial carcinoma treated with EVs with that of patients treated with chemotherapy.

(ii) Secondary Objectives

To compare the progression-free survival (PFS1) of subjects treated with EVs to those treated with chemotherapy according to Response Evaluation Criteria in Solid Tumors (RECIST) V1.1

To compare the overall response rate (ORR) of EV versus chemotherapy according to RECIST V1.1

To evaluate the duration of response (DOR) of EV and chemotherapy according to RECIST V1.1

To compare the disease control rate (DCR) of EV versus chemotherapy according to RECIST V1.1

To assess the safety and tolerability of EVs

To assess quality of life (QOL) and patient-reported outcome (PRO) parameters

(iii) Research objectives

Exploratory genomic and/or other biomarkers in tumor tissue and peripheral blood that may correlate with treatment outcome, including connexin-4 expression

To evaluate the pharmacokinetics of EVs (TAb, ADC, and MMAE)

To assess the incidence of ATA

To assess PFS as assessed by RECIST V1.1 (PFS2) in subjects treated with EV compared with docetaxel, paclitaxel, or vinflunine by investigator review

Healthcare Resource Utilization (HRU)

6.1.3.2 Study Design and Dosing Rationale

(i) Study design

This is a global, open-label, randomized, Phase 3 study in adult subjects with locally advanced or metastatic urothelial carcinoma who have received platinum-containing chemotherapy and have experienced disease progression or recurrence during or after treatment with an immune checkpoint inhibitor. Subjects who interrupted CPI treatment due to toxicity were eligible if they had evidence of disease progression after the interruption.

Approximately 600 subjects will be randomized in a 1:1 ratio to EV (Arm A) or chemotherapy (Arm B). Subjects will be stratified according to: Eastern Cooperative Oncology Group performance status (ECOG PS), world region, and liver metastases.

OS was the primary endpoint. OS was defined as the time from randomization to the date of death. Secondary endpoints included PFS1, ORR, DOR, DCR, safety, and QOL/PRO.

Subjects in Group A will receive EV on Day 1, Day 8, and Day 15 of each 28-day cycle. Subjects in Group B will receive docetaxel, paclitaxel, or vinflunine on Day 1 of each 21-day cycle, as determined by the investigators before randomization (vinflunine is only an option for comparators in countries where it has been approved for use in urothelial cancer). Within the control group, the upper limit of the overall proportion of subjects receiving vinflunine will be approximately 35%. Subjects will continue to receive study treatment until they meet radiological disease progression or other interruption criteria as determined by the investigator's assessment, or at the time of termination or completion of the study, whichever occurs first. Crossover in the study will not be allowed. During the study treatment, subjects assigned to the chemotherapy group will not be allowed to switch to different chemotherapy treatments. This study will consist of three phases: screening, treatment, and follow-up.

Screening will occur up to 28 days prior to randomization. Screening assessments may be repeated during the 28-day screening period.

A subject does not have to IRT "Fail Screening" and re-enter Screening with a new subject ID, as long as the subject is enrolled within the 28-day window from signing the informed consent. If more than 28 days have passed from the date the informed consent was signed, the subject must IRT Fail Screening. A new consent form must be signed and the subject re-enters Screening with a new subject ID. A subject may only be re-screened once.

Subjects will begin in Cycle 1 and continue in subsequent 21-day or 28-day cycles until one of the discontinuation criteria is met or upon study termination or study completion, whichever occurs first. Treatment cycles are defined as 28 days for Group A and 21 days for Group B.

Subjects' responses will be assessed according to RECIST V1.1. Imaging of the two groups will be performed every 56 days (± 7 days) from the first dose of study treatment during baseline and throughout the study, until PFS1 is recorded by radiological disease progression or the subject loses tracking, dies, withdraws the study consent, or starts subsequent anticancer therapy. Baseline imaging performed as standard care before informed consent can be used, as long as it is performed within 28 days before randomization. All subjects will undergo bone scans (scintigraphy) at screening/baseline. Subjects with positive bone scans at baseline will undergo bone scans every 56 days (± 7 days) or more frequently (if clinically indicated) during the entire study. If clinically indicated to be unrelated to baseline status, the subject should undergo tracking bone scans. Brain scans (computed tomography (CT) with contrast/magnetic resonance imaging (MRI)) will only be performed clinically at screening/baseline, and will be repeated during the entire study by clinically indicated or according to standard care.

QOL assessments and PROs will be collected from all randomized subjects at protocol-specified time points. The following validated tools will be used: European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ-C30) and EuroQOL 5-Dimension 5-Level Questionnaire (EQ-5D-5L). HRU information will be collected at protocol-specified time points, with a particular focus on the number of subjects with no planned use of medical resources related to clinical or AEs from subjects assigned to treatment groups A and B.

Blood samples for pharmacokinetics and ATA will be collected throughout the study for subjects randomized to Group A. Concentrations of EV ADC and monomethyl aritampin E (MMAE) in serum or plasma will be measured and ATA will be assessed using validated assays. Pharmacokinetic samples will not be collected from subjects randomized to Group B. Samples for exploration of biomarkers will be collected at protocol-specified time points.

After discontinuation of study drug, subjects will be followed up for 30 days (+7 days) after their last dose for safety assessment. If a subject discontinues study drug before radiographic disease progression (i.e., PFS1), the subject should enter the post-treatment follow-up period and continue to undergo imaging assessments every 56 days (±7 days) until PFS1 is documented or the subject starts another anticancer treatment, whichever occurs earlier.

After PFS1, subjects will enter the long-term follow-up period and be followed according to institutional guidelines, but no less than every 3 months from the date of the follow-up visit for survival status and progression status on subsequent therapy (ie, PFS2).

Subjects will be followed until PFS2 is documented, or the subject starts another anticancer therapy, whichever occurs first. All subsequent anticancer therapies, including the date of PFS2 and site of progression, will be recorded on the case report form.

After PFS2, subjects will enter the survival follow-up period and will be followed every 3 months for survival status until death, loss of follow-up, withdrawal of study consent, or termination of the study by the sponsor. This study is expected to end once the final survival analysis is completed. Subjects will be eligible to continue treatment in this study until they meet the discontinuation criteria as outlined in Section 6.1.7 Discontinuation, or upon study termination or study completion, whichever occurs first.

The IDMC will be licensed to supervise safety and planned interim efficacy analyses, which will occur after at least 285 OS events (about 65% of the total planned events) are observed. The primary analysis will be conducted at 439 OS events. The IDMC can recommend to the sponsor whether the trial should be terminated, modified or continued unchanged based on the continuous review of safety data and interim efficacy analysis. Other details will be outlined in the IDMC concession.

(ii) Dosage rationale

(a) Ennosumab vedotin

EV will be administered at a dose of 1.25 mg/kg as an approximately 30-minute intravenous infusion on days 1, 8, and 15 of each 28-day cycle. This dose and regimen has demonstrated an acceptable safety profile and encouraging clinical activity in a Phase 1 study that evaluated escalating dose levels of 0.5, 0.75, 1, and 1.25 mg/kg, with the expansion group at 0.75, 1, and 1.25 mg/kg dose levels. The maximum tolerated dose (MTD) was not reached in this study. At the 1 mg/kg dose level, 2 dose-limiting toxicities (DLTs) were observed: Grade 3 anal pain (later changed to Grade 2 by the investigator) believed to be related to irradiation recall, and Grade 4 hyperuricemia without clinical sequelae. No DLTs were observed at 1.25 mg/kg, and doses higher than 1.25 mg/kg were not tested.

The incidence of some of the most frequent drug-related AEs (eg, diarrhea and rash), although primarily grade 1 to 2 and clinically manageable, increased with increasing dose level.

Additionally, dose reductions due to AEs were more frequent for 1.25 mg/kg versus lower dose levels. Safety assessments in patients with metastatic urothelial carcinoma and patients with non-metastatic urothelial carcinoma showed that the frequencies of all TEAEs, AEs leading to withdrawal, and Grade 3 to 4 TEAEs were comparable across all dose levels.

Although all EV doses demonstrated activity, the 1.25 mg/kg dose was associated with the highest activity and had an acceptable safety profile.

Based on the pharmacokinetic data of the Phase 1 study, the half-life of EV is ~1 to 2 days. No significant (<30%) intra-cycle ADC accumulation was observed with the current dosing regimen (on days 1, 8, and 15 of each 28-day cycle) at any dose level. Minimal (<50%) intra-cycle MMAE accumulation was observed. It is expected that the dosing schedule in the study will maintain ADC exposure within each 28-day cycle, thereby facilitating a good balance of activity and safety, as observed in the Phase 1 trial.

In summary, the proposed dosing regimen of 1.25 mg/kg on days 1, 8, and 15 of each 28-day cycle in this study has demonstrated an acceptable safety profile and encouraging clinical activity above that observed at lower dose levels.

(b) Comparator

There is no accepted standard of care following CPI treatment for locally advanced or mUC; however, prior to CPI approval, taxanes were commonly used following platinum-based therapy, as recommended in treatment guidelines. Additionally, vinflunine is approved in Europe for the treatment of mUC after failure of a prior platinum-based regimen.

KEYNOTE-045, a multicenter, randomized, active-controlled trial comparing pembrolizumab with investigator's choice of paclitaxel, docetaxel, or vinflunine every 3 weeks in patients with locally advanced or mUC with disease progression on or after platinum-containing chemotherapy. Bellmunt J et al., N Engl J Med. 2017;376:1015-26. DOI:10.1056/NEJMoa1613683.

Although no labeled taxane dosing guidelines are available in this setting, this is the only large randomized trial for which combined survival data for these agents have been reported, and therefore the current Phase 3 enroku vs. chemotherapy study will use the same comparator selection and its corresponding dose. Patients randomized to the chemotherapy arm of the KEYNOTE-045 study were treated with paclitaxel (175 mg/m ² ), docetaxel (75 mg/m ² ), or vinflunine (320 mg/m ² ) administered intravenously every 3 weeks. The upper limit of the overall proportion of subjects in the control group who received vinflunine was approximately 35% (KEYNOTE protocol).

6.1.3.3 End point

(i) Primary endpoint

OS

(ii) Secondary endpoints

By RECIST V1.1, PFS1

ORR (CR+PR) according to RECIST V1.1

Pass RECIST V1.1, DCR (CR+PR+SD)

Through RECIST V1.1, DOR

Safety variables (eg, AEs, laboratory tests, vital sign measurements, 12-lead ECG, and ECOG PS)

QOL and PRO parameters (QLQ-C30 and EQ-5D-5L)

(iii) Exploration endpoint

Exploratory genomic and/or other biomarkers in tumor tissue and peripheral blood that may correlate with treatment outcome, including connexin-4 expression

Plasma or serum concentrations of TAb, ADC, and MMAE

The incidence of ATA for EV

PFS2

HRU

6.1.4 Study population

6.1.4.1 Selection of study population

Patients with locally advanced or metastatic urothelial carcinoma previously treated with platinum-based chemotherapy and an immune checkpoint inhibitor (CPI).

6.1.4.2 Inclusion criteria

A subject is eligible for the study if all of the following apply:

Written informed consent and privacy language approved by the Institutional Review Board (IRB)/Independent Ethics Committee (IEC) in accordance with national regulations (e.g., Health Insurance Portability and Accountability Act authorization for research sites in the United States) must be obtained from subjects before any research-related procedures (including withdrawal of contraindicated medications, if applicable).

The subjects were legal adults according to local regulations when signing the informed consent.

Subjects have histologically or cytologically confirmed urothelial carcinoma (ie, bladder, renal pelvis, ureter, or urethra). Subjects with urothelial carcinoma (transitional cell) with squamous differentiation or mixed cell types are eligible.

Subjects must have experienced radiographic progression or recurrence of locally advanced or metastatic disease during or after a CPI (programmed cell death protein-1 (PD-1) or anti-programmed cell death-ligand 1 (PD-L1)). Subjects who discontinued CPI therapy due to toxicity were eligible provided they had evidence of disease progression after the discontinuation. The CPI did not have to be the most recent therapy. Subjects were eligible if their most recent therapy had been a non-CPI-based regimen and progressed/relapsed during or after their most recent therapy. Locally advanced disease must not be resectable with curative intent by the treating physician.

Subjects must have received a platinum-containing regimen (cisplatin or carboplatin) in the metastatic/locally advanced, neoadjuvant or adjuvant setting. If platinum was administered in the adjuvant/neoadjuvant setting, subjects must have progressed within 12 months of completion.

Subjects had radiographically documented metastatic or locally advanced disease at baseline.

Archived tumor tissue samples should be available for presentation to the central laboratory prior to study treatment. If archived tumor tissue samples are not available, fresh tissue samples should be provided. If fresh tissue samples cannot be provided due to safety concerns, participation in the study must be discussed with the Medical Monitor.

The subjects had an ECOG PS of 0 or 1.

The subjects had the following baseline laboratory data:

Absolute neutrophil count (ANC) ≥1500/mm ³

Platelet count ≥100 × ¹⁰⁹ /L

Hemoglobin ≥9 g/dL

Serum total bilirubin ≤1.5× upper limit of normal (ULN)* or ≤3× ULN for subjects with Gilbert's disease

Creatinine clearance (CrCl) ≥30 mL/min as estimated by institutional criteria or as measured by a 24-hour urine collection (glomerular filtration rate (GFR) may also be used in lieu of CrCl)

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 2.5 × ULN or ≤ 3 × ULN for subjects with liver metastases*

*If total bilirubin > ULN, or if AST and/or ALT > 1.5× ULN with alkaline phosphatase > 2.5× ULN, docetaxel should not be selected as a comparator.

Female subjects must meet any of the following criteria:

●No reproductive potential:

Postmenopausal (defined as the absence of any menstrual periods for at least one year without other obvious pathological or physiological etiology) prior to screening, or

● Documented infertility by surgery (eg, hysterectomy, bilateral salpingectomy, bilateral oophorectomy).

NOTE: Those patients who are postmenopausal and attribute their menopause to alternative medical causes are not considered and must follow the criteria for subjects of childbearing potential.

●Or, if of childbearing potential:

Agree not to attempt to become pregnant during the study and for at least 6 months after the last dose of study drug,

● and had a negative urine or serum pregnancy test within 7 days prior to Day 1 (women with verification of a false-positive result and documented negative pregnancy status were eligible to participate),

● and if heterosexual, agree to use condoms plus 1 form of highly effective birth control* according to locally accepted standards starting at Screening and throughout the study period and for at least 6 months after the last study drug administration.

Female subjects must agree not to breastfeed or donate eggs starting at screening and throughout the study period and for at least 6 months after the last study drug administration.

Sexually active male subjects with female partners of reproductive potential are eligible if:

Agree to use male condoms starting at Screening and continuing throughout study treatment and for at least 6 months after the last study drug administration. If the male subject has not had a vasectomy or is not infertile as defined below, his female partner is to utilize 1 form of highly effective birth control* according to locally accepted standards starting at Screening and continuing throughout study treatment and for at least 6 months after the male subject receives his final study drug administration.

*Highly effective forms of birth control include:

● Consistent and correct use of established hormonal contraceptives that suppress ovulation,

●An established intrauterine device (IUD) or intrauterine hormone-releasing system (IUS).

Bilateral fallopian tube occlusion

●Vasectomy (Vasectomy is a highly effective method of contraception, provided that the absence of sperm has been confirmed. Otherwise, an additional highly effective method of contraception should be used)

● Male infertility due to bilateral orchiectomy or radical cystoprostatectomy/removal of the seminal vesicles

Abstinence is considered highly effective only when it is defined as abstention from heterosexual intercourse throughout the risk period associated with study treatment. The reliability of abstinence needs to be assessed with respect to the duration of the study and the preferred and common lifestyle of the participants.

Warning: Abstinence is not a sufficient contraceptive method in Switzerland

Beginning at screening and throughout the study period, and for at least 6 months after the final study drug administration, male subjects were not allowed to donate sperm.

Male subjects with pregnant or breastfeeding partners must agree to abstain from sex or use condoms during pregnancy or during the entire study period during which the partner breastfeeds and for at least 6 months after the final study drug administration.

The subjects agreed not to participate in another intervention study while receiving the treatment in this study.

No waivers of the inclusion criteria were permitted.

6.1.4.3 Exclusion criteria

Subjects will be excluded from participation if any of the following apply:

Subjects had pre-existing sensory or motor neuropathy of ≥ Grade 2.

Subjects with active central nervous system (CNS) metastases. Subjects with treated CNS metastases are allowed into the study if all of the following are true:

●CNS metastases have been clinically stable for at least 6 weeks prior to screening

If steroids are required for CNS metastases, subjects are on a stable dose of ≤20 mg/day of prednisone or equivalent for at least 2 weeks

Baseline scans show no evidence of new or enlarging brain metastases

●The subject does not have leptomeningeal disease

Subjects are experiencing clinically significant toxicity (Grade 2 or higher, excluding alopecia) related to prior treatment, including systemic therapy, radiation therapy, or surgery. Subjects with ≤ Grade 2 immunotherapy-related hypothyroidism or panhypopituitarism are eligible if adequately maintained/controlled on stable doses of hormone replacement therapy (if indicated). Patients experiencing ≥ Grade 3 immunotherapy-related hypothyroidism or panhypopituitarism are excluded. Subjects experiencing immunotherapy-related colitis, uveitis, myocarditis, or pneumonitis or with other immunotherapy-related AEs requiring high doses of steroids (>20 mg/day of prednisone or equivalent) are excluded.

Subjects were previously treated with EV or other MMAE-based ADCs.

In the control group, subjects had received prior chemotherapy for urothelial carcinoma and all available study therapies (i.e., both prior paclitaxel and docetaxel in regions where vinflunine is not an approved therapy; or prior paclitaxel, docetaxel, and vinflunine in regions where vinflunine is an approved therapy). Note: Subjects who have received both docetaxel and paclitaxel will be excluded after the vinflunine cap is reached.

Subjects have received more than 1 prior chemotherapy regimen for locally advanced or metastatic urothelial carcinoma, including chemotherapy for adjuvant or neoadjuvant disease if relapse occurred within 12 months of completing therapy. Substitution of carboplatin for cisplatin does not constitute a new regimen, provided no new chemotherapeutic agent is added to the regimen.

Subjects had a history of another malignancy within 3 years prior to the first dose of study drug, or any signs of residual disease from a previously diagnosed malignancy. Subjects with non-melanoma skin cancer, localized prostate cancer that has undergone curative therapy and has no signs of progression, low-risk or very-low-risk (according to standard guidelines) localized prostate cancer that is under active surveillance/watchful waiting with no intent to treat, or any type of carcinoma in situ (if complete resection was performed) are permitted.

Subjects were currently receiving systemic antimicrobial therapy for viral, bacterial, or fungal infection at the time of the first dose of EV. Conventional antimicrobial prophylaxis was permitted.

