Method of treating muscle-invasive urothelium cancer or muscle-invasive bladder cancer with antibody drug conjugate (ADC) that binds to 191P4D12 protein

Abstract

Provided herein are methods of treating urinary tract epithelial cancer or bladder cancer with an antibody drug conjugate (ADC) that binds to 191P4D12 protein (connexin-4).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of the Chinese invention patent application filed on January 4, 2023, with the invention name “Method for treating muscle-invasive urothelial carcinoma or muscle-invasive bladder cancer with an antibody-drug conjugate (ADC) bound to 191P4D12 protein” and application number 202380023405.4.

[0003] This application claims the benefit of U.S. Application No. 63 / 296,831, filed on January 5, 2022, and U.S. Application No. 63 / 304,136, filed on January 28, 2022, the disclosures of each of which are incorporated herein by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a computer-readable sequence listing, which has been submitted with this application in XML file format, and its entire content is incorporated herein by reference. The sequence listing XML file submitted with this application is named "14369-279-228_SequenceListing.xml", created on December 15, 2022, and is 34,925 bytes in size. Field of the Invention

[0006] Provided herein are methods for treating urothelial carcinoma or bladder cancer, such as muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC), using an antibody drug conjugate (ADC) that binds to the 191P4D12 protein (connexin-4). Background Art

[0007] 191P4D12 (also known as connexin-4) is a 66 kDa type I transmembrane protein belonging to the connexin family of adhesion molecules. It consists of an extracellular domain (ECD) containing three immunoglobulin (Ig)-like domains, a transmembrane helix, and an intracellular region (Takai et al., Annu Rev Cell Dev Biol (2008); 24: 309-42). Connexin is believed to mediate Ca2+ binding via both homophilic and heterophilic trans-interactions at adherens junctions. 2+Independent cell-cell adhesion, where it can recruit cadherins and regulate cytoskeletal rearrangements at the adherens junctions (Rikitake et al., Cell Mol Life Sci (2008); 65(2): 253-63.). Connexin-4 has low sequence identity with other connexin family members, and in the ECD, the sequence identity ranges between 25%-30% (Reymond et al., Biol Chem (2001); 276(46): 43205-15).

[0008] The three Ig-like domains in the ECD of connexin-4 are called V, C1 and C2. The C1 domain is responsible for cis interactions (homodimerization), while the V domain of most connexin molecules contributes to trans interactions and cell-cell adhesion (Mandai et al., Curr Top Dev Biol (2015); 112: 197-231; Takai et al., Nat Rev Mol Cell Biol (2008); 9 (8): 603-15.).

[0009] Connexin-4 was originally identified bioinformatically and cloned from human trachea (Reymond et al., J Biol Chem (2001) 276(46):43205-15.). Connexin-4 was identified as significantly upregulated in urothelial carcinoma using suppression subtractive hybridization on a pool of urothelial carcinoma specimens. Characterization of expression in multiple tumor specimens at the RNA level and by immunohistochemistry (IHC) also showed that higher levels of connexin-4 are present in breast, pancreatic, lung, and other cancers (Challita-Eid et al., Cancer Res (2016); 76(10):3003-13.).

[0010] Connexin-4 has been found to be expressed in a variety of cancers, particularly urothelial, breast, lung, pancreatic and ovarian cancers. Higher expression levels are associated with disease progression and / or poor prognosis (Fabre-Lafay et al., BMC Cancer (2007); 7:73).

[0011] Urothelial carcinoma

[0012] Urothelial carcinoma is the most common form of cancer in the genitourinary tract and a leading cause of morbidity and mortality. Approximately 151,000 new cases of bladder cancer are diagnosed in Europe each year, with bladder cancer being the most common primary site of urothelial cancer (Ervik 2016). The U.S. National Cancer Institute estimates that more than 79,000 new cases of bladder cancer were diagnosed in 2017, with more than 16,800 deaths from the disease (National Cancer Institute 2018).

[0013] Bladder cancer

[0014] Bladder cancer accounts for approximately 5% of all new cancer cases in the United States in men (the fifth most common neoplasm) and 3% in women (the eighth most common neoplasm). The incidence is slowly increasing with the increasing ageing 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 in the United States for both men and women. An estimated 17,980 deaths (13,050 in men and 4,930 in women) occur each year from bladder cancer, accounting for 3% of cancer-related deaths. Bladder cancer incidence and mortality rates increase substantially with age and will become an increasing problem as the population becomes more elderly. Globally, approximately 580,000 people will be diagnosed with bladder cancer in 2020, and bladder cancer will cause approximately 210,000 deaths worldwide.

[0015] Most bladder cancers recur in the bladder. Bladder cancer is managed via 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 ways to eliminate cancer, but have an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that benefit bladder cancer patients.

[0016] There is a significant need for additional treatment approaches for urothelial carcinoma and bladder cancer. These approaches include the use of antibodies and antibody drug conjugates as therapeutic modalities.

[0017] Muscle-invasive bladder cancer (MIBC) and muscle-invasive urothelial carcinoma (MIUC)

[0018] Muscle-invasive urothelial carcinoma (MIUC) includes cancers that originate in all parts of the urinary tract (renal pelvis or upper urinary tract, bladder, and lower urinary tract (urethra)). Muscle-invasive bladder cancer (MIBC) refers to cancer of the bladder, excluding the upper and lower urinary tract. Up to 25% of all patients diagnosed with urothelial cancer have muscle-invasive disease, and these patients are at considerable risk for progression or metastasis. The five-year mortality rate for muscle-invasive bladder cancer (MIBC) is approximately 69% (American Cancer Society (ACS), Survival rates for bladder cancer, 2019, www.cancer.org / cancer / bladder-cancer / detection-diagnosis-staging / survival-rates.html. Accessed: February 23, 2018). Prognosis and recurrence vary depending on the stage of disease as well as other prognostic features, including lymph node involvement, lymphovascular invasion, tumor stage, presence of variant histology, and molecular subtype (Lotan et al., J. Clin. Oncol., 2005, 23(27):6533-9; Karakiewicz et al., J Urol., 2006, 176(4 Pt 1):1354-61; Choi 2014).

[0019] The treatment of MIBC is complex and requires multidisciplinary collaboration among surgeons, radiotherapists, and medical oncologists (Aragon-Ching et al., Am Soc Clin Oncol Educ Book, 2018, 38: 307-18). Over the past 20 years, cisplatin-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy (RC) and lymph node dissection has become the SOC for MIBC, as evidenced by multiple randomized trials and a large meta-analysis, which was originally published by the Advanced Bladder Cancer Meta-Analysis Collaboration in 2003 and updated in 2005 (Advanced Bladder Cancer Meta-analysis, Eur Urol., 2005, 48(2): 202-5). The final version of the meta-analysis included 11 randomized trials with 3005 patients and compared platinum-based NAC (all but one trial involved cisplatin) plus definitive local therapy (cystectomy or radiation therapy) with the same local therapy alone. Results showed a significant benefit for patients receiving cisplatin-based combination NAC, with a 14% reduction in the risk of death (HR, 0.86; 95% confidence interval [CI], 0.77-0.95; P = .003), which translated into a 5% improvement in 5-year OS and a 9% improvement in disease-free survival (DFS). For patients receiving cisplatin-based NAC, the 5-year OS was 50%, compared with 45% for patients receiving definitive local therapy alone. A survival benefit has not been shown for patients receiving carboplatin-based combinations. Recently, additional NAC regimens, including accelerated or dose-dense MVAC and combinations of gemcitabine (gemcitabine) and cisplatin (GC), have been investigated with the goal of improving patient tolerability and shortening treatment duration with comparable pathological response rates. Approximately 30% to 40% of patients achieve pCR rates, and 50% of patients achieve pathological downstaging with accelerated or dose-dense MVAC (Choueiri et al., J Clin Oncol., 2014, 32(18): 1889-94; Plimack et al., J Clin Oncol., 2014, 32(18): 1895-901).

[0020] Although NAC before RC is effective, more than 50% of patients still have residual high-risk disease (≥pT2) and have a poor prognosis. In addition, many patients are not suitable for surgery or cisplatin, and bladder-preserving strategies are increasingly considered not only as the best treatment alternative, but should also be part of the range of treatment options offered to patients at diagnosis (Aragon-Ching et al., Am Soc Clin Oncol Educ Book, 2018, 38:307-18). However, these studies have been limited to patients selected by biomarkers who are expected to respond well to neoadjuvant therapy.

[0021] In addition to chemotherapy, immunotherapy has been successfully used to treat locally advanced and metastatic bladder cancer and is entering the MIBC field as a monotherapy or in combination with chemotherapeutic agents. Two recent investigator-initiated studies aimed to obtain biomarker results and evaluate the efficacy of single-agent programmed cell death 1 / programmed cell death ligand 1 (PD1 / PDL1) inhibitors in patients with MIBC in the neoadjuvant setting. Both have published preliminary data. In the PURE-01 study, patients with T2T3bN0M0 disease, whether or not eligible for cisplatin, received 3 cycles of 200 mg pembrolizumab before RC (Necchi et al., J Clin Oncol., 2018, 36(34): 3353-60). Pembrolizumab can be safely administered, with a pCR rate of 42% in 50 treated patients, a pCR rate of 54.3% in patients with PDL1-positive disease, and a pathological downstaging rate of 65.7%. Immune-mediated adverse events (imAEs) were reported but did not delay planned surgery. Postoperative complications were similar to those reported in the literature and were related to open or robotic surgery. Neoadjuvant pembrolizumab did not affect intraoperative performance measures such as the number of lymph nodes resected, surgical margins, and operative time. In the ABACUS study, 69 patients with T2T4N0M0 MIBC who were not eligible for cisplatin received 2 cycles of 1200 mg of neoadjuvant atezolizumab before RC, and no significant safety issues were found (Powles et al., 2018, J Clin Oncol., 36(Suppl. 15):Abstract 4506).

[0022] pCR was observed in 29% of patients and 40% of those with PD-L1-positive disease, compared with only 16% in the PD-L1-negative cohort. A total of 39% of patients had downstage to non-muscle-invasive disease. Both studies lacked the long-term follow-up data needed to assess background PFS and OS after surgery in patients who received neoadjuvant immunotherapy.