The subject had known active hepatitis B (eg, HBsAg reactivity) or active hepatitis C (eg, HCV RNA detected (qualitative)).

Subjects had a known history of human immunodeficiency virus (HIV) infection (HIV 1 or 2).

Subjects had a documented history of cerebrovascular events (stroke or transient ischemic attack), unstable angina, myocardial infarction, or cardiac symptoms consistent with New York Heart Association Class III-IV (including congestive heart failure) within 6 months prior to the first dose of study drug.

Subjects had received radiation therapy or major surgery within 4 weeks prior to the first dose of study drug.

Subjects were receiving chemotherapy, biologics, investigational agents, and/or anti-neoplastic treatment with immunotherapy and had not completed 2 weeks prior to the first dose of study drug.

The subject is known to be allergic to EVs or to any excipients contained in EV drug formulations (including histidine, trehalose dihydrate, and polysorbate 20); or the subject is known to be allergic to biological drugs prepared in Chinese hamster ovary (CHO) cells.

The subject was known to be allergic to:

Docetaxel or any other excipient listed in the product label, including polysorbate 80;

●paclitaxel or any other excipient listed in the product label, including polyethylene glycol glycerol ricinoleate 35 (Ph. Eur.); and

Vinflunine or any other excipient listed in the product label, including other vinca alkaloids (vinblastine, vincristine, vindesine, vinorelbine).

Subjects are known to have active keratitis or corneal ulcers. Subjects with superficial punctate keratitis are permitted if, in the opinion of the investigator, their condition is adequately treated.

The subject has other underlying medical conditions that, in the opinion of the investigator, will impair the subject's ability to receive or tolerate the planned treatment and follow-up.

History of uncontrolled diabetes within 3 months of the first dose of study drug. Uncontrolled diabetes was defined as hemoglobin A1c (HbA1c) ≥ 8% or HbA1c between 7% and < 8% with associated symptoms of diabetes (polyuria or polydipsia) not otherwise explained.

No waivers of the exclusion criteria are permitted.

6.1.5 Treatment

6.1.5.1 Identification of drug products

(i) Study Drugs

The drug product EV (ASG-22CE) is a sterile, preservative-free, white to off-white lyophilized powder to be reconstituted for intravenous administration. The drug product is supplied by the sponsor in single-use glass vials containing 30 mg EV (ASG-22CE) in each vial. The drug product should be stored at 2-8°C. Details of drug product receipt, labeling, storage and preparation are provided in the supplemental pharmacy guide.

(ii) Comparison of drugs

The comparator drug will be supplied by the responsible site pharmacy at each clinical trial site. If the site is unable to procure or utilize a local supply of the comparator drug, the sponsor may provide the supply centrally, as appropriate. The sites are permitted to use generic docetaxel and paclitaxel approved by the respective regulatory agencies. Refer to the product labels for docetaxel, paclitaxel, and vinflunine for storage and handling conditions and warning statements.

For specific dosing instructions, the studies refer to Section 6.1.6.1 Dosing and Administration of Study Drugs and Other Medications.

6.1.5.2 Packaging and labeling

The EVs (ASG-22CE) used in this study will be prepared, packaged, and labeled by qualified staff in accordance with Standard Operating Procedures (SOP), Good Manufacturing Practice (GMP) guidelines, ICH GCP guidelines, and applicable local laws/regulations.

Each carton and vial will be labeled to identify the contents as a drug product in compliance with regulatory guidelines, GMPs, and local laws and regulations.

If supplied by the Sponsor, the comparator drug used in this study will be labeled in accordance with SOP GMP guidelines, ICH GCP guidelines and applicable local laws/regulations.

The qualified person will carry out the final release of the medicine in accordance with the requirements of Annex 13 of EU Directive 2003/94/EC.

6.1.5.3 Study Drug Disposal

Current ICH GCP guidance requires the investigator to ensure that investigational drug delivered from the sponsor is received by the investigator and/or designee, and:

● record such deliveries,

●Handle and store study drugs according to labeled storage conditions,

● Study medications have appropriate expiration/retesting and are administered to study subjects only according to the protocol, and

● Standard procedures for returning any unused study drug to the Sponsor, or tracking alternative disposition of unused study drug.

The person in charge of the research site or the storage manager of the research drug shall assume the following responsibilities for the research drug:

●The research drug storage manager should store the research drugs in accordance with the sponsor's written research drug handling procedures and assume its responsibilities.

The Study Drug Storage Manager should prepare and maintain records of study drug receipt, study site inventory, usage for each subject, and return of unused study drug to the Sponsor or alternative disposal. These records should include date, quantity, batch/serial number, expiration date (if applicable), and unique codes assigned to study drugs and subjects.

• The Study Drug Stock Manager should prepare and maintain records that appropriately document the delivery of protocol-specified doses to subjects and reconciliation of all study drug supplied by the Sponsor.

6.1.5.4 Blinding

This was an open-label study.

Although the study is an open label study, in order to maintain the integrity of the trial, the analysis or summary by randomized treatment assignment or actual treatment assignment will be limited and recorded while the study is ongoing and before the primary hard lock. Interim analysis will be performed externally by an independent data analysis center (IDAC). Details will be included in the SAP.

6.1.5.5 Designation and allocation

Subjects will be randomized to treatment groups in a 1:1 ratio via interactive response technology (IRT) according to the randomization schedule. All subjects who meet the eligibility criteria will be randomized. Field personnel will dispense treatments according to the assignment of the IRT system. Specific procedures for randomization via IRT are contained in the study procedure manual. Researchers must select a treatment from the group B selection to be used in the case of randomization of subjects to group B before randomization. Within the control group, the upper limit of the overall proportion of subjects receiving vinflunine will be approximately 35%.

6.1.6 Treatment and evaluation

6.1.6.1 Administration and administration of study drugs and other medications

(i) Dosage/dosing regimen and administration period

(a) Ennosumab vedotin

EV will be administered at a dose of 1.25 mg/kg as an approximately 30-minute intravenous infusion on Days 1, 8, and 15 of each 28-day cycle. In the absence of an IRR, the infusion rate for all subjects should be calculated to achieve an approximately 30-minute infusion period. EV must not be administered as an intravenous push or bolus. EV should not be mixed with other medications. At least 1 week must pass between each EV dose.

Weight-based dosing is calculated using the subject's actual body weight on Day 1 of each cycle. Unless required by institutional standards, Group A does not require recalculation of doses based on actual body weight on Days 8 and 15 of each cycle. Weight-based dosing is not applicable except for subjects weighing more than 100 kg; the dose for these subjects is based on 100 kg. The maximum dose allowed for this study is 125 mg.

Subject body weight must be measured during all relevant assessment time points as described in the event schedule.

Subjects should be observed during enroku administration and for at least 60 minutes after the infusion period for the first 3 cycles.All supportive measures consistent with optimal patient care should be administered throughout the study according to institutional standards.

Closely monitor the injection site for redness, swelling, pain, and infection during and at any time after administration. Subjects should be advised to report any redness or discomfort during or immediately after administration.

(b) Comparison of drugs

Chemotherapy agents will be administered following local product labeling or Summary of Product Characteristics (SmPC) and institutional guidelines, and precautions will be taken to prevent extravasation according to institutional standards and as described in: “Chemotherapy and Biotherapy Guidelines and Recommendations for Practice” (Polovich M et al., Chemotherapy and biotherapy guidelines and recommendations for practice. 4th ed. Pittsburgh: Oncology Nursing Society; 2014. 473p.) and “Management of Chemotherapy Extravasation: ESMO-EONS Clinical Practice Guidelines.” Fidalgo JA et al., Ann Oncol. 2012; 23(7): 167-73.

Male subjects randomized to docetaxel, vinflunine, or paclitaxel should seek medical advice regarding sperm cryopreservation due to possible infertility prior to receiving treatment.

Docetaxel

Docetaxel study treatment should be administered as an intravenous infusion on Day 1 of each 21-day cycle after all procedures/assessments are completed, including required premedication according to local standard of care prior to Day 1. Docetaxel will be administered at 75 mg/ ^m2 unless otherwise specified in Sections 6.1.6.1(i)(b) and 6.1.6.1(ii)(c). Refer to the centrally supplied local product labeling or SmPC and institutional guidelines for additional guidance on docetaxel dosing.

Docetaxel will be administered within 1 hour or according to local guidelines. Subjects should be observed during docetaxel administration and for at least 30 minutes after the infusion period for the first 3 cycles. All supportive measures consistent with optimal patient care should be given throughout the study according to institutional standards.

All subjects should be premedicated with corticosteroids, such as dexamethasone 16 mg/day orally (e.g., 8 mg twice daily) for 3 days according to local standard of care, starting 1 day before docetaxel administration to reduce the incidence and severity of fluid retention and the severity of allergic reactions. Appropriate premedication regimens may be determined by the investigator.

Vinflunine

Vinflunine study treatment should be administered as an intravenous infusion on Day 1 of each 21-day cycle after all procedures/assessments are completed. Vinflunine will be administered at 320 mg/ ^m2 unless otherwise specified in Section 6.1.6.1(ii)(d). For guidance on initial dose adjustments, see 6.1.6.1(ii)(d) Vinflunine Dose Adjustment. Subjects should be observed during vinflunine administration and for at least 30 minutes after the infusion period for the first 3 cycles. All supportive measures consistent with optimal patient care should be given throughout the study in accordance with institutional standards. For additional guidance on vinflunine administration, refer to local product labeling or SmPC and institutional guidelines for vinflunine.

Pacific Taxol

Pacific paclitaxel study treatment should be administered as an intravenous infusion on Day 1 of each 21-day cycle after all procedures/assessments are completed. Pacific paclitaxel will be administered at 175 mg/ ^m2 unless otherwise specified in Section 6.1.6.1(ii)(e). For initial dose adjustments, see Section 6.1.6.1(ii)(e) Pacific paclitaxel dose adjustments. Pacific paclitaxel should be administered within 3 hours or as per local guidelines. Subjects should be observed during paclitaxel administration and for at least 30 minutes after the infusion period for the first 3 cycles. All supportive measures consistent with optimal patient care should be given throughout the study according to institutional standards. For additional guidelines on the administration of paclitaxel, refer to local product labeling or institutional guidelines for SmPC and paclitaxel.

All subjects should be premedicated prior to paclitaxel administration to prevent severe allergic reactions. Such premedication may consist of: dexamethasone 20 mg orally approximately 12 and 6 hours before paclitaxel; diphenhydramine (or its equivalent) 50 mg intravenously 30 to 60 minutes before paclitaxel; and cimetidine (300 mg) or ranitidine (50 mg) intravenously 30 to 60 minutes before paclitaxel. Appropriate premedication regimens may be determined by the investigator.

(ii) Increasing or decreasing the dose of study drug

(a) Ennosumab vedotin

Dose reductions to 1 mg/kg (dose level -1) and 0.75 mg/kg (dose level -2) will be permitted, depending on the type and severity of toxicity. Subjects who require a dose reduction may be escalated by 1 additional dose level (e.g., subjects who are escalated to 0.75 mg/kg may only be escalated to 1 mg/kg), provided that the toxicity does not require interruption of study drug and has returned to baseline or ≤ Grade 1. If toxicity recurs, no further escalation will be permitted. Subjects who experience a corneal AE of ≥ Grade 2 will not be permitted to escalate the dose.

EV should not be administered to subjects with CrCl <30 mL/min. Dose adjustment recommendations for EV-related toxicity are presented in Tables 13 and 14. Researchers at the study site may allow interruptions in dosing due to other EV-related toxicities at their discretion. Interruptions in dosing may last up to 8 weeks (2 cycles). If the subject's toxicity does not otherwise require permanent interruption of dosing, interruptions in dosing for subjects who have obtained clinical benefit from treatment may be extended for more than 8 weeks. If there is a dosing interruption, the time course of the response assessment will not be adjusted.

Table 13 Recommended dose adjustments for enroku vedotin-related hematologic toxicities*

*Note: Hematologic toxicity refers to anemia, thrombocytopenia, neutropenia, and febrile neutropenia.

Table 14 Recommended dose adjustments for non-hematologic toxicities associated with enroku vedotin

AE: adverse event; EV: enrofloxacin

*Grade 3/4 electrolyte imbalances/laboratory abnormalities (except hyperglycemia) that are not associated with clinical sequelae and/or corrected with supplementation/appropriate management within 72 hours of their onset do not require interruption of treatment (e.g., Grade 4 hyperuricemia). Grade 3 rash that does not limit self-care activities of daily living or is associated with infection and requires systemic antibiotics does not require interruption of treatment, provided that symptoms are not severe and can be managed with supportive care.

** Eye examinations should be performed by an ophthalmologist. In countries where optometrists can perform the examination and prescribe, optometrists may be used for the actual examination.

Enrotumab vedotin-related rash

In Phase 1 studies, rash and dermatologic-like AEs were common in patients treated with EV and were more frequently seen at the highest dose. Although the exact etiology of EV-related cutaneous virulence is unclear at this time, rash may be a target toxicity due to the expression of connexin-4 in the skin. The most common types of skin AEs reported in ASG-22CE-13-2 were maculopapular rash, rash, skin exfoliation, and skin pigmentation disorders. Most occurred early (during cycle 1) and some were accompanied by itching. Mild rashes due to EV should be treated with local supportive care as needed. For itching, topical corticosteroids and antihistamines have been used as needed. Grade 3 rashes that do not limit self-care activities of daily living or are accompanied by infection and require systemic antibiotics do not require interruption of treatment, provided that the symptoms are not severe and can be managed with supportive care.

Management of hyperglycemia

Investigators should monitor blood glucose levels and if any symptoms of hyperglycemia are observed, additional assessments, including a thorough evaluation for infection, are recommended. In addition, if steroids are used to treat any other medical condition, blood glucose levels may require additional monitoring. If elevated blood glucose levels are observed, subjects should be treated according to local standards of care and referral to an endocrinologist may be considered.

It is recommended that a subject (especially one with a history of or currently suffering from diabetes or hyperglycemia) notify his or her physician promptly if his or her glucose levels become uncontrollable or if he or she experiences symptoms suggestive of hyperglycemia (e.g., frequent urination, increased thirst, blurred vision, fatigue, and headaches).

Subjects entering the study with elevated HbA1c (≥6.5%) at baseline should be referred to an appropriate provider for glucose management during Cycle 1. Blood glucose should be checked prior to each dose, and dosing should be withheld for blood glucose >250 mg/dL (13.9 mmol/L) (Grade 3 or higher). Dosing may continue once the subject's blood glucose has improved to ≤Grade 2 and the subject is clinically and metabolically stable. Blood glucose >500 mg/dL (27.8 mmol/L) (Grade 4) considered related to EV requires interruption of treatment. If the subject experiences new-onset diabetes, the subject will be evaluated with a metabolic panel, urine ketones, glycosylated hemoglobin, and C-peptide to assess new-onset type 1 diabetes in the setting of a previous CPI.

Management of infusion-related reactions (IRRs) associated with enrofloxacin

IRRs can occur during infusions of study treatment. Infusions should be performed in a setting that is appropriately equipped and staffed to manage anaphylaxis if it occurs. All supportive measures consistent with optimal patient care should be administered throughout the study, according to institutional standards. Supportive measures may include administration of medications for IRRs.

Subjects who have experienced IRR may be premedicated for subsequent infusions. Premedication may include analgesics (e.g., acetaminophen or equivalent), antihistamines (e.g., diphenhydramine hydrochloride), and corticosteroids administered approximately 30 to 60 minutes before each infusion or according to institutional standards. If a subject experiences an IRR in the context of premedication, continued treatment with Ennosumab Vedotin must be discussed with the medical monitor before the next planned dose.

If anaphylaxis occurs, study treatment administration should be immediately and permanently discontinued.

(b) Dose adjustment for treatment with docetaxel, paclitaxel, or vinflunine

In general, for grade 4 drug-related hematological toxicity and ≥ grade 3 non-hematological toxicity, treatment with chemotherapy comparator (docetaxel, paclitaxel or vinflunine) should be stopped, and subsequent doses should be adjusted according to Table 15 below. Dose adjustment will apply to all subsequent doses. Specific dose adjustment guidelines for docetaxel, paclitaxel and vinflunine can be found in sections 6.1.6.1 (ii) (b), 6.1.6.1 (ii) (c) and 6.1.6.1 (ii) (d) below. Dose adjustment should also be considered according to local product labels or SmPC and institutional guidelines. For docetaxel, paclitaxel or vinflunine-related ≥ grade 3 hematological toxicity, blood transfusion or growth factors can be used as indicated according to institutional guidelines.

(c) Docetaxel dose adjustment

Docetaxel should not be given to subjects with total bilirubin > ULN, or to subjects with AST and/or ALT > 1.5×ULN and concomitant alkaline phosphatase > 2.5×ULN. Subjects with elevated bilirubin or abnormal transaminases and alkaline phosphatase are at increased risk for developing grade 4 neutropenia, febrile neutropenia, infection, severe thrombocytopenia, severe stomatitis, severe skin toxicity, and toxic death. Docetaxel should also not be given to subjects with neutrophil counts < 1500 cells/ ^mm3 .

Severe fluid retention has been reported after docetaxel therapy. Subjects should be pre-administered corticosteroids before each docetaxel injection to reduce the incidence and severity of fluid retention. Subjects with pre-existing effusions should be closely monitored for possible worsening of effusions from the first dose. Subjects who develop peripheral edema can be treated with standard measures, such as salt restriction and oral diuretics. Dosing interruption can last up to 6 weeks (2 cycles). If the toxicity of the subject does not otherwise require permanent dosing interruption, the interruption of dosing for subjects who respond to treatment can be extended for more than 6 weeks. The recommended dose adjustment guidelines for subjects receiving docetaxel are detailed in Table 16 below. Dose adjustments (e.g., severe or cumulative skin reactions) and starting doses not specified below should also consider local product labels or SmPC and institutional guidelines.

Table 16 Suggested guidelines for docetaxel dose adjustment

ANC: absolute neutrophil count; N/A: not applicable; T: temperature

(d) Vinflunine dosage adjustment

In the setting of a WHO/ECOG PS of 1 or an ECOG PS of 0 and prior pelvic irradiation, vinflunine treatment should be initiated at a dose of 280 mg/m ^2. In the absence of any hematologic toxicity causing treatment delay or dose reduction during the first cycle, the dose for subsequent cycles may be increased to 320 mg/m ² every 21 days. In subjects with moderate renal impairment (40 mL/min ≤ CrCl ≤ 60 mL/min), the recommended dose is 280 mg/m ² given once every 21-day cycle. In subjects with renal impairment (30 mL/min ≤ CrCl < 40 mL/min), the recommended dose is 250 mg/m ² given once every 21-day cycle.

In subjects with mild hepatic impairment (Child-Pugh class A), the recommended dose of vinflunine is 250 mg/ ^m2 given once every 21-day cycle. Refer to local product labeling or SmPC and institutional guidelines for other dose adjustments.