[0023] There are currently no standard neoadjuvant or perioperative treatment options for patients with MIBC who are ineligible for cisplatin. Therefore, newer treatment approaches, including novel drug combination strategies, are needed for this patient population. Summary of the invention

[0024] Provided herein are methods for treating a variety of cancers in human subjects, such as urothelial carcinoma and bladder cancer, including cisplatin-ineligible subjects with muscle-invasive bladder cancer (MIBC) and muscle-invasive urothelial carcinoma (MIIUC), using antibody drug conjugates (ADCs) that bind 191P4D12.

[0025] Embodiment 1. A method for treating muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) in a human subject, the method comprising (a) administering to the subject an effective amount of an antibody drug conjugate (ADC); wherein the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE); wherein the subject is not suitable for treatment with cisplatin (cisplatin ineligible); and wherein the pathological complete response rate (pCRR) is at least 30%.

[0026] Embodiment 2. A method for treating muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) in a human subject, the method comprising administering to the subject an effective amount of an antibody drug conjugate (ADC); wherein the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE); wherein the subject is not suitable for treatment with cisplatin (cisplatin ineligible); and wherein the pathological downstaging rate (pDSR) is at least 50%.

[0027] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the method comprises administering 3 cycles of the ADC to the subject.

[0028] Embodiment 4. The method of any one of Embodiments 1 to 3, wherein the treatment of the cancer further comprises radical cystectomy and pelvic lymph node dissection (RC+PLND).

[0029] Embodiment 5. The method of Embodiment 4, wherein the human subject receives the RC+PLND about four (4) weeks to about twelve (12) weeks after administration of the ADC to the human subject.

[0030] Embodiment 6. The method of embodiment 1 or 2, further comprising (b) performing radical cystectomy and pelvic lymph node dissection (RC+PLND) on the subject.

[0031] Embodiment 7. The method of Embodiment 6, wherein (b) is performed from about four (4) weeks to about twelve (12) weeks after (a).

[0032] Embodiment 8. A method of treating muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) in a human subject, the method comprising: administering to the subject an effective amount of an antibody drug conjugate (ADC) for multiple cycles, wherein

[0033] (a) the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE);

[0034] (b) the subject is not suitable for treatment with cisplatin (cisplatin-ineligible); and

[0035] (c) the number of ADC treatment cycles administered to the cisplatin-ineligible subject is equal to or less than the number of cycles of standard of care (SOC) therapy used to treat cisplatin-eligible subjects with MIUC or MIBC.

[0036] Embodiment 9. The method of embodiment 8, wherein the SOC therapy for a cisplatin-appropriate subject comprises cisplatin.

[0037] Embodiment 10. The method of embodiment 8, wherein the SOC therapy for the cisplatin-appropriate subject comprises (i) methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC), or (ii) gemcitabine plus cisplatin.

[0038] Embodiment 11. The method of embodiment 8, wherein the SOC therapy for the cisplatin-appropriate subject comprises administration of a programmed cell death 1 (PD-1) or programmed cell death ligand 1 (PD-L1) inhibitor.

[0039] Embodiment 12. The method of any one of Embodiments 8 to 11, wherein the number of ADC treatment cycles administered to the cisplatin-ineligible subject is 3 or 4.

[0040] Embodiment 13. The method of any one of Embodiments 8 to 11, wherein the number of ADC treatment cycles administered to the cisplatin-ineligible subject is 3.

[0041] Embodiment 14. A method of administering neoadjuvant or perioperative therapy to treat muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) in a human subject, the method comprising: administering to the subject a plurality of cycles of an effective amount of an antibody drug conjugate (ADC), wherein

[0042] (a) the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE);

[0043] (b) the subject is not suitable for treatment with cisplatin (cisplatin-ineligible); and

[0044] (c) the subject is still suitable for radical cystectomy and pelvic lymph node dissection (RC+PLND) surgery after multiple treatment cycles with the ADC.

[0045] Embodiment 15. The method of embodiment 14, wherein the ADC is administered as neoadjuvant therapy prior to the surgery.

[0046] Embodiment 16. The method of embodiment 14, wherein the ADC is administered before and after the surgery.

[0047] Embodiment 17. A method of treating muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) in a human subject, the method comprising: administering to the subject a plurality of cycles of an effective amount of an antibody drug conjugate (ADC), wherein

[0048] (a) the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE);

[0049] (b) the subject is not suitable for treatment with cisplatin (cisplatin-ineligible); and

[0050] (c) the efficacy of treating the subject with the effective amount of the ADC is similar to the therapeutic efficacy observed for cisplatin-eligible patients treated with standard of care (SOC) therapy for treating cisplatin-eligible subjects with MIUC or MIBC.

[0051] Embodiment 18. The method of embodiment 17, wherein the efficacy of the treatment is at least as effective as the efficacy of the treatment observed for SOC in cisplatin-appropriate subjects.

[0052] Embodiment 19. The method of embodiment 17 or 18, wherein the measure of therapeutic efficacy is one or more of the following: pathological complete response rate (pCRR), pathological downstaging rate (pDSR), disease-free survival (DFS), event-free survival (EFS), overall survival (OS), progression-free survival (PFS) and duration of response (DoR).

[0053] Embodiment 20. The method of embodiment 19, wherein the pCRR of the cisplatin-ineligible subject is at least 30%.

[0054] Embodiment 21. The method of embodiment 19, wherein the pDSR of the cisplatin-ineligible subject is at least 50%.

[0055] Embodiment 22. The method of any one of Embodiments 18 to 21, wherein the SOC therapy for a cisplatin-appropriate subject comprises cisplatin.

[0056] Embodiment 23. The method of embodiment 22, wherein the SOC therapy for the cisplatin-appropriate subject comprises (i) methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC), or (ii) gemcitabine plus cisplatin.

[0057] Embodiment 24. The method of embodiment 18, wherein the SOC therapy for the cisplatin-appropriate subject comprises a programmed cell death 1 (PD-1) or programmed cell death ligand 1 (PD-L1) inhibitor.

[0058] Embodiment 25. The method of embodiment 24, wherein the PD-1 inhibitor is nivolumab or pembrolizumab.

[0059] Embodiment 26. The method of embodiment 24, wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0060] Embodiment 27. The method of any one of embodiments 1 to 26, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, the 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, the 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.

[0061] Embodiment 28. The method of any one of embodiments 1 to 27, 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

[0062] 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.

[0063] Embodiment 29. The method of any one of embodiments 1 to 27, 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

[0064] The antibody or its antigen-binding fragment comprises: CDR-H1 composed of the amino acid sequence SEQ ID NO: 16, CDR-H2 composed of the amino acid sequence SEQ ID NO: 17, CDR-H3 composed of the amino acid sequence SEQ ID NO: 18; CDR-L1 composed of the amino acid sequence SEQ ID NO: 19, CDR-L2 composed of the amino acid sequence SEQ ID NO: 20 and CDR-L3 composed of the amino acid sequence SEQ ID NO: 21.

[0065] Embodiment 30. The method of any one of embodiments 1 to 29, 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.

[0066] Embodiment 31. The method of any one of embodiments 1 to 30, 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.

[0067] Embodiment 32. The method of any one of embodiments 1 to 30, wherein the antigen binding fragment is Fab, F(ab')2, Fv or scFv.

[0068] Embodiment 33. The method of any one of embodiments 1 to 31, wherein the antibody is a fully human antibody.

[0069] Embodiment 34. The method of any one of Embodiments 1 to 31 and 33, wherein the antibody is IgG1 and the light chain is a kappa light chain.

[0070] Embodiment 35. The method of any one of embodiments 1 to 34, wherein the antibody or antigen-binding fragment thereof is recombinantly produced.

[0071] Embodiment 36. The method of any one of embodiments 1 to 35, wherein the antibody or antigen-binding fragment is coupled to each unit of MMAE via a linker.

[0072] Embodiment 37. The method of embodiment 36, 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.

[0073] Embodiment 38. The method of embodiment 36 or 37, wherein the linker has the 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 in the range of 0 to 12; and -Y- is a spacer unit, y is 0, 1 or 2.

[0074] Embodiment 39. The method of embodiment 38, wherein the stretcher unit has the structure of the following formula (1); the amino acid unit is valine-citrulline; and the spacer unit is a PAB group comprising the structure of the following formula (2):

[0075]

[0076] Embodiment 40. The method of embodiment 38 or 39, wherein the Stretcher unit 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.

[0077] Embodiment 41. The method of any one of Embodiments 1 to 40, wherein the ADC comprises 1 to 20 MMAE units per antibody or antigen-binding fragment thereof.

[0078] Embodiment 42. The method of any one of Embodiments 1 to 41, wherein the ADC comprises 1 to 10 MMAE units per antibody or antigen-binding fragment thereof.

[0079] Embodiment 43. The method of any one of Embodiments 1 to 42, wherein the ADC comprises 2 to 8 MMAE units per antibody or antigen-binding fragment thereof.

[0080] Embodiment 44. The method of any one of Embodiments 1 to 43, wherein the ADC comprises 3 to 5 MMAE units per antibody or antigen-binding fragment thereof.

[0081] Embodiment 45. The method of any one of Embodiments 1 to 42, wherein the ADC has the following structure:

[0082]

[0083] wherein L- represents the antibody or antigen-binding fragment thereof, and p is 1 to 10.

[0084] Embodiment 46. The method of Embodiment 45, wherein p is 2 to 8.

[0085] Embodiment 47. The method of Embodiment 45 or 46, wherein p is 3 to 5.

[0086] Embodiment 48. The method of any one of Embodiments 45 to 47, wherein p is 3 to 4.

[0087] Embodiment 49. The method of any one of Embodiments 45 to 48, wherein p is about 4.

[0088] Embodiment 50. The method of any one of Embodiments 45 to 48, wherein the effective amount of the antibody drug conjugate has a mean p-value of about 3.8.

[0089] Embodiment 51. The method of any one of Embodiments 1 to 50, wherein the ADC is formulated as a pharmaceutical composition comprising L-histidine, polysorbate-20 (TWEEN-20), and trehalose dehydrate.