The recommended doses for subjects ≥75 years of age are as follows:

- In subjects who are at least 75 years of age but less than 80 years of age, the dose of vinflunine to be administered is 280 mg/ ^m2 per 21 day cycle.

• In subjects 80 years of age and older, the dose of vinflunine to be administered is 250 mg/ ^m2 per 21 day cycle.

In subjects who initiate vinflunine at 280 mg/ ^m2 and experience an AE requiring dose adjustment, the dose should be reduced to 250 mg/ ^m2 after the first occurrence and resolution and interrupted after the second occurrence. In subjects who initiate vinflunine at 250 mg/ ^m2 and experience an AE requiring dose adjustment, vinflunine should be interrupted.

Cases of reversible posterior encephalopathy syndrome (PRES) have been observed following administration of vinflunine. Typical clinical symptoms (with varying degrees) are: neurological (headache, confusion, seizures, visual symptoms), systemic (hypertension), and gastrointestinal (nausea, vomiting). Radiological signs are white matter abnormalities in the posterior region of the brain. In subjects who develop neurological signs of PRES, vinflunine must be interrupted. Dose adjustments for subjects receiving vinflunine are detailed in Table 17 below. Interruption of dosing may last up to 6 weeks (2 cycles). If the subject's toxicity otherwise does not require permanent interruption of dosing, the interruption of dosing for subjects who derive clinical benefit from treatment may be extended for more than 6 weeks. For additional information, refer to vinflunine. SmPC.

Table 17 Changchun Fluorine Dosage Adjustment

ANC: absolute neutrophil count; N/A: not applicable; T: temperature

¹ Adverse Events The National Cancer Institute Common Terminology Criteria defined Grade 2 constipation as requiring laxatives, Grade 3 as obstipation requiring manual evacuation or enema, and Grade 4 as obstruction or toxic megacolon. Grade 2 mucositis was defined as "moderate", Grade 3 as "severe", and Grade 4 as "life-threatening".

(e) Pacific Taxol dose adjustment

Paclitaxel should not be administered to subjects with a baseline neutrophil count of less than 1500 cells/ ^mm3 . Subjects should not be retreated with paclitaxel in subsequent cycles until neutrophils recover to levels >1500 cells/ ^mm3 and platelets recover to levels >100000/ ^mm3 . Severe conduction abnormalities have been documented in <1% of subjects during paclitaxel therapy and in some cases have required placement of an atrial pulse generator. If a subject develops significant conduction abnormalities during paclitaxel infusion, appropriate therapy should be administered and continuous cardiac monitoring should be performed during subsequent paclitaxel therapy, provided that the subject does not require interruption.

In cases of mild hepatic impairment (total bilirubin ≥ 1.25 ULN), paclitaxel should be started at a dose of 135 mg/m ^2. Dose adjustments for subjects receiving paclitaxel are detailed below. Dosing interruptions may last up to 6 weeks (2 cycles). If the subject's toxicity does not otherwise require permanent dosing interruption, dosing interruptions for subjects who respond to treatment may be extended beyond 6 weeks. The recommended dose adjustment guidelines for subjects receiving paclitaxel are detailed in Table 18 below. For other dose adjustments, refer to the local product label or SmPC and institutional guidelines.

Table 18 Suggested guidelines for paclitaxel dose adjustment

ANC: absolute neutrophil count; N/A: not applicable; T: temperature

(iii) Previous and concomitant treatment (drug and non-drug therapies)

Subjects must withdraw from further study treatment administration if the investigator determines that any of the following medications are required to provide adequate medical support to the subject:

Other research drugs

● Chemotherapy or other drugs intended to provide anti-tumor activity. This does not apply to subjects with a history of breast cancer in the setting of adjuvant endocrine therapy, or to subjects using agents intended for the treatment of bone metastases (e.g., bisphosphonates or RANK ligand inhibitors).

Radiation therapy

NOTE: Radiation therapy to symptomatic solitary lesions or to bone may be considered on a case-by-case basis in special circumstances after consultation with the sponsor. The irradiated lesions must be non-target lesions according to RECIST V1.1, and the subject must have clearly measurable disease outside the irradiated area.

(a) Group A (ennolumab vedotin)

Subjects receiving potent cytochrome P450 (CYP) 3A4 inhibitors or P-pg inhibitors concomitantly with EV should be closely monitored for adverse reactions.

(b) Group B (docetaxel)

Concomitant use of drugs that strongly inhibit or induce CYP3A4 may affect docetaxel exposure and should be avoided.

(c) Group B (Vinflunine)

The use of strong inhibitors of the CYP3A4 enzyme should be avoided in subjects receiving vinflunine.

Medicinal products that prolong the QT/QTc interval should be avoided.

(d) Group B (paclitaxel)

Caution should be used when administering paclitaxel with strong inhibitors or inducers of CYP3A4 and CYP2C8.

Refer to local drug inserts for concomitant medication restrictions or requirements for docetaxel, paclitaxel, and vinflunine. All concomitant treatments will be recorded in the eCRF or electronic data source unless otherwise specified.

(iv) Treatment compliance

The dose and schedule of EV, paclitaxel, docetaxel, and vinflunine administered to each subject will be recorded on the appropriate electronic case report form (eCRF) at each cycle. The reasons for dose delays, reductions, or omissions will also be recorded.

If toxicity or adverse events occur on Day 1 of any cycle and EV cannot be administered, the start of the cycle may be delayed. If toxicity occurs on Day 8 or Day 15 of any cycle and the dose needs to be maintained for >3 days, the dose must be eliminated rather than delayed. If a subject receives only Day 1 and needs to skip Days 8 and 15, the subject may restart the next cycle as early as Day 22 (new Day 1) if the toxicity subsequently subsides.

6.1.6.2 Demographic data and baseline characteristics

(i) Demographics

Demographic information will be collected for all subjects according to local regulations and will include date of birth, sex, race, ethnicity, and smoking history (pack-years).

(ii) Medical history

Medical history will include all significant medical conditions other than urothelial carcinoma that resolved prior to informed consent or were ongoing at the time of consent. If an ongoing condition is applicable, details will be collected including onset and recovery dates and Common Terminology Criteria for Adverse Events (CTCAE) grade.

(iii) Diagnosis, severity and duration of the target disease

For urothelial carcinoma, information including, but not limited to, the following will be collected during the Screening Period and entered into the eCRF:

●Date of initial diagnosis of the primary cancer, histologic type, and date of histopathology or cytopathology diagnosis

Date of diagnosis of locally advanced, metastatic, or recurrent disease

●TNM classification and disease stage at screening

● Sampling methods and tumor tissue types for connexin-4 mutation analysis

Prior treatment for the underlying condition (including medications, radiation therapy, and surgery)

(iv) Behavioral status

The ECOG PS scale will be used to assess performance status. See Section 6.1.9.9. Oken et al., Am J Clin Oncol. 1982; 5:649-55.

6.1.6.3 Efficacy evaluation

Response and progression will be assessed using RECIST V1.1 6.1.9.8.

Imaging of the two groups will be performed at screening/baseline and every 56 days (± 7 days) from the first dose of study treatment during the entire study. Baseline imaging performed as standard care before informed consent can be used as long as it is performed within 28 days before randomization. All subjects will undergo bone scans (scintigraphy) at screening/baseline. Subjects with positive bone scans at baseline will undergo bone scans every 56 days (± 7 days) or more frequently (if clinically indicated) during the entire study. If clinically indicated throughout the study, even if not positive at baseline, subjects with negative bone scans at baseline should undergo bone scans. Only if clinically indicated at screening/baseline, a brain scan (CT with contrast/MRI) will be performed, and repeated as clinically indicated or according to the standard of care during the entire study. If the subject interrupts the study drug before radiological disease progression, the subject should continue to undergo imaging assessments every 56 days (± 7 days) until disease progression is recorded, or the subject starts another anticancer treatment, whichever occurs earlier.

CT scans (chest and abdomen) with contrast are the preferred mode for tumor assessment. If local standard practice or if the subject is contraindicated for CT scanning (e.g., the subject is allergic to contrast agents), magnetic resonance imaging is acceptable. All other approved RECIST scanning methods, such as x-rays, are optional. Additional instructions for imaging assessment can be found in the study procedure manual.

The assessment will include tumor measurements of target lesions, non-target lesions, and any new lesions. Characterization of the overall assessment will be evaluated for established time points.

At the end of the subject study, the best overall response to the study protocol will be characterized. In order to ensure comparability, the same technology should be used for subsequent assessments after screening and response. If possible, the image of any 1 subject should be assessed by the same researcher during the duration of the study. For subjects with known brain metastases when entering the study, it is recommended that repeated imaging also include the brain, and the same method used to detect brain lesions at baseline is used to track lesions during the entire study.

Sites of disease progression including target, non-target, and/or new lesions should be documented in the eCRF. Additional imaging may be performed at any time to confirm suspected progression of disease.

This study will be analyzed based on the results of local (investigator site) radiological assessments, including dates of progression and death. Since imaging scans may be required for future regulatory purposes or independent review of all or representative samples scanned may be considered after completion of PFS1 analysis, copies of all scans will be collected throughout the study and stored centrally by the coordinating vendor. Images of all randomized subjects will be sent to the imaging vendor according to the frequency of Table 10.

(i) Assessment of target lesions

(a) Complete reaction

CR was defined as the disappearance of all target and non-target lesions. Any pathological lymph node (whether target or non-target) had to be reduced to <10 mm in the short axis compared with the baseline measurement.

(b) Partial reaction

PR was defined as a decrease of at least 30% in the sum of target lesion diameters (longest axis for nonnodal lesions and short axis for nodal lesions), using the baseline sum of diameters as reference.

(c) Stable disease

SD was defined as neither a sufficient decrease to qualify as a PR nor a sufficient increase to qualify as progressive disease, taking the smallest sum of diameters at the time of study drug administration as reference.

(d) Progressive disease

PD is defined as an increase of at least 20% in the sum of target lesion diameters (longest axis for non-nodal lesions and lesions for nodal lesions) using the smallest sum on study (this includes the baseline sum if that is the smallest sum on study) as reference. In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5 mm. The appearance of 1 or more new lesions is also considered progression.

(ii) Assessment of non-target lesions

In order to achieve clear progression based on non-target lesions, there must be a significant worsening of the overall level of non-target disease, such that even in the presence of SD or PR of target lesions, the overall tumor burden has increased enough to warrant interruption of therapy. A modest increase in the size of 1 or more non-target lesions is generally not sufficient to define clear progression.

(a) Complete reaction

For CR of non-target lesions, a subject had to have disappearance of all non-target lesions and all lymph nodes had to be non-pathological in size (short axis <10 mm).

(b) Non-CR/non-PD

Non-CR/non-PD of non-target lesions was defined as the presence of 1 or more non-target lesions.

(c) Progressive disease

PD of non-target lesions was defined as unequivocal progression of existing non-target lesions or the appearance of 1 or more new lesions.

(iii) Assessment of time point response

The overall response status at each time point for subjects with measurable disease at baseline will be reported according to the tables in Sections 6.1.9.6 and 6.1.9.7.

6.1.6.4 Pharmacokinetic assessment

PK samples will be collected during the treatment period and at post-treatment follow-up visits for subjects receiving EV to determine ADC and MMAE concentrations. If a subject presents to the clinic but is not dosed, a pre-dose PK sample collection should be performed.

Blood samples (7 mL/sample) should be collected for pharmacokinetic analysis at the time points indicated in the assessment schedule (Table 10). Blood samples should be collected via a peripherally placed intravenous cannula or by direct venipuncture.

Blood should not be drawn from the arm or well used for study drug infusion.

Bioanalysis of TAb, ADC, and MMAE in serum or plasma was performed using validated methods in a bioanalytical laboratory specified by the sponsor.

6.1.6.5 Safety assessment

(i) Vital signs

Vital signs, including systolic and diastolic blood pressure (mmHg), radial pulse rate (beats/minute), and temperature will be obtained and recorded according to the assessment schedule (Table 10). All vital sign measurements will be obtained with the subject in a sitting or supine position.

If a clinically significant change in a vital sign compared to baseline (before treatment) is noted, the change will be recorded as an AE on the AE page of the eCRF. Clinical significance will be defined as a change in a vital sign with medical relevance that the investigator believes may lead to a change in medical care. The investigator will continue to monitor the subject until the parameter returns to ≤ Grade 1 or baseline (before treatment) values, or until the investigator determines that tracking is no longer medically necessary.

(ii) Adverse events

For information on AE collection and data disposition, see Section 6.1.6.6 (Adverse Events and Other Safety Aspects).

(a) Adverse events of possible hepatic origin

If an AE in a subject enrolled in the study and receiving study drug is accompanied by elevated liver function test values (LFTs, such as AST, ALT, bilirubin, etc.) or is suspected to be due to abnormal liver function, see Section 6.1.9.2 Hepatic Safety Monitoring and Assessment for detailed information on liver abnormalities, monitoring and assessment.

Subjects with AEs of hepatic origin accompanied by LFT abnormalities should be carefully monitored.

(iii) Laboratory Assessment

Table 19 below is a table of laboratory tests that will be performed during the conduct of the study. See Assessment Schedule for study visit collection dates.

Table 19

Laboratory tests will be performed before dosing according to the assessment schedule and sent to the central laboratory for analysis. All screening experiments must be sent to the central laboratory, but if the screening results from the central laboratory are not available at the time of planned randomization, local laboratory results can be used to determine eligibility. In the case where the central laboratory results received after randomization are not within the qualified parameters, if the local laboratory results meet the qualified standards, the subject will still be considered qualified and will not be considered as a protocol deviation. Local laboratory results supporting eligibility and dosing decisions must be entered into the clinical database. If a local laboratory is used to support dosing decisions, local laboratory tests will include complete blood count and classification, glucose, serum creatinine, ALT and AST. In the case of multiple laboratory data during this period, the most recent data should be used. Laboratory assessments collected outside the -7 days of randomization should be repeated. Additional assessments can be performed centrally or locally to monitor AEs or as required by dose adjustment requirements.

Creatinine clearance assessment or GFR estimation should be performed according to institutional guidelines prior to administration of vinflunine and EV, as recommended in Section 6.1.6.1(ii) (Dose Increments or Decreases of Study Drug).

Additional laboratory testing should be performed according to institutional standards of care. Clinical significance beyond the range of laboratory findings should be determined and documented by the Investigator/Investigator Assistant who is a qualified physician.

(iv) Physical Examination

Standard, complete physical examination will be conducted at screening to assess general appearance, skin, eyes, ears, nose, throat, neck, cardiovascular, chest and lungs, abdomen, musculoskeletal, neurological status, mental status and lymphatic system. For follow-up and end-of-treatment (EOT) visits, physical examinations may be more directional, but should include examinations of the lungs, abdomen, skin and cardiovascular system. Physical examinations will be performed at visits, as outlined in the assessment schedule (Table 10). Each physical examination will include weight; height is required only at screening. If clinically significant deterioration of the study results compared to baseline is noted at any study visit, the change will be recorded as an AE on the AE eCRF. Clinical significance is defined as any change in the physical study results with medical relevance that can lead to a change in medical care. Researchers will continue to monitor subjects until the parameters return to ≤ Grade 1 or baseline conditions, or until researchers determine that tracking is no longer medically necessary.

(v) Eye examination

An ophthalmological assessment is required for subjects with recent ocular discomfort (within 3 months of screening). The assessment should include the following: visual acuity, slit lamp, pressure measurement examination, and dilated fundus examination. A previous ophthalmological examination performed within 3 months of screening is acceptable, provided that the symptoms are not new since the examination. Ophthalmological assessments during treatment should be performed according to the standard of care or when clinically indicated (e.g., the subject develops new or worsening ocular symptoms). During the study, subjects who experience corneal adverse events are required to undergo an EOT slit lamp examination. The EOT slit lamp examination must be performed ≥4 weeks from the last dose.

Perform additional eye examinations as clinically indicated.

(vi) Electrocardiogram

A standard 12-lead ECG will be performed and assessed using local standard procedures according to the assessment schedule in Table 10. Clinically significant abnormal findings at Screening should be recorded as medical history.

6.1.6.6 Adverse events and other safety aspects

(i) Definition of adverse events

An AE is any untoward medical event in a subject that is temporally associated with the use of a medicinal product, whether or not considered to be related to the medicinal product. Thus, an AE may be any unfavorable and unexpected sign (including abnormal laboratory findings), symptom, or disease (new or exacerbated) temporally associated with the use of a medicinal product.

In order to identify any events that may be related to the study procedures and may lead to changes in the conduct of the study, the Sponsor collects AEs, even if the subject has not yet received study medication. AE collection begins after signing the informed consent form and will be collected until 30 days after the last dose of study medication.

(a) Abnormal laboratory findings

Any abnormal laboratory test result (e.g., hematology, clinical chemistry, or urinalysis) or other safety assessment (e.g., ECG, radiology scans, vital sign measurements, physical examination), including those results that are worse than baseline, which are considered clinically significant in the medical and scientific judgment of the Investigator and not related to the underlying disease, will be reported as an (S)AE.

Any clinically significant abnormal laboratory finding or other abnormal safety assessment related to the underlying disease need not be reported as an (S)AE unless judged by the investigator to be more severe than expected for the subject's condition.

In the absence of any of the above criteria, a repeat abnormal laboratory test or other safety assessment does not constitute an AE.Any abnormal test result determined to be an error does not need to be reported as an AE.

(b) Potential cases of drug-induced liver injury

For a detailed description of Drug Induced Liver Injury (DILI), refer to Section 6.1.9.2 Hepatic Safety Monitoring and Assessment. Abnormal values in aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT) that meet the criteria outlined in Section 6.1.9.2 Hepatic Safety Monitoring and Assessment in the absence of other causes of liver injury, concurrent or accompanied by abnormal elevations in total bilirubin, are considered potential cases of drug-induced liver injury (potential Hy's Law case) and are always considered important medical events and reported in accordance with Section 6.1.6.6 (v) (Reporting of Serious Adverse Events).

(c) Disease progression and study endpoints

The following events will not be considered (S)AEs under this protocol:

● Disease progression: Events including defined study endpoints that clearly conform to the expected pattern of progression of the underlying disease are not recorded as AEs unless they result in death. These data will be captured as efficacy assessment data as outlined in Section 6.1.6.3 Efficacy Assessments. If there is any uncertainty as to whether the event is attributable to expected disease progression and/or if there is evidence of a causal relationship between the study drug and the event, it should be reported as an (S)AE. All deaths within 30 days of the last dose of study drug must be reported as SAEs.

- Pre-planned and selected hospitalizations or procedures for diagnostic, therapeutic, or surgical procedures for pre-existing conditions that did not worsen during the course of the clinical trial. These procedures are collected according to the eCRF completion guidelines.

(ii) Definition of Serious Adverse Event (SAE)

An AE was considered “serious” if, in the opinion of the investigator or sponsor, it resulted in any of the following outcomes:

● Lead to death

Life-threatening (an AE is considered “life-threatening” if, in the opinion of the investigator or sponsor, its occurrence places the subject at immediate risk of death. It does not include AEs that, in their more severe form, could result in death)

● Causes persistent or significant incapacitation/disability or substantially disrupts the ability to carry out normal life functions

●Cause congenital abnormalities or birth defects

Required inpatient hospitalization (other than planned procedures permitted under the study) or resulted in a prolongation of hospitalization (other than a planned prolongation of hospitalization not caused by an AE). Hospitalization for treatment/observation/investigation caused by an AE was considered serious.

●Other medically significant events (defined in the following paragraphs)

Medical and scientific judgment may be used in deciding whether expedited reporting is appropriate for other situations, such as important medical events that may not be immediately life-threatening or result in death or hospitalization but that may endanger the subject or may require intervention to prevent one of the other outcomes listed in the definition above. These events, including those that may result in incapacitation/disability, are generally considered serious. Examples of such events are emergency room or home emergency treatment of allergic bronchospasm; blood dyscrasias or seizures that do not result in hospitalization; or the development of drug dependence or drug abuse.

The sponsor has a list of events that they classify as "Always Serious" events. If you report an AE that is considered an "Always Serious" event based on this classification, you can request additional information about the event.

(iii) Criteria for causal relationship with study drug

The investigator is obliged to assess the relationship between the study drug and each occurrence of each (S) AE. The investigator will use clinical judgment to determine the relationship. The investigator should also use the information provided herein and/or the product information of the commercially available product. The investigator is requested to provide an explanation of the causal relationship assessment for each SAE and this must be recorded on the SAE worksheet.

Causality assessment is one of the criteria used in determining regulatory reporting requirements. The investigator may revise his/her causality assessment based on new information about the SAE and should send an SAE follow-up report and update the eCRF with the new information and updated causality assessment.

After reviewing the relevant data, the causal relationship between the study drug and each (S)AE will be assessed by answering ‘yes’ or ‘no’ to the question “Do you think there is a reasonable possibility that the event was caused by the study drug?”

When making a causality assessment, the following factors should be considered when deciding whether there is evidence and/or assertion that there is a ‘reasonable possibility’ (rather than a relationship that cannot be excluded) that the (S)AE may have been caused by the study drug or whether there is evidence to reasonably reject a causal relationship:

● A plausible temporal relationship between exposure to study drug and the onset and/or resolution of the (S)AE. Did the subject actually receive the study drug? Was there a reasonable temporal relationship between the onset of the (S)AE and the administration of the study drug?

● Plausibility; i.e., is it possible that the event was caused by the study drug? Consider biological and/or pharmacological mechanisms, half-life, literature evidence, drug class, preclinical and clinical study data, etc.

●Dechallenge/dose reduction/rechallenge:

o Did the (S) AE resolve or improve after discontinuation of medication or reduction of the dose of the suspected medication? The effect of treatment on the event was also considered when evaluating the discontinuation experience.

o Did the (S)AE recur when the suspected drug was reintroduced after having been stopped?

● Laboratory or other test results; specific laboratory studies support the assessment of the relationship between the (S)AE and study drug (eg, based on values before, during, and after treatment)

● Available alternative explanations independent of study drug exposure; e.g., other concomitant medications, past medical history, concurrent or underlying illnesses, risk factors including medical and family history, season, location, etc., and the strength of alternative explanations

There may be situations where an SAE occurs and the investigator has minimal information to include in the initial report to the Sponsor. However, it is very important that the Investigator always assesses causality for each event prior to initial transmission of the SAE data to the Sponsor. A causality assessment of 'no' is considered in situations where there is limited or insufficient information about the event to make an informed judgment and in the absence of any indication or evidence to establish causality. In such situations, the Investigator is expected to obtain additional information about the event as soon as possible and reassess causality upon receipt of the additional information.

(iv) Criteria for defining the severity of adverse events

AEs involving abnormal clinical laboratory values will be graded using the NCI-CTCAE guidelines (version 4.03). Items not specified in the NCI-CTCAE version 4.03 will be assessed according to the criteria in Table 20 below and entered into the eCRF.

Table 20

level Evaluation Criteria 1- Mild Asymptomatic or mild symptoms, clinical, or diagnostic observations were noted; no intervention was indicated. 2-Moderate Local or noninvasive intervention is indicated. 3-Severe Medically significant but not immediately life-threatening, hospitalization or prolonged hospitalization. 4- Life-threatening Life-threatening outcome, urgent intervention indicated 5- Death Deaths related to AEs

The investigators will use the following definitions to rate the severity of each adverse event:

● Mild: does not interfere with normal daily activities

Moderate: Interferes with normal daily activities

Severe: Inability to carry out daily activities

(v) Reporting of Serious Adverse Events

Collection of AEs and expedited reporting of SAEs will begin after receipt of informed consent and will continue until 30 days after the last administration of study drug.

In the event of an SAE, the Investigator must contact the Sponsor promptly (within 24 hours of becoming aware) by fax or email.

The investigator must complete the SAE Worksheet containing all information required by local and/or regional regulations and submit it promptly (within 24 hours of awareness) to the Sponsor by email or fax. If the SAE Worksheet is not possible or cannot be faxed within 24 hours, the local drug safety contact should be notified by telephone.

The following minimum information is required:

●International Study Number (ISN)/Study Number,

●Subject number, gender and age,

●Report date,

Description of the SAE (Severity Standard Event),

● A causal relationship to the study drug (including cause), and

Medications provided (if any)

(vi) Tracking of adverse events

All AEs occurring during or after a subject has discontinued the study will be followed until resolved or judged to be no longer clinically significant, or until they become chronic to the extent that they can be adequately characterized by the Investigator.

The Investigator must promptly notify the Sponsor if, after the protocol-defined AE collection period (see Section 6.1.6.6(i) Definition of Adverse Events), an AE progresses to a SAE or the Investigator becomes aware of any (S)AE (including death) in which he/she believes there is a reasonable possibility that it is related to study drug treatment or study participation.

(vii) Monitoring of common serious adverse events

Common SAEs are SAEs that are usually expected to occur in the study population but are not related to drug exposure. SAEs classified as "common" are provided in the reference to common serious adverse events in section 6.1.9.3. The list does not change the reporting obligations of researchers, nor does it change their obligations to perform causal relationship assessments, or prevent the need to report AEs that meet the definition of SAEs as detailed above. The purpose of this list is to alert researchers that some events reported as SAEs may not need to be accelerated to the regulatory agency based on the "common SAE" classification as specified in section 6.1.9.3 common serious adverse events. The sponsor will monitor any frequency changes of these events throughout the study. Any changes in this list will be communicated to participating clinical trial sites. Researchers must report the individual occurrences of these events as stated in the report of serious adverse events in section 6.1.6.6 (v).

(viii) Special Circumstances

Certain special circumstances observed related to study drug (e.g., incorrect administration (e.g., wrong dose of study drug, comparator, or background therapy)) are collected in the eCRF as protocol deviations per Section 6.1.8.8 Major Protocol Deviations or may require special reporting, as described below. These special circumstances are not considered AEs but need to be communicated to the Sponsor according to the timelines defined below.

If a special circumstance is related to or contributes to the AE, the AE will be assessed separately from the special circumstance and captured as an AE in the eCRF. If the AE meets the definition of an SAE, the SAE is reported as described in Section 6.1.6.6(v) Reporting of Serious Adverse Events, and details of the relevant special circumstance will be included in the clinical description on the SAE worksheet.

Special circumstances are:

Pregnancy

Medication errors and overdosage

Misuse/abuse

Occupational exposure

Suspected drug-drug interactions

(a) Pregnancy

If a female subject becomes pregnant during the study dosing period or within 6 months of interruption of dosing, the Investigator will report the information to the Sponsor using the Pregnancy Reporting Form according to the timelines in Section 6.1.6.6(v) Reporting of Serious Adverse Events and report in the eCRF.

The Investigator will attempt to collect pregnancy information from any female partners of male subjects who become pregnant during the study dosing period or within 6 months of a dosing interruption and report the information to the Sponsor using the Pregnancy Reporting Form according to the timelines in Section 6.1.6.6(v) Reporting of Serious Adverse Events.

The due date or expected end of pregnancy, last menstrual period, estimated date of conception, pregnancy outcomes and neonatal data should be included in this information.

Although pregnancy itself is not considered an AE or SAE, any pregnancy complications or terminations (including elective terminations) of a female study subject's pregnancy will be reported as an AE in the eCRF or as an SAE in accordance with Section 6.1.6.6(v), Reporting of Serious Adverse Events. For (S)AEs experienced by female partners of male subjects, (S)AEs are reported via the Pregnancy Reporting Form.

Additional information regarding pregnancy outcomes when also classified as SAEs is as follows:

●“Spontaneous abortion” includes miscarriage, abortion and missed abortion.

●Neonatal or infant death within 1 month of life will be reported as a SAE regardless of its relationship to the study drug.

If the infant died more than 1 month after birth, report whether the relationship between the death and in utero exposure to the study drug was judged by the investigator to be “probable.”

Congenital anomalies (including those in aborted fetuses)

Unless a congenital anomaly is identified prior to spontaneous abortion or miscarriage, the embryo or fetus should be assessed for congenital defects by visual inspection. (S)AEs experienced by the neonate/infant should be reported via the pregnancy reporting form. In general, follow-up will be no longer than 6 to 8 weeks after the expected date of delivery.

(b) Medication Errors, Overmedication, and Off-Label Use

If a medication error, overmedication, or "off-label use" (i.e., use beyond that stated in the protocol) is suspected, refer to Section 6.1.8.8 Major Protocol Deviations. Any relevant (S)AEs should be reported in the eCRF. If the AE meets the definition of an SAE, the SAE and details of the medication error, overmedication, or "off-label use" should also be reported as described in Section 6.1.6.6(v) Reporting of Serious Adverse Events.

In case of suspected overdosing of EV or accidental infusion as a bolus, subjects should receive supportive care and monitoring. A medical monitor/specialist should be contacted as appropriate.

No specific procedures are available to treat EV overdosing or accidental infusion as a bolus, and only supportive care may be given. If EV is accidentally overdosed or accidentally infused as a bolus, the investigator/assistant investigator will provide emergency procedures and/or universal maintenance therapy based on symptoms to ensure the safety of the subject.

In cases of suspected overdosage of paclitaxel, docetaxel, or vinflunine, refer to the approved package insert, SmPC, or local product information provided by the manufacturer for each agent.

Overdosing events should be recorded in the eCRF along with the actual dose administered.

(c) Misuse/abuse

If misuse or abuse of study drug is suspected, the Investigator must immediately (within 24 hours of becoming aware) send a Special Circumstances Worksheet to the Sponsor by fax or email. Any relevant (S)AEs should be reported in the eCRF. If the AE meets the definition of an SAE, the SAE and details of the misuse or abuse of study drug should also be reported as described in Section 6.1.6.6(v) Reporting of Serious Adverse Events.

(d) Occupational exposure

If occupational exposure to study drug occurs (e.g., inadvertent exposure to study drug while site staff is preparing for administration to patients), the Investigator must immediately (within 24 hours of becoming aware) deliver the Special Situation Worksheet to the Sponsor by fax or email. Any relevant (S)AEs occurring in a subject that are related to or give rise to the Special Situation should be reported on the Special Situation Worksheet.

(e) Suspected drug-drug interactions

If a drug-drug interaction related to the study drug is suspected, the Investigator must immediately (within 24 hours of becoming aware) send a Special Circumstances Worksheet to the Sponsor by fax or email. Any relevant (S)AEs should be reported in the eCRF. If the AE meets the definition of an SAE, the SAE and details of the suspected drug-drug interaction should also be reported as described in Section 6.1.6.6(v) Reporting of Serious Adverse Events.

(ix) New information for supply impact studies

The sponsor will inform all investigators involved in the clinical investigation and regulatory agencies when new information necessary for the conduct of the clinical investigation becomes available, as appropriate. Investigators should inform the IRB/IEC of such information as required.

The investigator will also inform the subject that the subject will need to sign an updated informed consent form in order to continue the clinical study.

(x) Deviations from the protocol and other actions taken to avoid risk to the life of the subject

Researchers cannot deviate from the protocol except in emergency situations to avoid risk to subjects. When researchers do not follow the protocol to avoid emergency risk to subjects, researchers should take the following actions.

1. Describe the deviation and its reasons in a written notice and promptly send a document describing the deviation or correction and the reasons to the sponsor and study site director. Keep a copy of the notice.

2. Discuss the need for protocol amendments with the sponsor as early as possible. Obtain approval of the draft revised protocol from the IRB and site director and obtain written approval from the sponsor.

6.1.6.7 Test drug concentration

Blood samples for PK and ATA will be collected throughout the study according to the sample collection schedule provided in Table 11. Confirmatory assays will be used to measure the concentrations of ADC, total antibodies (TAb), and MMAE in serum or plasma. PK samples will be collected and archived for possible analysis of other EV-related species. Confirmatory assays will also be used to determine the level of ATA in serum.

Refer to the laboratory manual for information on sample collection, handling, storage, and shipping.

6.1.6.8 Other measurements, assessments or methods

(i) Exploratory biomarkers

Procedures for collecting, handling, and shipping samples will be specified in the laboratory manual.

Other biomarkers, including DNA, RNA, and protein, described in Sections 6.1.6.8(ii) Biomarkers in Blood and 6.1.6.8(iii) Biomarkers in Pre-treatment Tumor Tissue may be analyzed to study possible relationships to mechanisms of resistance or sensitivity to the study treatment, dynamic changes associated with the study treatment, and method development or validation of diagnostic assays related to the study treatment.

After the database is closed, samples may be stored at the study sponsor's facility or a contract laboratory facility for up to 15 years, at which time the samples will be destroyed.

(ii) Biomarkers in blood

Plasma and peripheral blood mononuclear cell (PBMC) samples collected at baseline and post-baseline time points can be analyzed for markers of immune function, immune cell subsets, and cytokines. Plasma and PBMC samples can be used for additional exploratory analyses, as described in Section 6.1.6.8 (i) Exploratory Biomarkers.

(iii) Biomarkers in tumor tissue before treatment

Submission of unstained charged slides (at least 10 and up to 15 slides) of FFPE tumor blocks or fresh sections at the time of screening should be provided (unless prior approval is obtained from the sponsor). Archived tissue or fresh tumor tissue before treatment (obtained from fresh biopsy) is acceptable. For details, see the laboratory manual. Tumor tissue samples can be analyzed for connexin-4 expression, markers of disease subtypes, and markers associated with the tumor immune microenvironment. Tumor tissue samples can be used for additional exploratory analysis, as described in Section 6.1.6.8 (i) Exploratory Biomarkers.

(iv) Blood samples for future pharmacogenomic analysis (retrospective)

Pharmacogenomic (PGx) studies may be performed in the future to analyze or measure genes associated with clinical response, pharmacokinetics, and toxicity/safety issues. After randomization (see Assessment Schedule), a 4 mL whole blood sample will be collected for possible retrospective PGx analysis. The sample will be shipped to the sponsor's designated reserve CRO.

Details regarding sample collection, labeling, storage, and shipping procedures will be provided in a separate laboratory manual.

See Section 6.1.9.4 Retrospective PGx Substudies for additional details on the reserve process.

(v) EORTC Core Quality of Life Questionnaire QLQ-C30

The QLQ-C30 was developed to measure aspects of QOL relevant to patients with a wide range of cancers participating in clinical trials. Sneeuw KC et al., J Clin Epidemiol. 1998; 51: 617-31; Aaronson NK et al., J Natl Cancer Inst. 1993; 85: 365-76. The core scale of the current version (QLQ-C30, version 3) is a 30-item questionnaire consisting of:

●Functional domain (physical, role, cognitive, emotional, social);

Three symptom scales (fatigue, pain, nausea, and vomiting);

Individual items for symptoms (shortness of breath, loss of appetite, sleep disturbances, constipation, diarrhea) and the financial impact of the illness; and

●2 global items (health, overall QOL).

(vi) EuroQOL-5 dimensions

EQ-5D-5L is a standardized tool developed by the EuroQOL Group, used as a general, preference-based measure of health status. It is applicable to many health statuses and treatments and provides a simple descriptive overview and a single index value for health status. EQ-5D-5L is a measure of 5 self-reports of function and health status, which assesses 5-dimensional health status, including mobility, self-care, daily activities, pain/discomfort and anxiety/depression. Each dimension contains 5 levels (no problem, minor problem, moderate temperature, severe problem, extreme problem). The unique EQ-5D-5L health status is defined by combining 1 level of each dimension in the 5 dimensions. This questionnaire also records the health status of the respondent's self-rating on a visual analog scale with a vertical scale (0 to 100). The response to the 5 items is also converted into a weighted health status index (utility score) based on the value derived from the general population sample. Herdman M et al., Qual Life Res. 2011; 20 (10): 1727-36.

(vii) Health Resource Utilization (HRU)

HRU information will be collected, specifically focusing on the number of subjects with planned use of medical resources related to clinical events or AEs for all subjects (Section 6.1.9.7).

6.1.6.9 Total blood volume

The total amount of blood collected for study assessments from each subject will vary depending on how long the subject remains on treatment.

At any time during the study, if any laboratory abnormalities are noted in the subject or disease assessments, additional blood may be drawn for monitoring.

Additional blood that will be drawn for this study beyond standard monitoring will include draws for: eligibility assessment; hematology and chemistry assessments at specific study-defined time points; pharmacokinetics; and bioanalytical sampling.

The maximum amount of blood collected was approximately 130.0 mL in Cycle 1 for subjects randomized to Group A and 37.0 mL in Cycle 1 for subjects randomized to Group B. The maximum amount of blood collected for subjects who participated in Cycles 2 through 6 and completed the EOT visit was 219.0 mL for subjects randomized to Group A and 164.0 mL for subjects randomized to Group B.

6.1.7 Interruptions

6.1.7.1 Discontinuation of Individual Subjects

A treatment interruptor is a subject who is enrolled in the study and permanently discontinues study treatment for any reason.

Subjects are free to discontinue study treatment and/or withdraw from the study for any reason and at any time, without being required to give a reason for doing so and without penalty or prejudice. Investigators are also free to discontinue study treatment or terminate a subject's participation in the study at any time if the subject's clinical condition permits.

All subjects who discontinue study treatment will remain on the study and must continue to be followed according to the protocol-specific follow-up procedures as outlined in the Assessment Schedule (Table 10) until the subject explicitly withdraws consent for any further contact from him/her or from persons the participant has previously authorized to provide this information.