[0090] Embodiment 52. The method of any one of Embodiments 1 to 51, wherein the ADC is formulated as 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.

[0091] Embodiment 53. The method of any one of Embodiments 1 to 51, wherein the ADC is formulated as 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.

[0092] Embodiment 54. The method of any one of Embodiments 1 to 53, wherein the ADC has the following structure:

[0093]

[0094] wherein L- represents the antibody or antigen-binding fragment thereof and p is from about 3 to about 4, the antibody comprising a heavy chain comprising an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7; and a light chain comprising an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8, wherein the ADC is administered at a dose of about 1.25 mg / kg body weight of the subject, and wherein the dose is administered by IV infusion on Days 1 and 8 of each three-week cycle.

[0095] Embodiment 55. The method of any one of Embodiments 1 to 54, wherein the subject has cT2-T4aN0M0 stage MIBC.

[0096] Embodiment 56. The method of any one of embodiments 1 to 55, wherein the subject is considered ineligible for cisplatin if one or more of the following criteria are met: (a) GFR <60 mL / min but ≥30 mL / min, (wherein the GFR is measured by Cockcroft-Gault criteria, Modification of Diet in Renal Disease (MDRD) equation, or 24-hour urine collection); (b) ECOG performance status of 2; (c) NCI CTCAE version 4.03 ≥ grade 2 hearing loss; and (d) NYHA class III heart failure.

[0097] Embodiment 57. The method of any one of Embodiments 1 to 56, wherein the subject has not previously received systemic treatment, chemoradiotherapy, and / or radiotherapy for MIBC.

[0098] Embodiment 58. The method of any one of Embodiments 1 to 57, wherein the subject has not received an immune checkpoint inhibitor (CPI).

[0099] Embodiment 59. The method of embodiment 58, wherein the CPI is a programmed cell death 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor.

[0100] Embodiment 60. The method of any one of Embodiments 1 to 59, wherein the subject has not received a CD137 agonist, a CTLA-4 inhibitor, or an OX-40 agonist.

[0101] Embodiment 61. The method of any one of Embodiments 1 to 60, wherein the effective amount of the ADC is about 1 to about 10 mg / kg of subject body weight, about 1 to about 5 mg / kg of subject body weight, about 1 to about 2.5 mg / kg of subject body weight, or about 1 to about 1.25 mg / kg of subject body weight.

[0102] Embodiment 62. The method of any one of Embodiments 1 to 60, wherein the effective amount of the ADC is 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.

[0103] Embodiment 63. The method of any one of Embodiments 1 to 60, wherein said effective amount of said ADC is about 1 mg / kg body weight of the subject.

[0104] Embodiment 64. The method of any one of Embodiments 1 to 60, wherein the effective amount of ADC is about 1.25 mg / kg body weight of the subject.

[0105] Embodiment 65. The method of any one of Embodiments 1 to 60, wherein in (a), the effective amount of the ADC is administered to the subject on Day 1 and Day 8 of each 3-week cycle for a total of 3 cycles.

[0106] Embodiment 66. The method of Embodiment 65, wherein said effective amount of said ADC is about 1.25 mg / kg body weight of the subject.

[0107] Embodiment 67. The method of Embodiment 4 or 5, wherein in (a), the effective amount of the ADC is administered to the subject on Day 1 and Day 8 of each 3-week cycle for a total of 3 cycles.

[0108] Embodiment 68. The method of any one of Embodiments 4, 5 or 67, wherein the method further comprises (b) administering to the subject the effective amount of the ADC on Day 1 and Day 8 of each 3-week cycle, for a total of 6 cycles, approximately 8 weeks after the subject receives the RC+PLND.

[0109] Embodiment 69. The method of Embodiment 67 or 68, wherein said effective amount of said ADC is about 1.25 mg / kg body weight of the subject.

[0110] Embodiment 70. The method of embodiment 6 or 7, wherein in (a), the effective amount of the ADC is administered to the subject on day 1 and day 8 of each 3-week cycle for a total of 3 cycles.

[0111] Embodiment 71. The method of any one of Embodiments 5, 7 or 70, further comprising (c) administering to the subject the effective amount of the ADC on day 1 and day 8 of each 3-week cycle, about 8 weeks after (b), for a total of 3 cycles.

[0112] Embodiment 72. The method of Embodiment 70 or 71, wherein said effective amount of said ADC is about 1.25 mg / kg body weight of the subject.

[0113] Embodiment 73. The method of any one of Embodiments 8 to 64, wherein said effective amount of said ADC is administered to said human subject on Day 1 and Day 8 of a 3 week cycle for a total of 3 cycles.

[0114] Embodiment 74. The method of any one of Embodiments 1 to 73, wherein the ADC is administered by intravenous (IV) injection or infusion.

[0115] Embodiment 75. The method of any one of Embodiments 1 to 74, wherein the cancer is muscle invasive urothelial carcinoma (MIUC).

[0116] Embodiment 76. The method of any one of Embodiments 1 to 74, wherein the cancer is muscle invasive bladder cancer (MIBC).

[0117] Embodiment 77. The method of any one of Embodiments 1 to 76, wherein the ADC is Enfortumab vedotin (EV).

[0118] Embodiment 78. The method of any one of Embodiments 1 to 77, wherein the overall survival of the subject is extended by at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, or at least 12 months.

[0119] Embodiment 79. The method of any one of Embodiments 1 to 78, wherein the ADC is administered as a monotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figures 1A-1E Depicted are the nucleotide and amino acid sequences of connexin-4 protein ( Figure 1A ), the heavy chain of Ha22-2(2.4)6.1 ( Figure 1B ) and light chain ( Figure 1C ) and the heavy chain of Ha22-2(2.4)6.1 ( Figure 1D ) and light chain ( Figure 1E )'s amino acid sequence.

[0121] Figure 2 Describes the overall study design of the clinical study described in Section 6.1. TURBT, transurethral resection of bladder tumor.

[0122] Figure 3 The European Organisation for Research and Treatment (EORTC) Core Quality of Life (QLQ-C-30) assessment (EORTC-QLQ-C-30, current version, version 3) was depicted as described in Section 6.1.

[0123] Figure 4 The EuroQol-5 dimensions (EQ-5D-5L) described in Section 6.1 are depicted.

[0124] Figure 5 Depicts the MIUC disease stages described in Section 6.2 with respect to the anatomy of the bladder and pelvic region ( Figure 5 , T4a, and T4b) (see, e.g., www.cancer.org / cancer / bladder-cancer / detection-diagnosis-staging (staging.html).

[0125] Figure 6 Depicts the event-free survival (EFS) observed in the clinical studies described in Section 6.2: MIBC EVMono, subjects with muscle-invasive bladder cancer treated with enrofloxacin monotherapy.

[0126] Figure 7Describes the overall study design of the clinical study described in Section 6.3. TURBT, transurethral resection of bladder tumor. DETAILED DESCRIPTION

[0127] 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.

[0128] 5.1 Definitions

[0129] 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 the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001); Current Protocols in Molecular Biology (Ausubel et al., eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering, Vols. 1 and 2 (Kontermann and Dübel eds., 2nd ed. 2010).

[0130] Unless otherwise defined herein, the technical and scientific terms used in this specification shall 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.

[0131] 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, the functional fragments referring to portions of antibody heavy or light chain polypeptides that retain 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 Fragment, F(ab′) 2Fragments, 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, for example, 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 can 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 antibodies can be agonistic antibodies or antagonistic antibodies.

[0132] 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.

[0133] An "antigen" is a structure to which an antibody can selectively bind. A 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, an antigen is associated with a cell, such as present on or in a cell (e.g., a cancer cell).

[0134] 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.

[0135] 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 impart its specificity and affinity to the antigen (e.g., CDR). As used herein, "antigen binding fragment" includes "antibody fragments" that contain a portion of an intact antibody, such as the antigen binding region or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') and 2 and Fv fragments; diabodies and di-diabodies (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.

[0136] The term "binds" or "binding" refers to interactions 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. A complex may also include the binding 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 dissociation rate (k) of a binding molecule (e.g., an antibody) from a monovalent antigen is the affinity of the antibody or functional fragment for the epitope. off ) and binding rate (k on ) ratio (k off / k on ) is the dissociation constant K D , which is negatively correlated with affinity. K D The lower the value, the higher the antibody affinity. D The value varies for different complexes of antibody and antigen and depends on the kon With k off Both. The dissociation constant K of the antibodies provided herein D Can use any method provided herein or any other method well-known to those skilled in the art to measure.The affinity at one binding site cannot always reflect the true interaction intensity between antibody and antigen.When a complex antigen (e.g., a multivalent antigen) containing multiple, repeated antigenic determinants contacts an antibody containing multiple binding sites, the interaction of antibody and antigen at one site will increase the reaction probability at a second site.The intensity of this type of multiple interactions between a multivalent antibody and an antigen is called avidity.

[0137] In conjunction with the antibodies or antigen-binding fragments thereof described herein, terms such as "bind to," "that specifically bind to," and similar terms are also used interchangeably herein to 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, for example, as determined by 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 measuring the binding of the assay molecule compared 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 measured 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 sufficient affinity so that the binding molecule can be used, for example, as a diagnostic agent targeting the antigen. In certain embodiments, the antibody or antigen-binding fragment that binds to the antigen has a dissociation constant (K) less than or equal to the following: D ): 1000nM, 800nM, 500nM, 250nM, 100nM, 50nM, 10nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM or 0.1nM. In certain embodiments, the antibody or antigen-binding fragment binds to an epitope of an antigen that is conserved between antigens from different species (e.g., between humans and cynomolgus monkey species).

[0138] "Binding affinity" generally refers to the overall 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 expressed by the dissociation constant (K D ) is represented. 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 to achieve the purposes of this disclosure. Specific illustrative embodiments include the following. In one embodiment, "K D ” or “K D The "value" can be measured by assays known in the art, such as by binding assays. D It can be measured by RIA, for example, using a Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81). D or K D The value 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.

[0139] 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).

[0140] In certain embodiments, the 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 include 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 at least one or more variable regions that are substantially all, 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.