If a subject discontinues the study with a persistent AE or unresolved laboratory result significantly outside the reference range, the investigator will attempt to provide follow-up until the condition stabilizes or is no longer clinically significant.

The following are the treatment discontinuation criteria for individual subjects:

●The subject develops radiographic disease progression.

●The subject needs to receive another systemic anticancer treatment for the underlying or new cancer.

●Unacceptable toxicity to subjects.

●Female subjects are pregnant.

The investigator decides that interrupting treatment is in the best interest of the subject

●The subject refuses further treatment.

- Subject is not complying with the protocol based on the assessment of the Investigator or Medical Monitor.

● Despite reasonable efforts by the investigators to locate the subject, the subject was lost to follow-up.

●Death.

Subjects who discontinue study drug before reaching PFS1 and enter the Post-Treatment Follow-up Period will discontinue the Post-Treatment Period if any of the following occurs:

●Subject develops radiographic progressive disease (ie, PFS1) based on investigator assessment

●Subjects starting new systemic anticancer therapy (first-line anticancer therapy after discontinuation of study drug)

Death

●Subject refuses further study participation (ie, withdraws consent)

●Subjects are lost to follow-up despite reasonable efforts by the investigators to locate them

Subjects will discontinue the long-term follow-up period (for PFS2) if any of the following occur:

●Subjects starting new systemic anticancer therapy (second-line anticancer therapy after discontinuation of study drug)

● Based on the investigator's assessment, the subject has evidence of PD

●Subject refuses further study participation (ie, withdraws consent)

● Despite reasonable efforts by the investigators to locate the subject, the subject was lost to follow-up.

Death

● The sponsor ends the long-term tracking collection period

Subjects will discontinue the survival follow-up period if any of the following occurs:

●Subject refuses further study participation (ie, withdraws consent)

● Despite reasonable efforts by the investigators to locate the subject, the subject was lost to follow-up.

Death

● The sponsor ends the survival tracking collection period

6.1.8 Statistical methods

A Statistical Analysis Plan (SAP) will be written to provide details of the analysis, as well as specifications for tables, lists, and figures to be prepared. The SAP will be completed prior to enrollment of the first subject. Any changes to the analyses planned in the SAP will be accommodated in the clinical study report.

In general, continuous data will be summarized using descriptive statistics (number of subjects, mean, SD, minimum, median, and maximum) and frequencies and percentages for categorical data.

6.1.8.1 Sample size

Approximately 600 subjects will be randomized in a 1:1 ratio to 2 treatment groups: EV (Group A) and chemotherapy (Group B). Randomization will be stratified by baseline ECOG PS (0 vs. 1), world region (Western Europe, the United States, or elsewhere in the world), and liver metastases (yes or no). Assuming HR=0.75 (median OS in Group A and Group B were 10.7 months and months, respectively), a 10% decline rate, a final analysis at a planned 439 deaths and 1 interim analysis at 65% of the total planned events (285 deaths), this sample size will provide 85% power to detect statistically significant differences at an overall type I error rate of 0.025 on one side.

The sample size was determined by the primary endpoint of OS; 600 subjects will provide more than 90% power to detect statistically significant differences with respect to the selected secondary endpoints: PFS1 (assuming a median PFS1 of 6 months in Arm A and 4 months in Arm B, respectively), ORR, and DCR (assuming a 15% treatment difference in ORR and DCR between Arm A and Arm B).

6.1.8.2 Analysis Set

Detailed criteria for analysis sets will be laid out in SAP or classification specifications, and assignment of subjects to analysis sets will be determined prior to hard lock of the database.

(i) Full analysis set

The full analysis set (FAS) will consist of all subjects who underwent randomization. This analysis set complies with the intent to treat (ITT) principle, which includes all randomized subjects, and the FAS is equivalent to the ITT population. This will be the primary analysis set for efficacy analyses, except for response-related efficacy endpoints.

(ii) Security Analysis Set

The Safety Analysis Set (SAF), which consists of all subjects who received any amount of study drug, will be used for safety analyses.

(iii) Evaluable response set

The response evaluable set (RES) is defined as all subjects who are in the FAS and have measurable disease at baseline and are followed for at least 6 months after randomization. The RES will be used for the primary efficacy analysis of response-related endpoints (e.g., ORR and DCR).

(iv) Pharmacokinetic analysis set

The pharmacokinetic analysis set (PKAS) includes subjects receiving active drug, from whom at least one blood sample was collected and measurements of TAb, ADC and MMAE serum/plasma concentrations were determined, and whose sampling time and dosing time on the day of sampling were known.

6.1.8.3 Demographic data and baseline characteristics

Demographic data and baseline characteristics will be summarized by treatment group and overall. Ethnic origin will be summarized in the demographic table only. Descriptive statistics will include the number of subjects, mean, standard deviation, minimum, median, and maximum values for continuous endpoints, and frequencies and percentages for categorical endpoints.

(i) Subject configuration

The number and percentage of subjects who completed and discontinued treatment and the reasons for discontinuation will be presented for all randomized subjects and subjects in the SAF by treatment group and overall. Similar tables for screening disposition, study period disposition, and follow-up disposition will also be presented for all randomized subjects by treatment group and overall. All disposition details and the dates of the first and last assessment for each subject will be listed.

(ii) Prior and concomitant medications

All previous and concomitant medications will be presented in a list. The frequency of concomitant medications (prescriptions, over-the-counter, and nutritional supplements) will be summarized by treatment group and SAF's preferred term (PT). Medications will be coded using the WHO drug dictionary. Medication will be counted by the number of subjects taking each medication. Subjects who take the same medication multiple times will be counted only once for the medication. Medication will be presented in descending frequency order based on the total number of subjects taking each medication.

(iii) Medical history

The medical history of each subject will be presented in a table. A detailed medical history of each subject will be obtained during the screening period and will be summarized by treatment group and overall.

6.1.8.4 Efficacy analysis

Efficacy analyses of OS and PFS were performed for FAS. Results of statistical tests were interpreted based on FAS. Efficacy analyses of response-related endpoints, such as ORR and DCR, were performed for RES.

The family-by-family type I error rate of this study was strongly controlled at 2.5% (one-sided), which allowed the study to state that the primary endpoint OS for the FAS population was positive. OS was formally tested at both the interim analysis and the final analysis. At the interim or final analysis, only when the OS analysis was rejected, the grading (according to the order of PFS1->ORR->DCR) performed a formal hypothesis test for the selected secondary endpoints (including PFS1, ORR and DCR). The details of the significance level at the interim analysis and final analysis of each efficacy endpoint (OS, PFS1, ORR and DCR) are described in SAP.

(i) Analysis of the primary endpoint

(a) Preliminary analysis

The primary efficacy endpoint of OS was analyzed using the log-rank test stratified by ECOG PS (0 vs. 1), world region (Western Europe, United States, or rest of the world), and liver metastasis (yes or no). The analysis was performed using the randomized treatment and the stratum used for randomization. In addition, the stratified Cox proportional hazards model was used to estimate the hazard ratio and the corresponding 95% confidence interval. Median OS was estimated using the Kaplan-Meier method and will be reported by treatment group with the corresponding 95% confidence interval.

Primary analysis was performed using FAS.

(b) Sensitivity analysis

Additional analyses, such as unstratified log-rank tests and OS analyses adjusting for crossover effects, were performed as appropriate. Details are described in the SAP.

(ii) Analysis of secondary endpoints

(a) Progression-free survival

For each subject, PFS1 is defined as the time from the date of randomization until the date of radiological disease progression (according to RECIST V1.1) or until death due to any cause. If the subject has neither progressed nor died, the subject is censored on the last radiological assessment date. Before the start of anticancer therapy, subjects who received any other anticancer therapy for the disease before radiological progression were examined on the last radiological assessment day.

The efficacy endpoint of PFS1 was analyzed using the log-rank test stratified by ECOG PS (0 vs. 1), world region (Western Europe, United States, or rest of the world), and liver metastases (yes or no). The analysis was performed using the randomized treatment and the stratum used for randomization. In addition, the stratified Cox proportional hazards model was used to estimate the hazard ratio and the corresponding 95% confidence interval. The median PFS1 was estimated using the Kaplan-Meier method and reported by treatment group with the corresponding 95% confidence interval.

Primary analysis was performed using FAS. Additional sensitivity analysis targeting PFS1 was also performed. Details are described in SAP.

(b) Overall response rate

The overall response rate (ORR) is defined as the proportion of subjects with a complete or partial objective response based on RECIST V1.1. The ORR between Group A and Group B was compared using a stratified CMH test. The same stratification factors used in the event analysis time will be used for the stratified CMH test. The difference in response rate between the treatment groups and the corresponding 95% confidence intervals will be estimated. In addition, the ORR for each group will be estimated, and the corresponding 95% confidence intervals will be constructed. Primary analysis was performed using RES.

(c) Reaction duration

Duration of response (DOR) is defined as the time from the date of the first response CR/PR (according to RECISTV1.1) (whichever is recorded first) of the subject who achieved CR or PR (subsequently confirmed, as assessed by the researcher) to the date of radiological progression or death. If the subject did not progress or die, the subject was examined on the day of the last radiological assessment or on the day of the first CR/PR (if no other baseline radiological assessment was available after the first CR/PR). The median DOR was estimated using the Kaplan-Meier method and reported by treatment group together with the corresponding 95% confidence interval.

(d) Disease Control Rate (DCR)

DCR was defined as the proportion of subjects with complete or partial objective response or stable disease based on RECIST V1.1. The DCR between Group A and Group B was compared using a stratified CMH test. The same stratification factors used in the event analysis time were used for the stratified CMH test. The difference in response rate between the treatment groups and the corresponding 95% confidence interval were estimated. In addition, the DCR of each group was estimated, and the corresponding 95% confidence interval was constructed. The primary analysis was performed using RES.

(e) QOL and PRO parameters

Descriptive QOL and PRO analyses were performed on the FAS. Completion rates for each questionnaire are summarized. Additional analyses are detailed in the statistical analysis plan.

(iii) Subgroup analysis

Analyses for OS, PFS1, and ORR were repeated separately by ECOG PS (0 vs. 1), world region, and liver metastasis using FAS.

In addition, subgroup analysis was performed for the selected endpoints to determine whether the treatment effect was constant. Subgroups may include, but are not limited to, the following:

Age group (<65 years vs. ≥65 years; <75 years vs. ≥75 years)

Gender (female vs. male)

Prior platinum (cisplatin and carboplatin and both)

Number of prior lines of systemic therapy in the locally advanced or metastatic setting (1 to 2 vs ≥3)

Best response to most recent CPI (responder vs. nonresponder) Most recent CPI treatment (yes vs. no)

Baseline hemoglobin (≥10 versus <10 g/dL)

Histology (urothelial carcinoma/transitional cell and mixed urothelial carcinoma vs.

Primary tumor site (upper urinary tract vs bladder/other)

●Smoking status (never vs. ever vs. current)

Brain metastasis status (previous brain metastases vs. no previous brain metastases)

Investigator's choice of paclitaxel/docetaxel or vinflunine

Baseline connexin-4 IHC score (<150 vs 150-225 vs >225)

●Prior taxane (yes or no)

PD-L1 CPS (<10 versus ≥10)

(iv) Analysis of exploratory endpoints

Exploratory analyses of efficacy endpoints are described in the statistical analysis plan.

Using the PK analysis set, summarize serum or plasma TAb, ADC, and MMAE concentrations with descriptive statistics for each PK sampling time point. These data can be combined with data from population PK and PK/PD analyses from previous studies. Explore the relationship between TAb, ADC, MMAE and PD endpoints, safety, or efficacy.

6.1.8.5 Using the safety analysis set, summarize the incidence of ATA by visit and overall.

6.1.8.6 Security Analysis

The SAF was used to perform all safety analyses. All treated subjects were analyzed according to the treatment they received.

(i) Adverse events

AEs were coded using the Medical Dictionary for Regulatory Activities (MedDRA).

TEAEs were defined as AEs observed or worsened after initiation of study drug.

The number and percentage of subjects with treatment-emergent AEs, SAEs, AEs leading to treatment discontinuation, and AEs related to study drug are summarized by system organ class, preferred term, and treatment group. The number and percentage of AEs are also summarized by severity. All AEs are listed.

Study drug-related TEAEs were defined as any TEAE for which the investigator considered the causal relationship to be yes. AEs of interest as categorized by custom MedDRA queries and/or standard MedDRA queries were also summarized.

(ii) Laboratory Assessment

For quantitative laboratory measurements, descriptive statistics were used to summarize the outcomes and changes from baseline for subjects in SAF by treatment group and time point.

Changes in laboratory tests compared to normal ranges from baseline to each time point during treatment are also tabulated. Laboratory data are presented in the table.

(iii) Vital signs

Vital sign results and changes from baseline at the scheduled visits were summarized by treatment group using descriptive statistics for subjects in the SAF. Vital sign data are presented in a table.

(iv) Conventional 12-lead electrocardiogram

The 12-lead ECG results were summarized by treatment group and time point.

(v) ECOG performance status

Summary statistics (number and percentage of subjects) for each category of ECOG PS at each assessment are provided. Changes from baseline to final visit or early termination are also summarized. Negative change scores indicate improvement. Positive scores indicate a decline in performance.

(vi) Exposure-response relationship analysis

The relationship between TAb, ADC and MMAE concentrations and certain efficacy or safety endpoints can be analyzed in an exploratory manner. Additional details of these analyses are described in the exposure-response analysis plan.

6.1.8.7 Pharmacokinetic Analysis

Individual and summary tables of serum TAb and ADC and plasma MMAE concentrations are provided, as well as a listing of blood collection times and concentrations.

Summary statistics are provided, including n, mean, SD, geometric mean, minimum, median, maximum, and CV%. If all values are below the lower limit of quantification (BLOQ), the BLOQ value is set to not calculated. In the case where more than half of the individual data in the group are BLOQ, the geometric mean is not calculated. Additional model-based analyses can be performed and are described in the separate population PK analysis plan.

6.1.8.8 Significant plan deviations and other analyses

Major protocol deviations, as defined in Section 6.1.8.8 Major Protocol Deviations, will be summarized for all randomized subjects by treatment group and overall and by site. Tabulations of data will be provided by site and subject.

Significant protocol deviation criteria will be specifically identified in summary tables and listings. The unique identifier will be as follows:

PD1 - enter the study even if they do not meet the entry criteria,

PD2 - Developed withdrawal criteria during the study and did not withdraw,

PD3- received the wrong treatment or incorrect dose,

●PD4-receiving excluded concomitant therapy.

6.1.8.9 Interim Analysis (and Early Discontinuation of Clinical Studies)

The planned interim efficacy analysis occurs after approximately 285 OS events (approximately 65% of the total planned events) are observed. OS will be tested at a one-sided 0.00541 significance level according to the O'Brien-Fleming boundary as implemented by the Lan-DeMets α consumption function. Lan KKG, DeMets DL. Biometrika. 1983; 70: 659-63. The IDMC may recommend termination of the trial at the interim analysis based on statistically significant OS results in favor of EV. When the total number of deaths reaches 439, the final OS analysis will be performed at a one-sided 0.02332 significance level. If the exact number of events at the interim and final analyses is different from the plan, the significance level will be adjusted accordingly based on the O'Brien-Fleming method and the Lan-DeMets α consumption function.

Interim analysis will be conducted by IDAC and reviewed by IDMC. In addition, safety data review during the trial will be conducted regularly by IDMC. For example, IDMC will review safety data after the first 50 subjects have been randomized and taken study drug for approximately 3 months. The complete procedures for IDMC safety review and interim analysis will be described in a separate IDMC concession and interim analysis plan.

6.1.8.10 Handling of Missing Data, Outliers, Visit Windows, and Other Information

If applicable, the imputation method for missing data and the definition of the window to be used for analysis will be outlined in SAP.

6.1.9 Appendix to Clinical Research

6.1.9.1 List of Warning Concomitant Drugs

The following list describes drugs and foods that are common strong inhibitors/inducers of CYP3A, CYP2C8, and inhibitors of p-glycoprotein (P-gp) that should be avoided, used with caution, or closely monitored. This list should not be considered all-inclusive; consult individual drug labels for specific information. If there are concerns or questions about concomitant use of any of the drugs listed below, a discussion with the sponsor is encouraged.

Table 22 Summary of possible drug reactions

EV: Ennosumab vedotin; P-gp: p-glycoprotein

Additional information on inhibitors/inducers can be found in the FDA guidance (Drug Interactions and Labeling). fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm093664.html#table5-2

6.1.9.2 Hepatic safety monitoring and assessment

Any subject who is enrolled in a clinical study using active drug therapy and reveals an increase in serum transaminases (AT) to >3×ULN (>5×ULN in subjects with liver metastases) or bilirubin>2×ULN should undergo detailed testing of liver enzymes (including at least ALT, AST, ALP and TBL). The test should be repeated within 72 hours of notification of the test results. For studies using central laboratories, warnings about moderate and severe liver abnormalities are generated by the central laboratory to inform researchers, study monitors and research teams. If the subject has any symptoms suggestive of abnormal liver and gallbladder function, the subject should be asked.

(i) Definition of liver abnormalities

Confirmed abnormalities will be characterized as moderate and severe when the ULN is:

In addition, subjects should be considered to have severe hepatic abnormalities for any of the following:

ALT or AST >8×ULN.

ALT or AST > 5× ULN for more than 2 weeks (in the absence of liver metastases).

ALT or AST >3× ULN and International Normalized Ratio (INR) >1.5 (if INR testing is applicable/assessed).

ALT or AST >3× ULN with associated fatigue, nausea, vomiting, right upper quadrant abdominal pain or tenderness, fever, rash, and/or eosinophilia (>5 percent).

Abnormal liver function results other than those described above may be considered moderately or severely abnormal and require additional monitoring and follow-up at the discretion of the investigator.

(ii) Tracking procedures

Confirmed moderate and severe liver function abnormalities should be fully characterized by obtaining appropriate expert consultation, detailed relevant medical history, physical examination and laboratory tests. The site should complete the liver abnormality case report form (LA-CRF) that has been conducted globally and can be activated for any study or appropriate documentation. Subjects with confirmed abnormal liver function tests should be tracked as described below.

Confirmed moderately abnormal LFTs should be repeated 2 to 3 times weekly, then weekly or less frequently if the abnormality has stabilized or study drug has been discontinued and the subject is asymptomatic.

In the absence of another etiology, severe liver function abnormalities as defined above may be considered medically important events and may be reported as SAEs. The Sponsor should be contacted and all subjects informed that severe liver function abnormalities that may be attributable to study drug were observed.