[0141] In certain embodiments, the 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 whose amino acid sequence corresponds to the amino acid sequence of an antibody produced by a human and / or has been made using any technology 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 a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227: 381; Marks et al., 1991, J. Mol. Biol. 222: 581) and yeast display libraries (Chao et al., 2006, Nature Protocols 1: 755-68). Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boemer 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., a 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 TMTechnology). For human antibodies generated via human B cell hybridoma technology, see also, e.g., Li et al., 2006, Proc. Natl. Acad. Sci. USA, 103: 3557-62.

[0142] In certain embodiments, the antibody or antigen-binding fragment may comprise 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 cows) of human immunoglobulin genes (see, e.g., Taylor, LD et al. (1992) Nucl. Acids Res. 20: 6287-6295) or antibodies prepared, expressed, generated or isolated by any other means 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, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US 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.

[0143] 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 comprising 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 use in the present disclosure can be prepared by the hybridoma method first described by Kohler et al., 1975, Nature 256:495, or can be prepared using recombinant DNA methods in bacteria or eukaryotic animal 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).

[0144] 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 its 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., eds., 5th ed. 2001).

[0145] 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.

[0146] The term "variable region", "variable domain", "V region" or "V domain" refers to a portion of the light or heavy chain of an antibody, which is usually located at the amino terminus of the light or heavy chain, 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, more variable (e.g., extremely variable) region, called a "hypervariable region", each 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 loops connecting the β-folded structure and in some cases form a part of the β-folded structure. The hypervariable regions in each chain are tightly bound together by the FRs with the hypervariable regions from the other chains, promoting the formation of the antigen binding site of the antibody (see, e.g., 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 sequence between different antibodies. In a specific embodiment, the variable region is a human variable region.

[0147] 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 after residue 52 (residue 52a, according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). 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.

[0148] 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 α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu). 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.

[0149] 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 κ (kappa) or λ (lambda).

[0150] 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.

[0151] The CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems. For example, the 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 loops (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 is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). 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 Vol. 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 specific to immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complex (MHC) of humans and other vertebrates. Herein, CDRs are referred to according to the amino acid sequence and the position within the light or heavy chain. Since the "position" of CDRs within the immunoglobulin variable domain structure is conserved between species and is present in structures called loops, CDR and framework residues are easily identified by using a numbering system that aligns the variable domain sequences according to structural features. This information can be used to transplant and replace CDR residues from an immunoglobulin from one species into an acceptor framework typically from a human antibody. 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.

[0152] Table 1

[0153]

[0154] 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 of a given antibody, such as a variable region, and individual CDRs (such as CDR-H1, CDR-H2) or its region of an 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 Kabat, Chothia or Contact methods. In other cases, the specific amino acid sequence of a CDR is provided.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] In this article, 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. Therefore, the composition of the complete antibody may include an antibody population in which all K447 residues are removed, an antibody population in which the K447 residue is not 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 evaluated 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.

[0159] 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., bending, twisting, turning, or folding) to recognize and bind to the epitope.

[0160] The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear 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 the form of two or more related chains.

[0161] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopia, European Pharmacopia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0162] "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, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavoring agents, humectants, lubricants, fragrances, 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.

[0163] In one embodiment, each component is "pharmaceutically acceptable" in the sense that it is compatible with the other ingredients of the pharmaceutical formulation and suitable for use in contact with 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, e.g., 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.

[0164] The abbreviation "MMAE" refers to monomethyl auristatin E.

[0165] Unless the context indicates otherwise, a hyphen (-) indicates a point of attachment to a side molecule.

[0166] 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 mustard, 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.

[0167] 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 performed according to those substitutions set forth in Tables 2 and 3. For example, such changes include substitutions of any of isoleucine (I), valine (V) and leucine (L) for any other of these hydrophobic amino acids; substitutions of aspartic acid (D) for glutamic acid (E), and vice versa; substitutions of glutamine (Q) for asparagine (N), and vice versa; and substitutions of threonine (T) for serine (S), and vice versa. Other substitutions may also be considered conservative, depending on the environment of a particular amino acid and its role in the three-dimensional structure of a protein. 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 distinguishing feature of the amino acid residue is its charge, and the pK difference between the two amino acid residues is not significant. Still other changes can be considered "conservative" in certain circumstances (see, e.g., 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.

[0168] Table 2 Amino Acid Abbreviations

[0169] 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 Lvs Lysine V Val Valine A Ala Alanine D Asp Aspartic acid E Glu Glutamate G Gly Glycine

[0170] Table 3 Amino acid substitution or similarity matrix

[0171] Adapted from GCG software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix).

[0172] The higher the value, the more likely the substitution is to be found in the related native protein.

[0173]

[0174] The term "homology" or "homologous" is intended to mean the sequence similarity between two polynucleotides or between two polypeptides. Similarity can be determined by comparing the positions 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, for example, evaluating the similarity of the amino acids at the position according to Table 3. 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, comparison is performed using default parameters, examples of which are described below. 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.

[0175] 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.

[0176] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 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, wordlength=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, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To enable 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 that detects distant relationships between molecules (supra). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:1117. 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.

[0177] 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.

[0178] 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 enzymatically 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), auromycins, maytansinoids, ricin, ricin A chain, combrestatin, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxols, cisplatin, cc1065, ethidium bromide, Mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas aeruginosa exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitors and glucocorticoids and other chemotherapeutic agents, and radioactive isotopes such as At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 Or Bi 213 , P 32 and including Lu 177 The antibody can also be coupled to an anticancer prodrug activating enzyme that is capable of converting the prodrug into its active form.

[0179] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a binding molecule (eg, antibody) or pharmaceutical composition provided herein sufficient to produce a desired result.

[0180] 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.

[0181] "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.

[0182] 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 condition 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 management and improvement of the disease. The term "management" refers to the beneficial effects that a subject obtains from a therapy, which does not necessarily result in a cure of the disease.

[0183] 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).

[0184] 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.

[0185] As used herein, "locally advanced" cancer refers to cancer that has spread from where it started to nearby tissues or lymph nodes.

[0186] As used herein, "metastatic" cancer refers to cancer that has spread from where it started to different parts of the body.

[0187] As used herein, "nearly as effective" means that the treatment is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% as effective as SOC therapy.

[0188] 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.

[0189] As used in the disclosure and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0190] 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.

[0191] 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).

[0192] The term "variant" refers to a molecule that exhibits differences from a described type or norm, such as in a particular described protein (e.g. Figure 1A A protein having one or more different amino acid residues in corresponding positions of the 191P4D12 protein shown in FIG. 191P4D12. Analogs are examples of variant proteins. Splice isoforms and single nucleotide polymorphisms (SNPs) are other examples of variants.

[0193] 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 / generated 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, and 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.

[0194] 5.2 Methods for treating cancer

[0195] Urothelial carcinoma and bladder cancer, including muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC), are particularly difficult to treat diseases in patients who are not suitable for cisplatin. Typically, MIUC and MIBC patients who are not suitable for cisplatin are somewhat frail and suffer from multiple comorbidities in addition to urothelial carcinoma / bladder cancer. As described in the background section above, there are currently no standard neoadjuvant treatment options for MIBC or MIUC patients who are not suitable for cisplatin. Instead, these patients are usually treated directly with surgery. The present disclosure provides effective and safe neoadjuvant and perioperative methods for treating patients with urothelial carcinoma and / or bladder cancer, particularly patients with muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) and who are not suitable for cisplatin in this case.

[0196] For example, the results described herein demonstrate that, in some embodiments, the efficacy of treating a subject with an effective amount of ADC described herein is similar to the therapeutic efficacy observed for cisplatin-suitable patients (i.e., healthier patients) treated with standard of care (SOC) therapy for treating cisplatin-suitable subjects with MIUC or MIBC. The results described herein further demonstrate that, in certain embodiments, even after multiple cycles of treatment with the ADC described herein, the subject is still suitable for radical cystectomy and pelvic lymph node dissection (RC+PLND) surgery, i.e., the method has sufficient tolerance so that the subject remains healthy enough to receive surgery after neoadjuvant therapy. Finally, the results described herein further demonstrate that, in certain embodiments, the number of treatment cycles of the ADC described herein administered to a subject for whom cisplatin is not suitable may be equal to or less than the number of cycles of SOC therapy for treating cisplatin-suitable subjects (i.e., healthier subjects) with MIUC or MIBC. Prior to the results described herein, since this patient population has been shown to be difficult to treat historically, and there is currently no standard treatment for MIBC and MIBC patients for whom cisplatin is not suitable, there is considerable uncertainty as to whether the methods provided herein are effective and tolerable. Therefore, the levels of efficacy and safety achieved using the methods described herein are particularly remarkable and surprising.

[0197] 5.2.1 Methods of treating cancer in general and selected patients

[0198] Provided herein are methods of using antibody drug conjugates (ADCs) that bind 191P4D12 (connexin-4) to treat a variety of cancers in a subject, including a subject with urothelial carcinoma.

[0199] In one aspect, provided herein is a method for treating cancer in a human subject using an ADC that binds to 191P4D12 (connexin-4). In some embodiments, the cancer is muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC). In some embodiments, the method comprises administering an effective amount of an antibody drug conjugate (ADC) to the subject. In some embodiments, the ADC comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4). In some embodiments, the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of a cytotoxic agent or a cell growth inhibitor. In some embodiments, the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of an auristatin agent. In some embodiments, the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of a monomethyl auristatin E (MMAE). Section 5.3.2 provides additional examples of suitable ADCs that can be used in the methods disclosed herein. In some embodiments, the subject is not suitable for cisplatin treatment (cisplatin is not suitable). In some embodiments, the pathological down-stage rate (pDSR) is at least 50%. In some embodiments, the pathological down-stage rate (pDSR) is at least 50%. In some embodiments, an effective amount of ADC is administered to the subject on the 1st and 8th day of each 3-week cycle, for a total of 3 cycles. In some embodiments, the effective amount of ADC is about 1.25 mg / kg subject body weight. In some embodiments, the effective amount of ADC is about 1.0 mg / kg subject body weight.

[0200] In some embodiments, after the administering step, the subject receives radical cystectomy and pelvic lymph node dissection (RC+PLND) as part of cancer treatment (ie, the treatment of cancer also includes radical cystectomy and pelvic lymph node dissection (RC+PLND)). In some embodiments, the method comprises administering an effective amount of an antibody drug conjugate (ADC) to the subject for 2 or 3 cycles. In some embodiments, the method comprises administering an effective amount of an antibody drug conjugate (ADC) to the subject for 2 cycles. In some embodiments, the method comprises administering an effective amount of an antibody drug conjugate (ADC) to the subject for 3 cycles. In some embodiments, the subject suffers from urothelial carcinoma or bladder cancer. In some embodiments, the subject suffers from muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC). In some embodiments, the subject is not suitable for cisplatin treatment (cisplatin is not suitable).

[0201] In one aspect, the method comprises administering to a subject an effective amount of an antibody drug conjugate (ADC) for 3 cycles; wherein after the administering step, the subject undergoes radical cystectomy and pelvic lymph node dissection (RC+PLND) as part of cancer treatment; wherein the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE); wherein the subject has muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC); wherein the subject is not suitable for treatment with cisplatin (cisplatin-ineligible).

[0202] In one aspect, the method comprises (i) administering an effective amount of ADC to the subject on the 1st and 8th day of each 3-week cycle, wherein the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE); (ii) performing radical cystectomy and pelvic lymph node dissection (RC+PLND) on the subject; and (iii) administering an effective amount of ADC to the subject on the 1st and 8th day of each 3-week cycle for a total of 6 cycles approximately 8 weeks after step (ii); wherein the subject is not suitable for cisplatin treatment (cisplatin is not suitable). In some embodiments, the effective amount of ADC is 1.25 mg / kg. In some embodiments, in step (iii), an effective amount of ADC is administered for a total of 5 cycles. In some embodiments, in step (iii), an effective amount of ADC is administered for a total of 4 cycles. In some embodiments, in step (iii), an effective amount of ADC is administered for a total of 3 cycles. In some embodiments, in step (iii), an effective amount of ADC is administered for a total of 2 cycles. In some embodiments, in step (iii), an effective amount of the ADC is administered for a total of 1 cycle.

[0203] In one aspect, the method comprises (i) administering an effective amount of ADC to the subject on the 1st and 8th day of each 3-week cycle, wherein the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE), wherein after the administering step, the subject receives RC+PLND as part of cancer treatment; and (ii) administering an effective amount of ADC to the subject on the 1st and 8th day of each 3-week cycle after the subject receives RC+PLND, for a total of 6 cycles, about 8 weeks after the subject receives RC+PLND; wherein the subject is not suitable for cisplatin treatment (cisplatin is not suitable). In some embodiments, the effective amount of ADC is 1.25 mg / kg. In some embodiments, in step (ii), an effective amount of ADC is administered for a total of 5 cycles. In some embodiments, in step (ii), an effective amount of ADC is administered for a total of 4 cycles. In some embodiments, in step (ii), an effective amount of ADC is administered for a total of 3 cycles. In some embodiments, in step (ii), an effective amount of ADC is administered for a total of 2 cycles. In some embodiments, in step (ii), an effective amount of the ADC is administered for a total of 1 cycle.

[0204] In some embodiments of the methods provided herein, the human subject receives RC+PLND about four (4) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about five (5) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about six (6) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about seven (7) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about eight (8) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about nine (9) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about ten (10) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about eleven (11) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about four (4) to about eleven (11) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about four (4) to about ten (10) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about four (4) to about nine (9) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about four (4) to about eight (8) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about four (4) to about seven (7) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about four (4) to about six (6) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about four (4) to about five (5) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about six (6) weeks to about eight (8) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about seven (7) weeks to about nine (9) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about eight (8) weeks to about ten (10) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about nine (9) weeks to about eleven (11) weeks after the ADC is administered to the human subject.

[0205] In some embodiments of the methods provided herein, the human subject receives RC+PLND about four (4) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about five (5) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about six (6) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about seven (7) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about eight (8) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about nine (9) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about ten (10) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about eleven (11) weeks after the ADC is administered to the human subject. In some embodiments, the human subject receives RC+PLND about twelve (12) weeks after the ADC is administered to the human subject.

[0206] In some embodiments, the method further comprises step (b) performing radical cystectomy and pelvic lymph node dissection (RC+PLND) on the subject. In some embodiments, step (b) is performed about four (4) weeks to about twelve (12) weeks after step (a). In some embodiments, step (b) is performed about five (5) weeks to about twelve (12) weeks after step (a). In some embodiments, step (b) is performed about six (6) weeks to about twelve (12) weeks after step (a). In some embodiments, step (b) is performed about seven (7) weeks to about twelve (12) weeks after step (a). In some embodiments, step (b) is performed about eight (8) weeks to about twelve (12) weeks after step (a). In some embodiments, step (b) is performed about nine (9) weeks to about twelve (12) weeks after step (a). In some embodiments, step (b) is performed about ten (10) weeks to about twelve (12) weeks after step (a). In some embodiments, step (b) is performed about eleven (11) weeks to about twelve (12) weeks after step (a). In some embodiments, step (b) is performed about four (4) to about eleven (11) weeks after step (a). In some embodiments, step (b) is performed about four (4) to about ten (10) weeks after step (a). In some embodiments, step (b) is performed about four (4) to about nine (9) weeks after step (a). In some embodiments, step (b) is performed about four (4) to about eight (8) weeks after step (a). In some embodiments, step (b) is performed about four (4) to about seven (7) weeks after step (a). In some embodiments, step (b) is performed about four (4) to about six (6) weeks after step (a). In some embodiments, step (b) is performed about four (4) to about five (5) weeks after step (a). In some embodiments, step (b) is performed about six (6) weeks to about eight (8) weeks after step (a). In some embodiments, step (b) is performed about seven (7) weeks to about nine (9) weeks after step (a). In some embodiments, step (b) is performed about eight (8) weeks to about ten (10) weeks after step (a). In some embodiments, step (b) is performed about nine (9) weeks to about eleven (11) weeks after step (a).

[0207] In some embodiments of the methods provided herein, step (b) is performed about four (4) weeks after step (a). In some embodiments, step (b) is performed about five (5) weeks after step (a). In some embodiments, step (b) is performed about six (6) weeks after step (a). In some embodiments, step (b) is performed about seven (7) weeks after step (a). In some embodiments, step (b) is performed about eight (8) weeks after step (a). In some embodiments, step (b) is performed about nine (9) weeks after step (a). In some embodiments, step (b) is performed about ten (10) weeks after step (a). In some embodiments, step (b) is performed about eleven (11) weeks after step (a). In some embodiments, step (b) is performed about twelve (12) weeks after step (a).

[0208] In some embodiments of the methods provided herein, the pathological complete remission rate (pCRR) of the subject is at least 30%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 35%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 40%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 45%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 50%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 55%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 60%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 65%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 70%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 75%.

[0209] In some embodiments of the method provided herein, the subject population is treated by the method, and the pathological complete remission rate (pCRR) in the treated population is at least 30%. In some embodiments, the subject population is treated by the method, and the pathological complete remission rate (pCRR) in the treated population is at least 35%. In some embodiments, the subject population is treated by the method, and the pathological complete remission rate (pCRR) in the treated population is at least 40%. In some embodiments, the subject population is treated by the method, and the pathological complete remission rate (pCRR) in the treated population is at least 45%. In some embodiments, the subject population is treated by the method, and the pathological complete remission rate (pCRR) in the treated population is at least 50%. In some embodiments, the subject population is treated by the method, and the pathological complete remission rate (pCRR) in the treated population is at least 55%. In some embodiments, the subject population is treated by the method, and the pathological complete remission rate (pCRR) in the treated population is at least 60%. In some embodiments, the subject population is treated by the method, and the pathological complete remission rate (pCRR) in the treated population is at least 65%. In some embodiments, the subject population is treated by the method, and the pathological complete remission rate (pCRR) in the treated population is at least 70%. In some embodiments, the subject population is treated by the method, and the pathological complete remission rate (pCRR) in the treated population is at least 75%.

[0210] In some embodiments of the methods provided herein, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 30%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 35%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 40%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 45%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 50%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 55%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 60%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 65%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 70%. In some embodiments, the pathological complete response rate (pCRR) in the population of subjects treated with the ADC is at least 75%.

[0211] In some embodiments of the methods provided herein, the pathological downstaging rate (pDSR) of the subject is at least 50%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 55%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 60%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 65%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 70%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 75%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 80%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 85%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 90%.

[0212] In some embodiments of the method provided herein, the subject population is treated by the method, and the pathological down-stage rate (pDSR) in the treated population is at least 50%. In some embodiments, the subject population is treated by the method, and the pathological down-stage rate (pDSR) in the treated population is at least 55%. In some embodiments, the subject population is treated by the method, and the pathological down-stage rate (pDSR) in the treated population is at least 60%. In some embodiments, the subject population is treated by the method, and the pathological down-stage rate (pDSR) in the treated population is at least 65%. In some embodiments, the subject population is treated by the method, and the pathological down-stage rate (pDSR) in the treated population is at least 70%. In some embodiments, the subject population is treated by the method, and the pathological down-stage rate (pDSR) in the treated population is at least 75%. In some embodiments, the subject population is treated by the method, and the pathological down-stage rate (pDSR) in the treated population is at least 80%. In some embodiments, the subject population is treated by the method, and the pathological downstaging rate (pDSR) in the treated population is at least 85%. In some embodiments, the subject population is treated by the method, and the pathological downstaging rate (pDSR) in the treated population is at least 90%.

[0213] In some embodiments of the methods provided herein, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 50%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 55%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 60%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 65%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 70%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 75%.