To further evaluate abnormal liver laboratory findings, researchers are expected to:

● Obtain a more detailed history of symptoms and prior or concurrent illnesses. Symptoms and new onset illnesses are recorded as "AEs" in the (e)CRF. Attention should be paid to ailments and conditions that may lead to secondary liver abnormalities, such as hypotensive events and decompensated cardiac disease. Nonalcoholic steatohepatitis is seen in obese patients with hyperlipoproteinemia and/or diabetes and may be associated with fluctuating AT levels. The investigator should ensure that the medical history form captures any ailments prior to study entry that may be relevant when assessing liver function.

● Obtain a history of concomitant medication use (including over-the-counter medications, complementary and alternative medicines), alcohol use, recreational drug use, and special diets. Enter medications (including dosages) into the (e)CRF. Information on alcohol, other substance use, and diet should be entered on the LA-CRF or appropriate documentation.

● Obtain a history of exposure to environmental chemicals.

●Based on the subject's medical history, other testing may be appropriate and include:

○Acute viral hepatitis (A, B, C, D, E or other infectious agents),

○Ultrasound or other imaging to assess biliary disease,

○ Other laboratory tests, including INR, direct bilirubin.

●Consider gastroenterology or hepatology consultation.

●Submit results of any additional testing and possible etiologies on the LA-CRF or appropriate documentation.

(iii) Research interruption

Subjects may discontinue the study in the absence of an increase in LFTs, such as viral hepatitis, pre-existing or acute liver disease, presence of liver metastases, or exposure to other agents associated with liver damage. The investigator may determine that continued study participation is not in the best interest of the subject. Treatment interruption should be considered if:

ALT or AST >8×ULN.

ALT or AST >5×ULN for more than 2 weeks (in subjects without liver metastases).

ALT or AST >3×ULN and TBL >2×ULN or INR >1.5 (if INR testing is applicable/assessed).

ALT or AST >5×ULN and (TBL >2×ULN in patients with liver metastases).

ALT or AST >3× ULN with associated fatigue, nausea, vomiting, right upper quadrant abdominal pain or tenderness, fever, rash, and/or eosinophilia (>5 percent).

Additionally, if close monitoring of subjects with moderate or severe liver laboratory tests is not possible, the drug should be interrupted.

* Definition of Ho's Law:

1. Evidence that the drug can cause hepatocellular carcinoma damage, generally shown by a higher rate of transaminase elevations 3× and greater above the upper limit of normal in the population compared with controls (2× elevations are too common to be distinguished between treated and untreated patients).

2. Cases of increased bilirubin (to at least 2×ULN) in a population with elevated transaminases to at least 3×ULN (but they are almost always higher) and without evidence of intra- or extra-hepatic bilirubin blockage (elevated alkaline phosphatase) or Gilbert's syndrome. Temple R. Hy's law: predicts severe hepatotoxicity. Pharmacoepidemiol Drug Saf. 2006; 15(S4): 241-3

3. Drug-induced hepatocellular injury, usually demonstrated by more frequent elevations of ALT or AST 3 times or more above the ULN compared with a (non-hepatotoxic) control drug or placebo.

4. Among trial subjects who showed such AT elevations (usually AT well above 3×ULN), one or more subjects also showed elevations in serum TBL to >2×ULN without initial findings of cholestasis (elevated serum ALP).

5. No other possible explanation for the combination of elevated AT and TBL was found, such as hepatitis A, B, or C virus; pre-existing or acute liver disease; or another drug that could cause the observed injury. Guidance for Industry. “Drug-Induced Liver Injury: Premarketing Clinical Evaluation”, issued by FDA in July 2009.

6.1.9.3 Common serious adverse events

The following is a list of SAEs that the Sponsor believes are relevant to the disease condition being studied. The list does not change our reporting obligations or prevent the need to report AEs that meet the definition of SAEs as detailed in Section 6.1.6.6(ii), Definition of Serious Adverse Event. The purpose of this list is to alert investigators that some events reported as SAEs may not require expedited reporting to regulatory agencies based on the classification of "Common SAEs." Investigators need to follow the requirements detailed in Section 6.1.6.6(v), Reporting of Serious Adverse Events.

Urinary tract pain

Bladder disorders

Dysuria

Urinary tract bleeding (hematuria)

Urinary tract obstruction

6.1.9.4 Retrospective PGx Substudies

(i) Introduction

PGx studies are designed to provide information about how naturally occurring changes in a subject's genes and/or expression may affect which treatment options are best for the subject based on genetic variation. By studying PGx through techniques such as genotyping, gene sequencing, statistical genetics, and genome-wide association studies, the relationship between genetic profiles and the kinetics, efficacy, or toxicity of a drug can be better understood. Since many diseases may be affected by 1 or more genetic variations, PGx studies can identify which genes are involved in determining how a subject may or may not respond to a drug.

(ii) Objectives

Future PGx studies that may be performed using the blood samples obtained will be exploratory. The goal of this study will be to analyze or measure genes associated with clinical response, pharmacokinetics, and toxicity/safety issues.

By analyzing genetic variation, it may be possible to predict how an individual subject will respond to treatment in terms of efficacy and/or toxicity.

(iii) Participation of subjects

Subjects who have consented to participate in this study may participate in this PGx substudy. As part of this substudy, subjects must provide written consent prior to providing any blood samples that may be used later for genetic analysis.

(iv) Sample collection and storage

Subjects who agree to participate in this sub-study will provide a tube of approximately 4-6 mL of whole blood according to the sponsor's instructions. Each sample will be identified by a unique subject number (first code).

(v) PGx analysis

Sponsors will initiate PGx analysis if evidence suggests that a genetic variant may affect the kinetics, efficacy, and/or safety of a drug.

(vi) Placement of PGx samples/data

All PGx samples collected will be stored for up to 15 years after the study database hard lock. Whole blood samples will be destroyed after the planned storage period if no analysis is required. Subjects have the right to withdraw consent at any time. PGx samples will be destroyed when withdrawal notification is received from the subject.

(vii) Disclosure of information to subjects

If applicable, exploratory PGx analysis may be performed after the conclusion of the clinical study. The results of the genetic analysis will not be provided to any investigator or subject, nor can they be requested at a later date.

6.1.9.5EORTC-QLQ-C30 (version 3)

We are interested in a few things about you and your health. Please answer all questions by circling the number that best applies to you. There are no "right" or "wrong" answers. The information you provide will be kept strictly confidential.

Please fill in your initials:

Your birthday (year, month, day):

Today's date (year, month, day):

For the following questions, please circle the number between 1 and 7 that best applies to you.

29. How would you rate your overall health during the past week?

30. How would you rate your overall quality of life during the past week?

6.1.9.6EQ-5D-5L

EQ-5D-5L is shown in Figure 2C.

6.1.9.7 Utilization of health resources

Have you been to the Emergency Room (ER) since your last study visit?

□No (If no, go to 4)

□Yes (if yes, go to 2)

How many times have you been to the emergency department since your last study visit?

For each emergency room visit, complete the following:

Since your last study visit, have you been admitted to the hospital (staying longer than 24 hours) without first going to the emergency room (ER)?

□No (If no, go to 7)

□Yes (If yes, go to 5)

How many admissions (stay longer than 24 hours; no prior ER referral)?

For each inpatient (>24-hour stay; no prior ER referral) visit, complete the following:

Length of stay in hospital (days) Hospital visit 1 Hospital visit 2 Hospital visit 3

Have you seen your general practitioner (primary physician) since your last study visit?

□No (If no, go to 9)

□Yes (if yes, go to 8)

How many times have you visited your general practitioner (primary physician)?

Have you been to a specialist physician (e.g., oncologist, rheumatologist, endocrinologist, plastic surgeon, etc.) since your last study visit?

□No

□Yes (If yes, go to 10)

How many times have you visited specialist physicians (e.g., oncologist, rheumatologist, endocrinologist, plastic surgeon, etc.)?

6.1.9.8RECIST V1.1

Table A - Time point responses: patients with target (+/- non-target) disease.

Table B - Time Point Responses: Patients with Non-Target Disease Only

Table C - Best overall response when confirmation of CR and PR was required.

Reproduced from: Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, et al. New response evaluation criteria in solid tumors: revised RECIST guidelines (version 1.1). Eur J Cancer. 2009;45:228-47.

6.1.9.9 ECOG Behavioral Status

ECOG: Eastern Cooperative Oncology Group

Reproduced from: Oken MM, Creech RH, Tormey DC, Horton J, Davis TE, McFadden ET, et al. Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol. 1982;5:649-55.

6.1.10 Description of terms or abbreviations used in clinical studies

6.1.11 Research Results

The treatment configurations for the clinical studies described herein are shown in Table 23 below:

Table 23 Treatment configuration

Data as of 2020-7-15 Ennosumab Vedotin Chemotherapy total Full analysis set 301 307 608 treat 296 291 587 Untreated 5 16 twenty one Deaths 134 167 301

The patient demographics for the clinical studies described herein are shown in Table 24 below:

Table 24 Demographic data and baseline characteristics, stratification factors and medical history

*Percentages may not total 100 due to rounding.

Eastern Cooperative Oncology Group (ECOG) performance status scores range from 0 to 4, with higher scores indicating greater disability.

The Bellmunt risk score ranges from 0 to 3 based on the presence of the following risk factors: hemoglobin level less than 10 g per deciliter, ECOG performance status score higher than 0, and liver metastases.

§Other histologic types include adenocarcinoma, squamous cell carcinoma, and pseudosarcomatous differentiation.

Best response in responding patients was defined as a confirmed complete or partial response; in nonresponding patients, best response was defined as stable disease or progressive disease.

The study exposures for the clinical studies described herein are shown in Table 25 below:

Table 25 Study Exposures

In an interim analysis that included a total of 301 deaths, overall survival (OS) for the clinical studies described herein was analyzed for the Full Analysis Set (FAS) population, as shown in Table 26 below:

Table 26 Summary of overall survival in FAS

*Predetermined efficacy margin = 0.00679 (adjusted for 301 observed deaths)

After a median follow-up of 11.1 months, the risk of death was 30% lower with enroku vedotin than with chemotherapy (hazard ratio, 0.70; 95% confidence interval [CI], 0.56 to 0.89; P=0.001), indicating that enroku vedotin had significantly longer overall survival.

Kaplan Meier curves—overall survival in FAS are shown in FIG4 .

The overall survival of the subgroups is shown in Figure 5. The median overall survival in the enroku vedotin group was 12.88 months (95% CI, 10.58 to 15.21), and in the chemotherapy group it was 8.97 months (95% CI, 8.05 to 10.74). The estimated percentage of patients alive at 12 months in the enroku vedotin group was 51.5% (95% CI, 44.6 to 58.0), and in the chemotherapy group it was 39.2% (95% CI, 32.6 to 45.6).

As an additional measure of efficacy, progression-free survival (PFS) was analyzed for the FAS population in an interim analysis that included a total of 432 PFS events, as shown in Table 27. In addition, PFS is shown in FIG6 .

Table 27 Overview of progression-free survival, investigator-assessed -FAS

The results of the subgroup analysis showed that there was a progression-free survival benefit with enroku vedotin across multiple subgroups, as shown in Figure 7. This forest plot shows the investigator-assessed progression-free survival in the predefined key subgroups of the intention-to-treat population consisting of all randomized patients. For "all subjects," the reported results are based on a stratified analysis with the following stratification factors: ECOG PS, region, and liver metastases.

The results of the sensitivity analysis were consistent with those of the primary analysis, as shown in Table 28.

Table 28 Sensitivity analysis of overall survival

*Stratified Cox proportional hazards models were analyzed for 1000 boot simulation data sets. Confidence intervals for the 1000 simulations ranged from 2.5% to 97.5%. P values were calculated based on 1000 simulations.

(95% CI) is based on a stratified Cox proportional hazards model with treatment as an explanatory variable. Stratification factors are ECOG PS, region, and liver metastasis. Weights are calculated according to two logarithmic models. One model includes only baseline covariates (age group, primary tumor site, and number of previous lines of therapy in a locally advanced or metastatic setting). The other model includes two baseline covariates and time-dependent variables (diameter sum and ECOG assessment). The one-sided P value is based on a weighted stratified log-rank test.

CI denotes confidence interval; ECOG PS: Eastern Cooperative Oncology Group performance status; HR: hazard ratio.

ORR and DOR were analyzed and are shown in Table 29.

Table 29 ORR and DOR, Investigator Assessment-Evaluable Responsiveness Population (RES)

*Disease control rate was defined as the proportion of patients with a confirmed complete response, confirmed partial response, or stable disease (at least 7 weeks) as best overall response.

In all patients with a confirmed complete or partial response.

The best overall response was defined according to RECIST v1.1. Complete or partial response was confirmed by two scans at least 4 weeks apart. The minimum duration of stable disease was 7 weeks. CI denotes confidence interval; and RECIST: Response Evaluation Criteria in Solid Tumors.

The results of the subgroup analysis are consistent with those of the preliminary analysis, as shown in Figure 8. This forest plot shows the total response rate assessed by researchers in a predetermined key subgroup of an evaluable response population, which is composed of all randomized patients with measurable disease at baseline. For "all subjects", the reported results are based on unstratified analysis. Complete responses were observed in 4.9% (14/288) of patients in the Ennomonab Vedotin group and in 2.7% (8/296) of patients in the chemotherapy group. Disease control was observed in 71.9% (95% CI, 66.3 to 77.0) and 53.4% (95% CI, 47.5 to 59.2), respectively (P < 0.001).

Among patients with a complete or partial response, the median duration of response was 7.39 months in the enrofloxacin group and 8.11 months in the chemotherapy group, as shown in FIG. 9 .

Grade 3 or higher treatment-related adverse events occurred in 51.4% of patients in the enroku vedotin group and in 49.8% of the chemotherapy group. After adjustment for treatment exposure, the rates were 2.4 and 4.3 events per patient-year in the enroku vedotin group and chemotherapy group, respectively. An overview of adverse events adjusted by patient-year is shown in Table 30.

Table 30 Overview of adverse events adjusted by patient-year

*TRAE indicates there is a reasonable possibility that the event may have been caused by study treatment as assessed by the investigator. If the relationship was missing, the adverse event was considered treatment-related.

E denotes number of events, PY denotes patient-years, TEAE denotes treatment-emergent adverse event, and TRAE denotes treatment-related adverse event.

Treatment-related adverse events leading to treatment adjustments are shown in Table 31. In particular, grade 3 or higher treatment-related adverse events occurring in at least 5% of patients included: in the enroku monoclonal antibody vedotin group, maculopapular rash (7.4%), fatigue (6.4%), and decreased neutrophil count (6.1%); and in the chemotherapy group, decreased neutrophil count (13.4%), anemia (7.6%), decreased white blood cell count (6.9%), neutropenia (6.2%), and febrile neutropenia (5.5%). Treatment-related adverse events leading to dose reduction, interruption of treatment, or discontinuation of treatment occurred in 32.4%, 51.0%, and 13.5% of patients in the enroku monoclonal antibody vedotin group, respectively, and in 27.5%, 18.9%, and 11.3% of the chemotherapy group, respectively.

Table 31 Treatment-related adverse events leading to treatment modification

Events with a percentage ≥ 2% in either treatment group are reported by preferred term (listed in order of highest frequency in the enroku vedotin group).

Treatment-emergent adverse events and deaths were analyzed and are shown in Table 32. All adverse events occurring during the treatment period are listed in Table 30.

Table 32 Overview of treatment-emergent adverse events and deaths

Adverse events of particular interest were analyzed and are shown in Table 33.

Table 33 Treatment-emergent adverse events of special safety concern (MedDRA v23.0)

The clinical study presented here compared PADCEV with chemotherapy in adult patients with locally advanced or metastatic urothelial carcinoma who had been previously treated with platinum-based chemotherapy and a PD-1/L1 inhibitor. Results from the clinical study showed that PADCEV had a significantly lower risk of recurrence compared to chemotherapy. (ennosumab vedotin-ejfv) met its primary endpoint of overall survival.

In clinical studies, PADCEV significantly improved overall survival (OS), with a 30% reduction in the risk of death (hazard ratio (HR) = 0.70; (95% confidence interval (CI): 0.56, 0.89); p = 0.001). PADCEV also significantly improved progression-free survival (PFS), a secondary endpoint, with a 39% reduction in the risk of disease progression or death (HR = 0.61 (95% CI: 0.50, 0.75); p < 0.00001).

For patients in the PADCEV arm of the trial, adverse events were consistent with those listed in the U.S. prescribing information, with rash, hyperglycemia, decreased neutrophil count, fatigue, anemia, and decreased appetite as the most frequent Grade 3 or higher adverse events occurring in greater than 5% of patients.

Treatment-related adverse events in the safety population are shown in Table 34. The incidence of treatment-related adverse events was high overall but similar in both groups (93.9% in the enrofloxacin group and 91.8% in the chemotherapy group).

Table 34 Treatment-related adverse events (safety population)

*The safety population includes all patients who received any amount of trial drug. Treatment-related adverse events occurring in at least 20% of patients in either treatment group, or grade 3 or higher occurring in at least 5% of patients in either treatment group were included. Treatment-related adverse events were those events for which there was a reasonable possibility that they were caused by the trial treatment, as assessed by the investigator. Events were considered treatment-related if data on relationship to treatment were missing.

A total of 113 patients (55 in the enroku vedotin group and 58 in the chemotherapy group) had pre-existing peripheral neuropathy.

Skin reactions and peripheral neuropathy were the most frequent treatment-related adverse events of special concern with enrofloxacin, as shown in Table 35.

The onset times of treatment-related adverse events of particular interest are shown in Table 36.

Table 36 Onset time of treatment-related adverse events of particular concern

*The median (range) time to first onset of grade ≥2 peripheral neuropathy in the enroku vedotin and chemotherapy groups was: 4.435 (0.36, 12.02) months; and 1.725 (0.07, 9.89) months, respectively.

NA means not applicable.

Treatment-induced peripheral neuropathy and hyperglycemia by baseline condition are shown in Table 37.

Table 37

*Baseline status indicates the health status (historical or ongoing) before the patient received any study treatment at the time of trial entry. Patients with a history of pre-existing grade ≥2 sensory or motor neuropathy or uncontrolled diabetes within 3 months of the first dose of study drug were excluded from the trial. Uncontrolled diabetes was defined as hemoglobin A1c ≥8% or between 7% and <8% with associated diabetic symptoms (polyuria or polydipsia) not otherwise explained. In addition to these exclusion criteria, patients with pre-existing neuropathy and hyperglycemia were allowed to enter the trial.

BMI denotes body mass index, and PN denotes peripheral neuropathy.

Regardless of the relationship to treatment, adverse events leading to death (excluding disease progression) during treatment occurred in 11 patients in each group; after adjustment for treatment exposure, the incidence remained the same. Treatment-related adverse events assessed by researchers leading to death occurred in 7 patients (2.4%) in the ennostrobin vedotin group (multiple organ dysfunction syndrome [in 2 patients], and liver function abnormalities, hyperglycemia, pelvic abscess, pneumonia, and septic shock [each in 1 patient]); and occurred in 3 patients (1.0%) in the chemotherapy group (neutropenic sepsis, sepsis, and pancytopenia [each in 1 patient]). The demographic characteristics of patients who died in the ennostrobin vedotin group are provided in Table 38.