[0214] In one aspect, 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 (ADC) for multiple cycles, wherein (a) the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof bound to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE); (b) the subject is not suitable for cisplatin treatment (cisplatin is not suitable); and (c) the number of ADC treatment cycles administered to the subject to whom cisplatin is not suitable is equal to or less than the number of cycles of the standard of care (SOC) therapy for the treatment of cisplatin-suitable subjects with MIUC or MIBC. In some embodiments, an effective amount of ADC is administered to the subject on the 1st and 8th day of each 3-week cycle, for a total of 3 cycles. In some embodiments, the effective amount of ADC is about 1.25 mg / kg of subject body weight. In some embodiments, the effective amount of ADC is about 1.0 mg / kg of subject body weight.

[0215] In one aspect, provided herein is a method for treating muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) in a human subject, the method comprising: administering to the subject multiple cycles of an effective amount of an antibody drug conjugate (ADC), wherein (a) the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE); (b) the subject is not suitable for cisplatin treatment (cisplatin-ineligible); and (c) the number of ADC treatment cycles administered to the cisplatin-ineligible subject is equal to or less than the number of cycles of standard of care (SOC) therapy used to treat cisplatin-eligible subjects with MIUC or MIBC.

[0216] In some embodiments of the methods provided herein, the SOC therapy for a subject to which cisplatin is suitable comprises cisplatin. In some embodiments, the SOC therapy for a subject to which cisplatin is suitable comprises (i) methotrexate, vinblastine, doxorubicin and cisplatin (MVAC), or (ii) gemcitabine plus cisplatin. In some embodiments, the SOC therapy for a subject to which cisplatin is suitable comprises methotrexate, vinblastine, doxorubicin and cisplatin (MVAC). In some embodiments, the SOC therapy for a subject to which cisplatin is suitable comprises gemcitabine plus cisplatin. In some embodiments, the SOC therapy for a subject to which cisplatin is suitable comprises administration of a programmed cell death 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor. In some embodiments, SOC therapy for a cisplatin-appropriate subject comprises: i. cisplatin; ii. methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC); iii. gemcitabine plus cisplatin; iv. administration of a programmed cell death 1 (PD-1) inhibitor; or v. administration of a programmed cell death ligand 1 (PD-L1) inhibitor.

[0217] In some embodiments of the methods provided herein, the number of ADC treatment cycles administered to a cisplatin-ineligible subject is 1, 2, 3, or 4. In some embodiments, the number of ADC treatment cycles administered to a cisplatin-ineligible subject is 3 or 4. In some embodiments, the number of ADC treatment cycles administered to a cisplatin-ineligible subject is 4. In some embodiments, the number of ADC treatment cycles administered to a cisplatin-ineligible subject is 3. In some embodiments, the number of ADC treatment cycles administered to a cisplatin-ineligible subject is 2. In some embodiments, the number of ADC treatment cycles administered to a cisplatin-ineligible subject is 1.

[0218] In some embodiments of the methods provided herein, the method further comprises performing radical cystectomy and pelvic lymph node dissection (RC+PLND) surgery on the subject. In some embodiments, RC+PLND is performed about four (4) weeks to about twelve (12) weeks after the ADC is applied to the human subject. In some embodiments, RC+PLND is performed about five (5) weeks to about twelve (12) weeks after the ADC is applied to the human subject. In some embodiments, RC+PLND is performed about six (6) weeks to about twelve (12) weeks after the ADC is applied to the human subject. In some embodiments, RC+PLND is performed about seven (7) weeks to about twelve (12) weeks after the ADC is applied to the human subject. In some embodiments, RC+PLND is performed about eight (8) weeks to about twelve (12) weeks after the ADC is applied to the human subject. In some embodiments, RC+PLND is performed about nine (9) weeks to about twelve (12) weeks after the ADC is applied to the human subject. In some embodiments, RC+PLND is performed about ten (10) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about eleven (11) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about eleven (11) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about ten (10) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about nine (9) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about eight (8) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about seven (7) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about six (6) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about five (5) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about six (6) weeks to about eight (8) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about seven (7) weeks to about nine (9) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about eight (8) weeks to about ten (10) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about nine (9) weeks to about eleven (11) weeks after the ADC is administered to the human subject.

[0219] In some embodiments, RC+PLND is performed about four (4) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about five (5) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about six (6) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about seven (7) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about eight (8) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about nine (9) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about ten (10) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about eleven (11) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about twelve (12) weeks after the ADC is administered to the human subject.

[0220] In one aspect, provided herein is a method for administering neoadjuvant or perioperative therapy to treat muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) in a human subject, the method comprising: administering to the subject an effective amount of an antibody drug conjugate (ADC) for multiple cycles, wherein (a) the ADC comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 (connexin-4) and a conjugate of one or more units of monomethyl auristatin E (MMAE); (b) the subject is not suitable for cisplatin treatment (cisplatin is not suitable); and (c) the subject is still suitable for radical cystectomy and pelvic lymph node dissection (RC+PLND) surgery after multiple treatment cycles with the ADC. In some embodiments, an effective amount of ADC is administered to the subject on the 1st and 8th day of each 3-week cycle, for a total of 3 cycles. In some embodiments, the effective amount of ADC is about 1.25 mg / kg of the subject's body weight. In some embodiments, the effective amount of ADC is about 1.0 mg / kg of the subject's body weight.

[0221] In some embodiments, the method further comprises performing radical cystectomy and pelvic lymph node dissection (RC+PLND) surgery on the subject. In some embodiments, ADC is administered as a neoadjuvant therapy before surgery. In some embodiments, ADC is administered before and after surgery. In some embodiments, ADC (i) is administered before surgery or (ii) is administered before and after surgery as a neoadjuvant therapy. In some embodiments, RC+PLND is performed about four (4) weeks to about twelve (12) weeks after ADC is administered to a human subject. In some embodiments, RC+PLND is performed about five (5) weeks to about twelve (12) weeks after ADC is administered to a human subject. In some embodiments, RC+PLND is performed about six (6) weeks to about twelve (12) weeks after ADC is administered to a human subject. In some embodiments, RC+PLND is performed about seven (7) weeks to about twelve (12) weeks after ADC is administered to a human subject. In some embodiments, RC+PLND is performed about eight (8) weeks to about twelve (12) weeks after ADC is administered to a human subject. In some embodiments, RC+PLND is performed about nine (9) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about ten (10) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about eleven (11) weeks to about twelve (12) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about eleven (11) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about ten (10) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about nine (9) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about eight (8) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about seven (7) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about six (6) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about four (4) to about five (5) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about six (6) weeks to about eight (8) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about seven (7) weeks to about nine (9) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about eight (8) weeks to about ten (10) weeks after the ADC is administered to the human subject.In some embodiments, RC+PLND is performed about nine (9) weeks to about eleven (11) weeks after administration of the ADC to the human subject.

[0222] In some embodiments, RC+PLND is performed about four (4) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about five (5) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about six (6) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about seven (7) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about eight (8) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about nine (9) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about ten (10) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about eleven (11) weeks after the ADC is administered to the human subject. In some embodiments, RC+PLND is performed about twelve (12) weeks after the ADC is administered to the human subject.

[0223] In some embodiments, the ADC is administered about one (1) week after surgery. In some embodiments, the ADC is administered about two (2) weeks after surgery. In some embodiments, the ADC is administered about three (3) weeks after surgery. In some embodiments, the ADC is administered about four (4) weeks after surgery. In some embodiments, the ADC is administered about five (5) weeks after surgery. In some embodiments, the ADC is administered about six (6) weeks after surgery. In some embodiments, the ADC is administered about two (2) months after surgery. In some embodiments, the ADC is administered about three (3) months after surgery. In some embodiments, the ADC is administered about four (4) months after surgery. In some embodiments, the ADC is administered about five (5) months after surgery. In some embodiments, the ADC is administered about six (6) months after surgery. In some embodiments, the ADC is administered about seven (7) months after surgery. In some embodiments, the ADC is administered about eight (8) months after surgery. In some embodiments, the ADC is administered about nine (9) months after surgery. In some embodiments, the ADC is administered about ten (10) months after surgery. In some embodiments, the ADC is administered about eleven (11) months after surgery. In some embodiments, the ADC is administered about twelve (12) months after surgery.

[0224] Standard of care (SOC) therapies for the treatment of muscle-invasive bladder cancer (MIIBC) include cisplatin-based neoadjuvant chemotherapy (NAC) (plus definitive local therapy (cystectomy or radiation)) (see, e.g., Advanced Bladder Cancer Meta-analysis, Eur Urol., 2005, 48(2):202-5), accelerated or dose-dense methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) (see, e.g., Choueiri et al., J Clin Oncol, 2014, 32(18):1889-94; Plimack et al., J Clin Oncol, 2014, 32(18):1895-901), gemcitabine plus cisplatin, programmed cell death 1 (PD-1) inhibitors (see, e.g., Necchi et al., J Clin Oncol, 2014, 32(18):1895-901), and cisplatin. Oncol., 2018, 36(34):3353-60) and programmed cell death ligand 1 (PD-L1) inhibitors (see, e.g., Powles et al., 2018, J Clin Oncol., 36(Suppl 15):Abstract 4506). Exemplary measures of therapeutic efficacy include pathological complete response rate (pCRR), pathological downstaging rate (pDSR), disease-free survival (DFS), event-free survival (EFS), overall survival (OS), progression-free survival (PFS) and / or duration of response (DoR). For example, the 5-year OS of patients treated with cisplatin-based NAC was 50%, compared with 45% for patients who received only definitive local treatment (see, e.g., Advanced Bladder Cancer Meta-analysis, Eur Urol., 2005, 48(2):202-5). For patients treated with accelerated or dose-dense MVAC, pCR rates were approximately 30% to 40% and pathological downstaging was achieved in 50% of patients (Choueiri 2014; Plimack 2014). For patients treated with pembrolizumab (a PD-1 inhibitor), a pCR rate of 42% was observed in 50 treated patients, a pCR rate of 54.3% in patients with PD L1-positive disease, and a pathological downstaging rate of 65.7% (Necchi 2018). For patients treated with atezolizumab (a PD-L1 inhibitor), a pCR was observed in 29% of patients and 40% of patients with PD-L1-positive disease, compared with only 16% in the PD-L1-negative cohort, with a total of 39% of patients downstaged to non-muscle-invasive disease (Powles 2018).

[0225] In one aspect, provided herein is a method for treating muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) in a human subject, the method comprising: administering to the subject multiple cycles of an effective amount of an antibody drug conjugate (ADC), wherein (a) the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE); (b) the subject is not suitable for cisplatin treatment (cisplatin ineligible); and (c) the efficacy of treating the subject with the effective amount of the ADC is nearly as effective as that observed for cisplatin-eligible patients treated with standard of care (SOC) therapy for treating cisplatin-eligible subjects with MIUC or MIBC.

[0226] In one aspect, provided herein is a method for treating muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) in a human subject, the method comprising: administering to the subject multiple cycles of an effective amount of an antibody drug conjugate (ADC), wherein (a) the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE); (b) the subject is not suitable for cisplatin treatment (cisplatin ineligible); and (c) the efficacy of treating the subject with the effective amount of the ADC is the same as the therapeutic efficacy observed for cisplatin-eligible patients treated with standard of care (SOC) therapy for treating cisplatin-eligible subjects with MIUC or MIBC.

[0227] In one aspect, provided herein is a method for treating muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) in a human subject, the method comprising: administering to the subject an effective amount of an antibody drug conjugate (ADC) for multiple cycles, wherein (a) the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE); (b) the subject is not suitable for cisplatin treatment (cisplatin is not suitable); and (c) the efficacy of treating the subject with an effective amount of ADC is similar to the therapeutic efficacy observed in cisplatin-suitable patients treated with standard of care (SOC) therapy for treating cisplatin-suitable subjects with MIUC or MIBC. In some embodiments, an effective amount of ADC is administered to the subject on days 1 and 8 of each 3-week cycle, for a total of 3 cycles. In some embodiments, the effective amount of ADC is about 1.25 mg / kg of subject body weight. In some embodiments, the effective amount of ADC is about 1.0 mg / kg of subject body weight.

[0228] In some embodiments, the therapeutic efficacy is at least as effective as the therapeutic efficacy observed for SOC for subjects suitable for cisplatin. In some embodiments, the measure of therapeutic efficacy is one or more of the following: pathological complete response rate (pCRR), pathological downstaging rate (pDSR), disease-free survival (DFS), event-free survival (EFS), overall survival (OS), progression-free survival (PFS), and duration of response (DoR). In some embodiments, the measure of therapeutic efficacy is pathological complete response rate (pCRR). In some embodiments, the measure of therapeutic efficacy is pathological downstaging rate (pDSR). In some embodiments, the measure of therapeutic efficacy is disease-free survival (DFS). In some embodiments, the measure of therapeutic efficacy is event-free survival (EFS). In some embodiments, the measure of therapeutic efficacy is overall survival (OS). In some embodiments, the measure of therapeutic efficacy is progression-free survival (PFS). In some embodiments, the measure of therapeutic efficacy is duration of response (DoR)

[0229] In some embodiments of the methods provided herein, the pathological complete remission rate (pCRR) of the subject is at least 30%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 35%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 40%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 45%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 50%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 55%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 60%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 65%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 70%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 75%.

[0230] In some embodiments of the methods provided herein, the pathological complete remission rate (pCRP) of the subject population treated with ADC is at least 30%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 35%. In some embodiments, the pathological complete remission rate (pCRP) of the subject population treated with ADC is at least 40%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 45%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 50%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 55%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 60%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 65%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 70%. In some embodiments, the pathological complete response rate (pCRR) in the population of subjects treated with the ADC is at least 75%.

[0231] In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 50%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 55%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 60%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 65%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 70%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 75%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 80%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 85%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 90%.

[0232] In some embodiments of the methods provided herein, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 50%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 55%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 60%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 65%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 70%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 75%.

[0233] In some embodiments of the methods provided herein, the SOC therapy for a subject to which cisplatin is suitable comprises cisplatin. In some embodiments, the SOC therapy for a subject to which cisplatin is suitable comprises (i) methotrexate, vinblastine, doxorubicin and cisplatin (MVAC), or (ii) gemcitabine plus cisplatin. In some embodiments, the SOC therapy for a subject to which cisplatin is suitable comprises methotrexate, vinblastine, doxorubicin and cisplatin (MVAC). In some embodiments, the SOC therapy for a subject to which cisplatin is suitable comprises gemcitabine plus cisplatin. In some embodiments, the SOC therapy for a subject to which cisplatin is suitable comprises a programmed cell death 1 (PD-1) inhibitor or a programmed cell death ligand 1 (PD-L1) inhibitor. In some embodiments, SOC therapy for cisplatin-suitable subjects comprises: i. cisplatin; ii methotrexate, vinblastine, doxorubicin and cisplatin (MVAC); iii. gemcitabine plus cisplatin; iv. programmed cell death 1 (PD-1) inhibitor; or v. programmed cell death ligand 1 (PD-L1) inhibitor. In some embodiments, SOC therapy for cisplatin-suitable subjects comprises programmed cell death 1 (PD-1) inhibitor. In some embodiments, SOC therapy for cisplatin-suitable subjects comprises programmed cell death ligand 1 (PD-L1) inhibitor. In some embodiments, the PD-1 inhibitor is nivolumab or pembrolizumab. In some embodiments, the PD-1 inhibitor is nivolumab. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of: atezolizumab, avelumab and durvalumab. In some embodiments, the PD-L1 inhibitor is atezolizumab, avelumab or durvalumab. In some embodiments, the PD-L1 inhibitor is atezolizumab. In some embodiments, the PD-L1 inhibitor is avelumab. In some embodiments, the PD-L1 inhibitor is durvalumab. In some embodiments, (i) the PD-1 inhibitor is nivolumab or pembrolizumab; or (ii) the PD-L1 inhibitor is selected from the group consisting of: atezolizumab, avelumab and durvalumab. In some embodiments of the methods provided herein, the subject suffers from cT2-T4aN0M0 stage MIBC stage MIBC.

[0234] In some embodiments of the methods provided herein, the effective amount of ADC is about 1.25 mg / kg.

[0235] In some embodiments of the methods provided herein, the cancer is urothelial or bladder cancer. In some embodiments, the cancer is urothelial cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is muscle-invasive urothelial carcinoma (MIUC). In some embodiments, the cancer is muscle-invasive bladder cancer (MIBC).

[0236] 5.2.1.1 Patients who are not suitable for cisplatin

[0237] For the methods provided herein, including but not limited to the methods in the preceding paragraphs, different conditions can be used to determine cisplatin unsuitability in a human subject.

[0238] In one embodiment, the conditions for determining cisplatin unsuitability include or consist of: GFR <60 mL / min but ≥30 mL / min, wherein GFR is measured by Croft-Gault, modified diet in renal disease equation (MDRD) or 24-hour urine collection. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: ECOG performance status of 2 points. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: NCI CTCAE version 4.03 ≥2 grade hearing loss. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: NYHA grade III heart failure. In some embodiments, a subject is considered unsuitable for cisplatin if one or more of the following criteria are met: (a) GFR <60 mL / min but ≥30 mL / min, (where GFR is measured by Croft-Gault, Modified Diet in Renal Disease (MDRD) or 24-hour urine collection); (b) ECOG performance status of 2; (c) NCI CTCAE version 4.03 ≥2 hearing loss; and (d) NYHA Class III heart failure. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: GFR <60 mL / min but ≥30 mL / min, where GFR is measured by Croft-Gault, Modified Diet in Renal Disease (MDRD) or 24-hour urine collection, and an ECOG performance status of 2. In some embodiments, the conditions for determining that cisplatin is unsuitable include or consist of: GFR<60mL / min but ≥30mL / min, wherein GFR is measured by Croft-Gault, modified diet in renal disease equation (MDRD) or 24-hour urine collection, and NCI CTCAE version 4.03≥2 grade hearing loss. In some embodiments, the conditions for determining that cisplatin is unsuitable include or consist of: GFR<60mL / min but ≥30mL / min, wherein GFR is measured by Croft-Gault, modified diet in renal disease equation (MDRD) or 24-hour urine collection, and NYHA grade III heart failure. In some embodiments, the conditions for determining that cisplatin is unsuitable include or consist of: 2 points of ECOG performance status and NCI CTCAE version 4.03≥2 grade hearing loss. In some embodiments, the conditions for determining that cisplatin is unsuitable include or consist of: 2 points of ECOG performance status and NYHA grade III heart failure. In some embodiments, the conditions for determining cisplatin unsuitability comprise or consist of: NCI CTCAE version 4.03 > 2 grade hearing loss and NYHA class III heart failure.In some embodiments, the human subjects for which the methods provided herein are directed may have any three of the following: GFR<60 mL / min but ≥30 mL / min, wherein GFR is measured by Croft-Gault, modified diet in renal disease equation (MDRD) or 24-hour urine collection, an ECOG performance status of 2 points, NCI CTCAE version 4.03 ≥2 hearing loss, and NYHA Class III heart failure. In some embodiments, the subject is considered unsuitable for cisplatin if one or more of the following criteria are met: (a) GFR<60 mL / min but ≥30 mL / min, wherein GFR is measured by Croft-Gault, modified diet in renal disease equation (MDRD) or 24-hour urine collection, (b) an ECOG performance status of 2 points, (c) NCI CTCAE version 4.03 ≥2 hearing loss, and (d) NYHA Class III heart failure. In some embodiments, the human subjects for which the methods provided herein are directed may have one or more of the following: GFR <60 mL / min but ≥30 mL / min, wherein GFR is measured by Croft-Gault formula, Modification of Diet in Renal Disease (MDRD) equation, or 24-hour urine collection, an ECOG performance status of 2, NCI CTCAE version 4.03 ≥2 hearing loss, and NYHA class III heart failure.