Table 38 Patient details on adverse events leading to death in the enrofloxacin vedotin group

*“Treatment-related” adverse events indicate a reasonable possibility that the event may have been caused by the study treatment, as assessed by the investigator. If the relationship was absent, the adverse event was considered treatment-related.

"Treatment-emergent" adverse events (n=11) also included all events considered to be treatment-related adverse events.

BMI stands for body mass index.

6.2 Example 2 - Analysis of difficult-to-treat subgroups according to the clinical study described in Section 6.1.

In the clinical studies described in Section 6.1, Enromax Vedotin (EV) showed superior overall survival (OS) compared to standard chemotherapy (SC) in patients with previously treated locally advanced or metastatic urothelial carcinoma (la/mUC). This subgroup analysis evaluated the efficacy/safety of EV in patients with poor prognostic factors, such as age ≥ 65 years, presence of liver metastases, primary upper urinary tract disease, and non-response to checkpoint inhibitors (CPIs).

Methods: In this open-label trial, patients with la/mUC who had previously received platinum-based chemotherapy and had disease progression during/after a PD-1/L1 inhibitor were randomized 1:1 to EV or investigator's choice of SC (docetaxel, paclitaxel, vinflunine). The primary endpoint of OS for subgroup analysis and secondary endpoints of progression-free survival (PFS) and overall response rate (ORR) assessed by the investigator according to RECISTv1.1 were prespecified. Kaplan-Meier analysis compared OS and PFS between treatments for the following difficult-to-treat subgroups selected: age ≥65 years, presence of liver metastases, primary upper urinary tract disease, and checkpoint inhibitor (CPI) non-response. ORR and safety within subgroups were also evaluated. Cox proportional hazards models were used to estimate hazard ratios. Cochran-Mantel-Haenszel tests were used to compare responses and disease control rates between groups.

Results: A total of 608 patients were randomized to EV (n = 301) or SC (n = 307); the median follow-up was 11.1 months. The OS benefit of EV was maintained across most subgroups (e.g., patients with liver metastases and patients who did not respond to previous immune checkpoint inhibitors (i.e., PD-1/L1; "CPI non-responsive" or "CPI non-responders")), with longer median OS observed in patients taking EV relative to patients taking SC, as shown in Table 39 and Figures 10A to 10D. For the subgroup of primary upper urinary tract disease, the median OS of EV was longer relative to SC and consistent with the median OS in the overall population, as shown in Table 39 and Figures 10A to 10D.

Compared with SC, PFS was also longer and ORR was higher in all subgroups of EV. In particular, similar benefits of PFS were observed in the subgroups of EV relative to SC, as shown in Table 39 and Figures 11A to 11D. For the subgroup of primary upper urinary tract disease, the median PFS of EV was longer relative to SC and consistent with the median PFS in the total population, as shown in Table 39 and Figures 11A to 11D. Among all randomized patients with measurable disease at baseline, the overall response rate (ORR) was higher in each subgroup of EV relative to SC, and was consistent between treatments in all subgroups, as shown in Table 39.

Safety and Tolerability: The overall rates of treatment-emergent and treatment-related adverse events (AEs) were similar between treatments in the subgroups. For example, the overall rate of AEs in patients ≥65 years of age treated with EV was 97.4%, and the overall rate of AEs in patients ≥65 years of age treated with SC was 98.9%. The overall rate of AEs in patients with liver metastases treated with EV was 97.8%, and the overall rate of AEs in patients with liver metastases treated with SC was 96.7%. The overall rate of AEs in patients with primary upper urinary tract disease treated with EV was 99.0%, and the overall rate of AEs in patients with primary upper urinary tract disease treated with SC was 99.0%. The overall rate of AEs in patients who had not responded to previous treatment with PD-1/L1 inhibitors and were treated with EV was 97.5%, and the overall rate of AEs in patients who had not responded to previous treatment with PD-1/L1 inhibitors and were treated with SC was 99.5%.

Treatment-related AEs were comparable between treatments in the subgroups, as shown in Figure 12. AEs were assessed in all patients receiving any amount of trial drug. When adjusted for treatment exposure, grade ≥3 treatment-related AEs occurred at a lower frequency in the EV group relative to the SC group in all four subgroups (i.e., in patients ≥65 years of age, in patients with liver metastases, in patients with primary upper urinary tract disease, and in patients unresponsive to prior treatment with PD-1/L1 inhibitors), as shown in Figure 13. The incidence of grade ≥3 treatment-related AEs in the difficult-to-treat subgroups was generally aligned and consistent with those observed in the overall EV-301 safety population, as shown in Table 40.

Conclusions: Patients with locally advanced or metastatic urothelial carcinoma (La/mUC) with poor prognostic factors who received EV had consistently longer OS, longer PFS, and higher ORR compared with those who received SC. The safety profile of the study treatment was consistent with those of previous studies and the overall study population; no new safety signals were observed. These data support the use of EV in previously treated patients with la/mUC, including those with poor prognostic factors.

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190 195 200

gcc gtc acc tca gag ttc cac ttg gtg cct agc cgc agc atg aat ggg 917

Ala Val Thr Ser Glu Phe His Leu Val Pro Ser Arg Ser Met Asn Gly

205 210 215

cag cca ctg act tgt gtg gtg tcc cat cct ggc ctg ctc cag gac caa 965

Gln Pro Leu Thr Cys Val Val Ser His Pro Gly Leu Leu Gln Asp Gln

220 225 230

agg atc acc cac atc ctc cac gtg tcc ttc ctt gct gag gcc tct gtg 1013

Arg Ile Thr His Ile Leu His Val Ser Phe Leu Ala Glu Ala Ser Val

235 240 245 250

agg ggc ctt gaa gac caa aat ctg tgg cac att ggc aga gaa gga gct 1061

Arg Gly Leu Glu Asp Gln Asn Leu Trp His Ile Gly Arg Glu Gly Ala

255 260 265

atg ctc aag tgc ctg agt gaa ggg cag ccc cct ccc tca tac aac tgg 1109

Met Leu Lys Cys Leu Ser Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp

270 275 280

aca cgg ctg gat ggg cct ctg ccc agt ggg gta cga gtg gat ggg gac 1157

Thr Arg Leu Asp Gly Pro Leu Pro Ser Gly Val Arg Val Asp Gly Asp

285 290 295

act ttg ggc ttt ccc cca ctg acc act gag cac agc ggc atc tac gtc 1205

Thr Leu Gly Phe Pro Pro Leu Thr Thr Glu His Ser Gly Ile Tyr Val

300 305 310

tgc cat gtc agc aat gag ttc tcc tca agg gat tct cag gtc act gtg 1253

Cys His Val Ser Asn Glu Phe Ser Ser Arg Asp Ser Gln Val Thr Val

315 320 325 330

gat gtt ctt gac ccc cag gaa gac tct ggg aag cag gtg gac cta gtg 1301

Asp Val Leu Asp Pro Gln Glu Asp Ser Gly Lys Gln Val Asp Leu Val

335 340 345

tca gcc tcg gtg gtg gtg gtg ggt gtg atc gcc gca ctc ttg ttc tgc 1349

Ser Ala Ser Val Val Val Val Gly Val Ile Ala Ala Leu Leu Phe Cys

350 355 360

ctt ctg gtg gtg gtg gtg gtg ctc atg tcc cga tac cat cgg cgc aag 1397

Leu Leu Val Val Val Val Val Leu Met Ser Arg Tyr His Arg Arg Lys

365 370 375

gcc cag cag atg acc cag aaa tat gag gag gag ctg acc ctg acc agg 1445

Ala Gln Gln Met Thr Gln Lys Tyr Glu Glu Glu Leu Thr Leu Thr Arg

380 385 390

gag aac tcc atc cgg agg ctg cat tcc cat cac acg gac ccc agg agc 1493

Glu Asn Ser Ile Arg Arg Leu His Ser His His Thr Asp Pro Arg Ser

395 400 405 410

cag ccg gag gag agt gta ggg ctg aga gcc gag ggc cac cct gat agt 1541

Gln Pro Glu Glu Ser Val Gly Leu Arg Ala Glu Gly His Pro Asp Ser

415 420 425

ctc aag gac aac agt agc tgc tct gtg atg agt gaa gag ccc gag ggc 1589

Leu Lys Asp Asn Ser Ser Cys Ser Val Met Ser Glu Glu Pro Glu Gly

430 435 440

cgc agt tac tcc acg ctg acc acg gtg agg gag ata gaa aca cag act 1637

Arg Ser Tyr Ser Thr Leu Thr Thr Val Arg Glu Ile Glu Thr Gln Thr

445 450 455

gaa ctg ctg tct cca ggc tct ggg cgg gcc gag gag gag gaa gat cag 1685

Glu Leu Leu Ser Pro Gly Ser Gly Arg Ala Glu Glu Glu Glu Asp Gln

460 465 470

gat gaa ggc atc aaa cag gcc atg aac cat ttt gtt cag gag aat ggg 1733

Asp Glu Gly Ile Lys Gln Ala Met Asn His Phe Val Gln Glu Asn Gly

475 480 485 490

acc cta cgg gcc aag ccc acg ggc aat ggc atc tac atc aat ggg cgg 1781

Thr Leu Arg Ala Lys Pro Thr Gly Asn Gly Ile Tyr Ile Asn Gly Arg

495 500 505

gga cac ctg gtc tga cccaggcctg cctcccttcc ctaggcctgg ctccttctgt 1836

Gly His Leu Val

510

tgacatggga gattttagct catcttgggg gcctccttaa acacccccat ttcttgcgga 1896

agatgctccc catcccactg actgcttgac ctttacctcc aacccttctg ttcatcggga 1956

gggctccacc aattgagtct ctcccaccat gcatgcaggt cactgtgtgt gtgcatgtgt 2016

gcctgtgtga gtgttgactg actgtgtgtg tgtggagggg tgactgtccg tggaggggtg 2076

actgtgtccg tggtgtgtat tatgctgtca tatcagagtc aagtgaactg tggtgtatgt 2136

gccacgggat ttgagtggtt gcgtgggcaa cactgtcagg gtttggcgtg tgtgtcatgt 2196

ggctgtgtgt gacctctgcc tgaaaaagca ggtattttct cagaccccag agcagtatta 2256

atgatgcaga ggttggagga gagaggtgga gactgtggct cagacccagg tgtgcgggca 2316

tagctggagc tggaatctgc ctccggtgtg agggaacctg tctcctacca cttcggagcc 2376

atgggggcaa gtgtgaagca gccagtccct gggtcagcca gaggcttgaa ctgttacaga 2436

agccctctgc cctctggtgg cctctgggcc tgctgcatgt acatattttc tgtaaatata 2496

catgcgccgg gagcttcttg caggaatact gctccgaatc acttttaatt tttttctttt 2556

ttttttcttg ccctttccat tagttgtatt ttttatttat ttttattttt attttttttt 2616

agagatggag tctcactatg ttgctcaggc tggccttgaa ctcctgggct caagcaatcc 2676

tcctgcctca gcctccctag tagctgggac tttaagtgta caccactgtg cctgctttga 2736

atcctttacg aagagaaaaa aaaaattaaa gaaagccttt agatttatcc aatgtttatact 2796

actgggattg cttaaagtga ggcccctcca acaccagggg gttaattcct gtgattgtga 2856

aaggggctac ttccaaggca tcttcatgca ggcagcccct tgggagggca cctgagagct 2916

ggtagagtct gaaattaggg atgtgagcct cgtggttatact gagtaaggta aaattgcatc 2976

caccattgtt tgtgatacct tagggaattg cttggacctg gtgacaaggg ctcctgttca 3036

atagtggtgt tggggagaga gagagcagtg attatagacc gagagagtag gagttgaggt 3096

gaggtgaagg aggtgctggg ggtgagaatg tcgcctttcc ccctgggttt tggatcacta 3156

attcaaggct cttctggatg tttctctggg ttggggctgg agttcaatga ggtttatttt 3216

tagctggccc accgatac actcagccag aatacctaga tttagtaccc aaactcttct 3276

tagtctgaaa tctgctggat ttctggccta agggagaggc tcccatcctt cgttccccag 3336

ccagcctagg acttcgaatg tggagcctga agatctaaga tcctaacatg tacattttat 3396

gtaaatatgt gcatatttgt acataaaatg atattctgtt tttaaataaa cagacaaaac 3456

ttgaaaaa 3464

<210> 2

<211> 510

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)...(510)

<223> 191P4D12

<400> 2

Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro Ala Gly

20 25 30

Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln Asp Ala

35 40 45

Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val Gly Gln

50 55 60

Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly Ala Gln Glu Leu Ala

65 70 75 80

Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr Glu Gly

85 90 95

Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu Asp Gly Ser Val

100 105 110

Leu Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg

115 120 125

Val Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Leu Arg Leu Arg

130 135 140

Val Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu

145 150 155 160

Glu Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser

165 170 175

Pro Ala Pro Ser Val Thr Trp Asp Thr Glu Val Lys Gly Thr Thr Ser

180 185 190

Ser Arg Ser Phe Lys His Ser Arg Ser Ala Ala Val Thr Ser Glu Phe

195 200 205

His Leu Val Pro Ser Arg Ser Met Asn Gly Gln Pro Leu Thr Cys Val

210 215 220

Val Ser His Pro Gly Leu Leu Gln Asp Gln Arg Ile Thr His Ile Leu

225 230 235 240

His Val Ser Phe Leu Ala Glu Ala Ser Val Arg Gly Leu Glu Asp Gln

245 250 255

Asn Leu Trp His Ile Gly Arg Glu Gly Ala Met Leu Lys Cys Leu Ser

260 265 270

Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp Thr Arg Leu Asp Gly Pro

275 280 285

Leu Pro Ser Gly Val Arg Val Asp Gly Asp Thr Leu Gly Phe Pro Pro

290 295 300

Leu Thr Thr Glu His Ser Gly Ile Tyr Val Cys His Val Ser Asn Glu

305 310 315 320

Phe Ser Ser Arg Asp Ser Gln Val Thr Val Asp Val Leu Asp Pro Gln

325 330 335

Glu Asp Ser Gly Lys Gln Val Asp Leu Val Ser Ala Ser Val Val Val

340 345 350

Val Gly Val Ile Ala Ala Leu Leu Phe Cys Leu Leu Val Val Val Val

355 360 365

Val Leu Met Ser Arg Tyr His Arg Arg Lys Ala Gln Gln Met Thr Gln

370 375 380

Lys Tyr Glu Glu Glu Leu Thr Leu Thr Arg Glu Asn Ser Ile Arg Arg

385 390 395 400

Leu His Ser His His Thr Asp Pro Arg Ser Gln Pro Glu Glu Ser Val

405 410 415

Gly Leu Arg Ala Glu Gly His Pro Asp Ser Leu Lys Asp Asn Ser Ser

420 425 430

Cys Ser Val Met Ser Glu Glu Pro Glu Gly Arg Ser Tyr Ser Thr Leu

435 440 445

Thr Thr Val Arg Glu Ile Glu Thr Gln Thr Glu Leu Leu Ser Pro Gly

450 455 460

Ser Gly Arg Ala Glu Glu Glu Glu Asp Gln Asp Glu Gly Ile Lys Gln

465 470 475 480

Ala Met Asn His Phe Val Gln Glu Asn Gly Thr Leu Arg Ala Lys Pro

485 490 495

Thr Gly Asn Gly Ile Tyr Ile Asn Gly Arg Gly His Leu Val

500 505 510

<210> 3

<211> 1432

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (32)...(1432)

<220>

<221> misc_feature

<222> (1)...(1432)

<223> Ha22-2(2,4)6.1 heavy chain

<400> 3

ggtgatcagc actgaacaca gaggactcac c atg gag ttg ggg ctg tgc tgg 52

Met Glu Leu Gly Leu Cys Trp

1 5

gtt ttc ctt gtt gct att tta gaa ggt gtc cag tgt gag gtg cag ctg 100

Val Phe Leu Val Ala Ile Leu Glu Gly Val Gln Cys Glu Val Gln Leu

10 15 20

gtg gag tct ggg gga ggc ttg gta cag cct ggg ggg tcc ctg aga ctc 148

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

25 30 35

tcc tgt gca gcc tct gga ttc acc ttc agt agc tat aac atg aac tgg 196

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Asn Met Asn Trp

40 45 50 55

gtc cgc cag gct cca ggg aag ggg ctg gag tgg gtt tca tac att agt 244

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser

60 65 70

agt agt agt agt acc ata tac tac gca gac tct gtg aag ggc cga ttc 292

Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

75 80 85

acc atc tcc aga gac aat gcc aag aac tca ctg tct ctg caa atg aac 340

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser Leu Gln Met Asn

90 95 100

agc ctg aga gac gag gac acg gct gtg tat tac tgt gcg aga gca tac 388

Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Tyr

105 110 115

tac tac ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc 436

Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

120 125 130 135

tca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc 484

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

140 145 150

aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac 532

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

155 160 165

tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc 580

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

170 175 180

agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac 628

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

185 190 195

tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag 676

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

200 205 210 215

acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg gac 724

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

220 225 230

aag aga gtt gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg 772

Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

235 240 245

tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc 820

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

250 255 260

cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca 868

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

265 270 275

tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac 916

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

280 285 290 295

tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg 964

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

300 305 310

gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc 1012

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

315 320 325

ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc 1060

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

330 335 340

aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa 1108

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

345 350 355

ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gag 1156

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

360 365 370 375

gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc 1204

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

380 385 390

tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag 1252

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

395 400 405

aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc 1300

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

410 415 420

ttc ctc tat agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg 1348

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

425 430 435

aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac 1396

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

440 445 450 455

acg cag aag agc ctc tcc ctg tcc ccg ggt aaa tga 1432

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

460 465

<210> 4

<211> 466

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)...(466)

<223> Ha22-2(2,4)6.1 heavy chain

<400> 4

Met Glu Leu Gly Leu Cys Trp Val Phe Leu Val Ala Ile Leu Glu Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Ser Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln

115 120 125

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

130 135 140

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

145 150 155 160

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

165 170 175

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

195 200 205

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

210 215 220

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

225 230 235 240

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Lys

465

<210> 5

<211> 735

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (25)...(735)

<220>

<221> misc_feature

<222> (1)...(735)

<223> Ha22-2(2,4)6.1 light chain

<400> 5

agtcagaccc agtcaggaca cagc atg gac atg agg gtc ccc gct cag ctc 51

Met Asp Met Arg Val Pro Ala Gln Leu

1 5

ctg ggg ctc ctg ctg ctc tgg ttc cca ggt tcc aga tgc gac atc cag 99

Leu Gly Leu Leu Leu Leu Trp Phe Pro Gly Ser Arg Cys Asp Ile Gln

10 15 20 25

atg acc cag tct cca tct tcc gtg tct gca tct gtt gga gac aga gtc 147

Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val

30 35 40

acc atc act tgt cgg gcg agt cag ggt att agc ggc tgg tta gcc tgg 195

Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Gly Trp Leu Ala Trp

45 50 55

tat cag cag aaa cca ggg aaa gcc cct aag ttc ctg atc tat gct gca 243

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile Tyr Ala Ala

60 65 70

tcc act ttg caa agt ggg gtc cca tca agg ttc agc ggc agt gga tct 291

Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

75 80 85

ggg aca gat ttc act ctc acc atc agc agc ctg cag cct gaa gat ttt 339

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

90 95 100 105

gca act tac tat tgt caa cag gct aac agt ttc cct ccc act ttc ggc 387

Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro Thr Phe Gly

110 115 120

gga ggg acc aag gtg gag atc aaa cga act gtg gct gca cca tct gtc 435

Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val

125 130 135

ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct 483

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

140 145 150

gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag 531

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

155 160 165

tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc 579

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

170 175 180 185

aca gag cag gac agc aag gac agc acc tac agc ctc agc agc acc ctg 627

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

190 195 200

acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa 675

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

205 210 215

gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg 723

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

220 225 230

gga gag tgt tag 735

Gly Glu Cys

235

<210> 6

<211> 236

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)...(236)

<223> Ha22-2(2,4)6.1 light chain

<400> 6

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Gly Ile Ser Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Phe Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ala Asn Ser Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 7

<211> 466

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)...(466)

<223> Ha22-2(2,4)6.1 heavy chain

<400> 7

Met Glu Leu Gly Leu Cys Trp Val Phe Leu Val Ala Ile Leu Glu Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Ser Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln

115 120 125

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

130 135 140

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

145 150 155 160

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

165 170 175

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

195 200 205

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

210 215 220

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

225 230 235 240

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Lys

465

<210> 8

<211> 236

<212> PRT

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)...(236)

<223> Ha22-2(2,4)6.1 light chain

<400> 8

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Gly Ile Ser Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Phe Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ala Asn Ser Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 9

<211> 5

<212> PRT

<213> Homo sapiens

<220>

<223> Heavy chain CDR1

<400> 9

Ser Tyr Asn Met Asn

1 5

<210> 10

<211> 17

<212> PRT

<213> Homo sapiens

<220>

<223> CDR2 of heavy chain

<400> 10

Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 11

<211> 8

<212> PRT

<213> Homo sapiens

<220>

<223> CDR3 of heavy chain

<400> 11

Ala Tyr Tyr Tyr Gly Met Asp Val

1 5

<210> 12

<211> 11

<212> PRT

<213> Homo sapiens

<220>

<223> CDR1 of light chain

<400> 12

Arg Ala Ser Gln Gly Ile Ser Gly Trp Leu Ala

1 5 10

<210> 13

<211> 7

<212> PRT

<213> Homo sapiens

<220>

<223> CDR2 of light chain

<400> 13

Ala Ala Ser Thr Leu Gln Ser

1 5

<210> 14

<211> 9

<212> PRT

<213> Homo sapiens

<220>

<223> CDR3 of light chain

<400> 14

Gln Gln Ala Asn Ser Phe Pro Pro Thr

1 5

<210> 15

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector

<400> 15

Gly Phe Leu Gly

1

<210> 16

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> VH CDR1 according to IMGT

<400> 16

Gly Phe Thr Phe Ser Ser Tyr Asn

1 5

<210> 17

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> VH CDR2 according to IMGT

<400> 17

Ile Ser Ser Ser Ser Ser Thr Ile

1 5

<210> 18

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> VH CDR3 according to IMGT

<400> 18

Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val

1 5 10

<210> 19

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> VL CDR1 according to IMGT

<400> 19

Gln Gly Ile Ser Gly Trp

1 5

<210> 20

<211> 3

<212> PRT

<213> Artificial Sequence

<220>

<223> VL CDR2 according to IMGT

<400> 20

Ala Ala Ser

1

<210> 21

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> VL CDR3 according to IMGT

<400> 21

Gln Gln Ala Asn Ser Phe Pro Pro Thr

1 5

<210> 22

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy chain variable region (VH), amino acid 20 (glutamic acid) to amino acid 136 (serine) of SEQ ID NO:7

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 23

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain variable region (VL), amino acid 23 (aspartic acid) to amino acid 130 (arginine) of SEQ ID NO: 8

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Gly Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105

Claims (98)

1. A method of treating urothelial cancer or bladder cancer in a human subject having liver metastasis, the method comprising administering to the subject having liver metastasis an effective amount of an antibody drug conjugate,

wherein the subject has received immune checkpoint inhibitor (CPI) therapy and

wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or More Monomethyl Auristatin E (MMAE) units, 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 a CDR of the heavy chain variable region shown 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 shown in SEQ ID NO. 23.

2. A method of treating urothelial cancer or bladder cancer in a human subject having a primary tumor site in the upper urinary tract, the method comprising administering to the subject having a primary tumor site in the upper urinary tract an effective amount of an antibody drug conjugate,

wherein the subject has received immune checkpoint inhibitor (CPI) therapy and

wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or More Monomethyl Auristatin E (MMAE) units, 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 a CDR of the heavy chain variable region shown 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 shown in SEQ ID NO. 23.

3. A method of treating urothelial cancer or bladder cancer in a human subject, the method comprising administering to the subject an effective amount of an antibody drug conjugate,

wherein the subject has received an immune checkpoint inhibitor (CPI) therapy,

wherein the subject has progression or recurrence of the cancer during or after the CPI therapy, and

wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or More Monomethyl Auristatin E (MMAE) units, 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 a CDR of the heavy chain variable region shown 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 shown in SEQ ID NO. 23.

4. The method of any one of claims 1-3, wherein the subject has a response duration of at least or about 7 months after the treatment.

5. The method of any one of claims 1-3, wherein the subject has a duration of response after the treatment in the range of 5-9 months.

6. The method of claim 1, wherein the subject has a progression free survival of at least or about 4 months after the treatment.

7. The method of claim 2 or 3, wherein the subject has a progression free survival of at least or about 5 months after the treatment.

8. The method of claim 1, wherein the subject has a progression free survival in the range of 4 to 9 months after the treatment.

9. The method of claim 2 or 3, wherein the subject has a progression free survival in the range of 5 to 9 months after the treatment.

10. The method of claim 1, wherein the subject has a total survival of at least or about 9 months after the treatment.

11. The method of claim 2, wherein the subject has a total survival of at least or about 12 months after the treatment.

12. The method of claim 3, wherein the subject has a total survival of at least or about 11 months after the treatment.

13. The method of any one of claims 1-3, wherein the subject has a total survival in the range of 9-19 months after the treatment.

14. The method of any one of claims 1-3, wherein the population of subjects is treated by the method, and wherein the percentage of subjects in the treated population that have a complete response is at least or about 4%.

15. The method of any one of claims 1-3, wherein the population of subjects is treated by the method, and wherein the percentage of subjects in the treated population that have a partial response is at least or about 35%.

16. The method of claim 1, wherein the population of subjects is treated by the method, and wherein the total response rate in the treated population is at least or about 35%.

17. The method of claim 2, wherein the population of subjects is treated by the method, and wherein the total response rate in the treated population is at least or about 43%.

18. The method of claim 3, wherein the population of subjects is treated by the method, and wherein the total response rate in the treated population is at least or about 39%.

19. The method of any one of claims 1-3, wherein the population of subjects is treated by the method, and wherein the percentage of subjects with stable disease in the treated population is at least or about 30%.

20. The method of any one of claims 1-3, wherein the population of subjects is treated by the method, and wherein the median response duration in the treated population is at least or about 7 months.

21. The method of any one of claims 1-3, wherein the population of subjects is treated by the method, and wherein the duration of response in the treated population is in the range of 5 to 9 months.

22. The method of claim 1, wherein the population of subjects is treated by the method, and wherein the median progression-free survival in the treated population is at least or about 4 months.

23. The method of claim 1, wherein the population of subjects is treated by the method, and wherein the progression-free survival in the treated population is in the range of 4 to 9 months.

24. The method of claim 2 or 3, wherein the population of subjects is treated by the method, and wherein the median progression-free survival in the treated population is at least or about 5 months.

25. The method of claim 2 or 3, wherein the population of subjects is treated by the method, and wherein the progression-free survival in the treated population is in the range of 5 to 9 months.

26. The method of claim 1, wherein the population of subjects is treated by the method, and wherein the median total survival in the treated population is at least or about 9 months.

27. The method of claim 2, wherein the population of subjects is treated by the method, and wherein the median total survival in the treated population is at least or about 12 months.

28. The method of claim 3, wherein the population of subjects is treated by the method, and wherein the median total survival in the treated population is at least or about 11 months.

29. The method of any one of claims 1-3, wherein the population of subjects is treated by the method, and wherein the total survival in the treated population is in the range of 9 to 19 months.

30. The method of any one of claims 1 to 3, wherein the complete response rate of the population of subjects treated with the method is at least or about 4%.

31. The method of any one of claims 1 to 3, wherein the partial response rate of the population of subjects treated with the method is at least or about 35%.

32. The method of claim 1, wherein the total response rate of the population of subjects treated with the method is at least or about 35%.

33. The method of claim 2, wherein the total response rate of the population of subjects treated with the method is at least or about 43%.

34. The method of claim 3, wherein the total response rate of the population of subjects treated with the method is at least or about 39%.

35. The method of any one of claims 1 to 3, wherein the median response duration of the population of subjects treated with the method is at least or about 7 months.

36. The method of any one of claims 1 to 3, wherein the duration of the response of the population of subjects treated with the method is from 5 to 9 months.

37. The method of claim 1, wherein the median progression-free survival of the population of subjects treated with the method is at least or about 4 months.

38. The method of any one of claims 1 to 3, wherein the progression free survival of the population of subjects treated with the method is 4 to 9 months.

39. The method of claim 2 or 3, wherein the median progression-free survival of the population of subjects treated with the method is at least or about 5 months.

40. The method of claim 2 or 3, wherein the progression free survival of the population of subjects treated with the method is from 5 to 9 months.

41. The method of claim 1, wherein the median total survival of the population of subjects treated with the method is at least or about 9 months.

42. The method of claim 2, wherein the median total survival of the population of subjects treated with the method is at least or about 12 months.

43. The method of claim 3, wherein the median total survival of the population of subjects treated with the method is at least or about 11 months.

44. The method of any one of claims 1 to 3, wherein the total survival of the population of subjects treated with the method is 9 to 19 months.

45. The method of any one of claims 1 to 44, wherein the subject is a subject receiving platinum-based chemotherapy.

46. The method of any one of claims 1-45, wherein the cancer is urothelial cancer, and wherein the human subject has locally advanced or metastatic urothelial cancer.

47. The method of any one of claims 1 to 46, wherein the subject has one or more conditions selected from the group consisting of:

(i) Absolute Neutrophil Count (ANC) of not less than 1500/mm ³ ；

(ii) Platelet count is not less than 100X 10 ⁹ a/L;

(iii) Hemoglobin is not less than 9g/dL;

(iv) Serum bilirubin does not exceed an Upper Limit of Normal (ULN) of 1.5 times or 3 times ULN for patients with gilbert disease;

(v) CrCl is not less than 30mL/min, and

(vi) Alanine aminotransferase and aspartate aminotransferase are no more than 3 times ULN.

48. The method of claim 47, wherein the subject has all of conditions (i) to (vi) of claim 47.

49. The method of claim 47 or 48, wherein the CrCl is measured by 24 hour urine collection or estimated by the Cockcroft-Gault standard.

50. The method of any one of claims 1-49, wherein the subject has no more than grade 2 sensory or motor neuropathy.

51. The method of any one of claims 1-50, wherein the subject does not have active central nervous system metastasis.

52. The method of any one of claims 1-51, wherein the subject does not have uncontrolled diabetes.

53. The method of claim 52, wherein the uncontrolled diabetes is determined by hemoglobin A1c (HbA 1 c) being no less than 8% or HbA1c being between 7% and 8% with associated diabetes symptoms that are not otherwise explained.

54. The method of claim 53, wherein the associated diabetic symptoms comprise or consist of: polyuria, polydipsia, or both polyuria and polydipsia.

55. The method of any one of claims 1 to 54, wherein the CPI therapy is a therapy of an inhibitor of programmed death receptor-1 (PD-1).

56. The method of any one of claims 1 to 54, wherein the CPI therapy is a therapy of a programmed death-ligand 1 (PD-L1) inhibitor.

57. The method of claim 55, wherein the PD-1 inhibitor is nivolumab or pamphlet Li Zhushan antibody.

58. The method of claim 56, wherein the PD-L1 inhibitor is selected from the group consisting of: alemtuzumab, avistuzumab and Dewaruzumab.

59. The method of any one of claims 1 to 58, wherein 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

Wherein 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.

60. The method of any one of claims 1 to 58, wherein 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

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.

61. The method of any one of claims 1 to 60, 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.

62. The method of any one of claims 1 to 61, wherein the antibody comprises: a heavy chain comprising an amino acid sequence in the range of amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID No. 7; and a light chain comprising an amino acid sequence in the range of amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO. 8.

63. The method of any one of claims 1 to 61, wherein the antigen binding fragment is a Fab, F (ab') 2, fv, or scFv.

64. The method of any one of claims 1 to 62, wherein the antibody is a fully human antibody.

65. The method of any one of claims 1 to 62 and 64, wherein the antibody is IgG1 and the light chain is a kappa light chain.

66. The method of any one of claims 1 to 65, wherein the antibody or antigen-binding fragment thereof is recombinantly produced.

67. The method of any one of claims 1 to 66, wherein the antibody or antigen binding fragment is conjugated to each unit of MMAE via a linker.

68. The method of claim 67, wherein the linker is an enzymatically cleavable linker, and wherein the linker forms a bond with a sulfur atom of the antibody or antigen binding fragment thereof.

69. The method of claim 67 or 68, wherein said linker is of the formula: -Aa-Ww-Yy-; wherein-A-is an extension subunit, a is 0 or 1; -W-is an amino acid unit, W is an integer ranging from 0 to 12; and-Y-is a spacer unit, Y is 0, 1 or 2.

70. The method of claim 69, wherein the extension subunit has the structure of formula (1); the amino acid unit is valine-citrulline; and the spacer unit is a PAB group comprising the structure of formula (2):

71. the method of claim 69 or 70, wherein the extension subunit forms a bond with a sulfur atom of the antibody or antigen binding fragment thereof; and wherein the spacer unit is linked to MMAE via a carbamate group.

72. The method of any one of claims 1 to 71, wherein the ADC comprises 1 to 20 MMAE units per antibody or antigen-binding fragment thereof.

73. The method of any one of claims 1 to 72, wherein the ADC comprises 1 to 10 MMAE units per antibody or antigen-binding fragment thereof.

74. The method of any one of claims 1 to 73, wherein the ADC comprises 2 to 8 MMAE units per antibody or antigen-binding fragment thereof.

75. The method of any one of claims 1 to 74, wherein the ADC comprises 3 to 5 MMAE units per antibody or antigen-binding fragment thereof.

76. The method of any one of claims 1 to 73, wherein the ADC has the structure:

wherein L-represents the antibody or antigen-binding fragment thereof, and p is 1 to 10.

77. The process of claim 76 wherein p is 2 to 8.

78. The method of claim 76 or 77, wherein p is 3 to 5.

79. The method of any one of claims 76 to 78, wherein p is 3 to 4.

80. The method of any one of claims 77 to 79, wherein p is about 4.

81. The method of any one of claims 76 to 79, wherein the effective amount of the antibody drug conjugate has an average p-value of about 3.8.

82. The method of any one of claims 1 to 81, wherein the ADC is administered at a dose of about 1 to about 10mg/kg of the subject body weight, about 1 to about 5mg/kg of the subject body weight, about 1 to about 2.5mg/kg of the subject body weight, or about 1 to about 1.25mg/kg of the subject body weight.

83. The method of any one of claims 1-82, wherein the ADC is administered at a dose of about 0.25mg/kg, about 0.5mg/kg, about 0.75mg/kg, about 1.0mg/kg, about 1.25mg/kg, about 1.5mg/kg, about 1.75mg/kg, about 2.0mg/kg, about 2.25mg/kg, or about 2.5mg/kg of the subject's body weight.

84. The method of any one of claims 1-83, wherein the ADC is administered at a dose of about 1mg/kg of body weight of the subject.

85. The method of any one of claims 1-83, wherein the ADC is administered at a dose of about 1.25mg/kg of body weight of the subject.

86. The method of any one of claims 1-85, wherein the ADC is administered by Intravenous (IV) injection or infusion.

87. The method of any one of claims 1-86, wherein the ADC is administered by IV injection or infusion three times every four week period.

88. The method of any one of claims 1-87, wherein the ADC is administered by IV injection or infusion on days 1, 8, and 15 of a four week cycle.

89. The method of any one of claims 1-88, wherein the ADC is administered by IV injection or infusion of about 30 minutes three times every four week period.

90. The method of any one of claims 1-89, wherein the ADC is administered by IV injection or infusion for about 30 minutes on days 1, 8, and 15 of every four week cycle.

91. The method of any one of claims 1-90, wherein the ADC is formulated in a pharmaceutical composition comprising L-histidine, polysorbate-20 (TWEEN-20), and trehalose dehydrate.

92. The method of any one of claims 1-91, wherein the ADC is formulated in a pharmaceutical composition comprising about 20mM L-histidine, about 0.02% (w/v) TWEEN-20, about 5.5% (w/v) trehalose dihydrate and hydrochloride, and wherein the pH of the pharmaceutical composition at 25 ℃ is about 6.0.

93. The method of any one of claims 1-91, wherein the ADC is formulated in a pharmaceutical composition comprising about 9mM histidine, about 11mM histidine monohydrochloride, about 0.02% (w/v) TWEEN-20 and about 5.5% (w/v) trehalose dihydrate, and wherein the pH of the pharmaceutical composition at 25 ℃ is about 6.0.

94. The method of any one of claims 1 to 93, wherein the ADC is an enrolment Shan Kangwei statin (EV) or a biological analogue thereof, wherein the EV is administered at a dose of about 1.25mg/kg of the subject's body weight, and wherein the dose is administered by IV injection or infusion for about 30 minutes on days 1, 8 and 15 of each four week cycle.

95. The method of any one of claims 1-94, wherein the population of subjects has a complete response after the treatment.

96. The method of any one of claims 1-94, wherein the population of subjects has a partial response after the treatment.

97. The method of any one of claims 1-94, wherein the population of subjects has a complete response or a partial response after the treatment.

98. The method of any one of claims 1-94, wherein the population of subjects has stable disease after the treatment.