[0239] In other embodiments, the conditions for determining cisplatin unsuitability include or consist of: an ECOG performance status of 2 points. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: impaired renal function. In certain embodiments, the conditions for determining cisplatin unsuitability include or consist of: hearing loss of no less than grade 2. In one embodiment, the conditions for determining cisplatin unsuitability include or consist of: an ECOG performance status of 2 points and impaired renal function. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: an ECOG performance status of 2 points and hearing loss of no less than grade 2. In other embodiments, the conditions for determining cisplatin unsuitability include or consist of: impaired renal function and hearing loss of no less than grade 2. In other embodiments, the conditions for determining cisplatin unsuitability include or consist of: an ECOG performance status of 2 points, impaired renal function, and hearing loss of no less than grade 2. In some embodiments, the conditions for determining cisplatin unsuitability comprise or consist of any one of an ECOG performance status of 2, impaired renal function, and hearing loss of no less than grade 2. In some embodiments, the conditions for determining cisplatin unsuitability comprise or consist of any combination or permutation of any two of an ECOG performance status of 2, impaired renal function, and hearing loss of no less than grade 2. In some embodiments, the conditions for determining cisplatin unsuitability comprise or consist of all three of an ECOG performance status of 2, impaired renal function, and hearing loss of no less than grade 2.

[0240] Impaired renal function can be determined using different methods known and available in the art. For the methods provided herein, including but not limited to the methods in the aforementioned paragraphs, various embodiments are provided herein to determine the impaired renal function of human subjects. In one embodiment, impaired renal function is determined according to a creatinine clearance (CrCl) less than 60mL / min. In some embodiments, impaired renal function is determined according to a CrCl less than 60mL / min but not less than 30mL / min. In certain embodiments, impaired renal function is determined according to a CrCl less than 30mL / min but not less than 15mL / min. In some embodiments of the method provided in this paragraph, CrCl is measured by 24-hour urine collection. In other embodiments of the method provided in this paragraph, CrCl is estimated according to the Croft-Gault standard.

[0241] Therefore, for the methods provided herein, including but not limited to the methods in the preceding paragraphs, some specific conditions based on creatinine clearance can be used to determine the cisplatin unsuitability of a human subject. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: a CrCl of less than 60 mL / min. In one embodiment, the conditions for determining cisplatin unsuitability include or consist of: an ECOG performance status of 2 points and a CrCl of less than 60 mL / min. In other embodiments, the conditions for determining cisplatin unsuitability include or consist of: a CrCl of less than 60 mL / min and a hearing loss of not less than grade 2. In other embodiments, the conditions for determining cisplatin unsuitability include or consist of: an ECOG performance status of 2 points, a CrCl of less than 60 mL / min, and a hearing loss of not less than grade 2. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: any one of an ECOG performance status of 2 points, a CrCl of less than 60 mL / min, and a hearing loss of not less than grade 2. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of any combination or arrangement of any two of an ECOG performance status of 2 points, a CrCl of less than 60 mL / min, and a hearing loss of no less than grade 2. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of all three of an ECOG performance status of 2 points, a CrCl of less than 60 mL / min, and a hearing loss of no less than grade 2. In some embodiments of the methods provided in this paragraph, CrCl is measured by 24-hour urine collection. In other embodiments of the methods provided in this paragraph, CrCl is estimated according to the Croft-Gault criteria.

[0242] Alternatively, for the methods provided herein, including but not limited to the methods in the preceding paragraphs, other specific conditions based on creatinine clearance can be used to determine the cisplatin unsuitability of a human subject. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: a CrCl of less than 60 mL / min but not less than 30 mL / min. In one embodiment, the conditions for determining cisplatin unsuitability include or consist of: an ECOG performance status of 2 points and a CrCl of less than 60 mL / min but not less than 30 mL / min. In other embodiments, the conditions for determining cisplatin unsuitability include or consist of: a CrCl of less than 60 mL / min but not less than 30 mL / min and a hearing loss of not less than grade 2. In other embodiments, the conditions for determining cisplatin unsuitability include or consist of: an ECOG performance status of 2 points, a CrCl of less than 60 mL / min but not less than 30 mL / min, and a hearing loss of not less than grade 2. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: an ECOG performance status of 2 points, a CrCl of less than 60 mL / min but not less than 30 mL / min, and any one of a hearing loss of not less than 2 grades. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: any combination or arrangement of any two of an ECOG performance status of 2 points, a CrCl of less than 60 mL / min but not less than 30 mL / min, and a hearing loss of not less than 2 grades. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: all three of an ECOG performance status of 2 points, a CrCl of less than 60 mL / min but not less than 30 mL / min, and a hearing loss of not less than 2 grades. In some embodiments of the methods provided in this paragraph, CrCl is measured by 24-hour urine collection. In other embodiments of the methods provided in this paragraph, CrCl is estimated according to the Croft-Gault criteria.

[0243] Similarly, for the methods provided herein, including but not limited to the methods in the preceding paragraphs, other specific conditions based on creatinine clearance can be used to determine the cisplatin unsuitability of a human subject. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: a CrCl of less than 30 mL / min but not less than 15 mL / min. In one embodiment, the conditions for determining cisplatin unsuitability include or consist of: an ECOG performance status of 2 points and a CrCl of less than 30 mL / min but not less than 15 mL / min. In other embodiments, the conditions for determining cisplatin unsuitability include or consist of: a CrCl of less than 30 mL / min but not less than 15 mL / min and a hearing loss of not less than grade 2. In other embodiments, the conditions for determining cisplatin unsuitability include or consist of: an ECOG performance status of 2 points, a CrCl of less than 30 mL / min but not less than 15 mL / min, and a hearing loss of not less than grade 2. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: an ECOG performance status of 2 points, a CrCl of less than 30 mL / min but not less than 15 mL / min, and any one of a hearing loss of not less than 2 grades. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: any combination or arrangement of any two of an ECOG performance status of 2 points, a CrCl of less than 30 mL / min but not less than 15 mL / min, and a hearing loss of not less than 2 grades. In some embodiments, the conditions for determining cisplatin unsuitability include or consist of: all three of an ECOG performance status of 2 points, a CrCl of less than 30 mL / min but not less than 15 mL / min, and a hearing loss of not less than 2 grades. In some embodiments of the methods provided in this paragraph, CrCl is measured by 24-hour urine collection. In other embodiments of the methods provided in this paragraph, CrCl is estimated according to the Croft-Gault criteria.

[0244] 5.2.1.2 Additional Patient Demographics

[0245] In addition, the human subjects for which the methods provided herein can be used are human subjects with various other conditions. In one embodiment, the human subjects for which the methods provided herein are directed may have histologically confirmed MIBC, with >50% urothelial histology as the main (formerly known as transitional cell carcinoma). In other embodiments, the human subjects suffer from cT2-T4aN0M0 stage MIBC. In other embodiments, the human subjects for which the methods provided herein are directed may suffer from clinical cT2-T4aN0M0 stages determined by TURBT within 90 days before the first dose of treatment and by CT of the chest with IV contrast and abdominal and pelvic CT urography. In other embodiments, the human subjects for which the methods provided herein are directed may have mixed cell types, with urothelial carcinoma as the main (>50%). In other embodiments, the human subjects for which the methods provided herein are directed may have an ECOG performance status of 0, 1 or 2. In other embodiments, the human subjects for which the methods provided herein are directed may have a life expectancy of ≥3 months. In other embodiments, the human subjects for which the methods provided herein are directed may be deemed suitable for RC+PLND by their urologists and / or oncologists. In some embodiments, the methods provided herein are directed to human subjects who have any two of the following: histologically confirmed MIBC with >50% urothelial histology as the predominant (formerly known as transitional cell carcinoma); clinical cT2-T4aN0M0 stage as determined by TURBT within 90 days prior to the first dose of treatment and by CT of the chest with IV contrast and CT urography of the abdomen and pelvis; mixed cell types with urothelial carcinoma as the predominant (>50%); an ECOG performance status of 0, 1, or 2; a life expectancy of ≥3 months; and being deemed a candidate for RC+PLND by their urologist and / or oncologist. In some embodiments, the methods provided herein are directed to human subjects who have any three of the following: histologically confirmed MIBC with >50% urothelial histology as the predominant (formerly known as transitional cell carcinoma); clinical cT2-T4aN0M0 stage as determined by TURBT within 90 days prior to the first dose of treatment and by CT of the chest with IV contrast and CT urography of the abdomen and pelvis; mixed cell types with urothelial carcinoma as the predominant (>50%); an ECOG performance status of 0, 1, or 2; a life expectancy of ≥3 months; and being deemed a candidate for RC+PLND by their urologist and / or oncologist.In some embodiments, the methods provided herein are directed to human subjects who have any four of the following: histologically confirmed MIBC with >50% urothelial histology as the predominant (formerly known as transitional cell carcinoma); clinical cT2-T4aN0M0 stage as determined by TURBT within 90 days prior to the first dose of treatment and by CT of the chest with IV contrast and CT urography of the abdomen and pelvis; mixed cell types with urothelial carcinoma as the predominant (>50%); an ECOG performance status of 0, 1, or 2; a life expectancy of ≥3 months; and being deemed a candidate for RC+PLND by their urologist and / or oncologist. In some embodiments, the methods provided herein are directed to human subjects who have any five of the following: histologically confirmed MIBC with >50% urothelial histology as the predominant (formerly known as transitional cell carcinoma); clinical cT2-T4aN0M0 stage as determined by TURBT within 90 days prior to the first dose of treatment and by CT of the chest with IV contrast and CT urography of the abdomen and pelvis; mixed cell types with urothelial carcinoma as the predominant (>50%); an ECOG performance status of 0, 1, or 2; a life expectancy of ≥3 months; and being deemed a candidate for RC+PLND by their urologist and / or oncologist. In some embodiments, the methods provided herein are directed to human subjects who have all five of the following: histologically confirmed MIBC with >50% urothelial histology as the predominant (formerly known as transitional cell carcinoma); clinical cT2-T4aN0M0 stage as determined by TURBT within 90 days prior to the first dose of treatment and by CT of the chest with IV contrast and CT urography of the abdomen and pelvis; mixed cell types with urothelial carcinoma as the predominant (>50%); ECOG performance status of 0, 1, or 2; life expectancy of ≥3 months; and are deemed suitable for RC+PLND by their urologist and / or oncologist. In some embodiments, the methods provided herein are directed to human subjects who have one or more of the following: histologically confirmed MIBC with >50% urothelial histology as the predominant (formerly known as transitional cell carcinoma); clinical cT2-T4aN0M0 stage as determined by TURBT within 90 days prior to the first dose of treatment and by CT of the chest with IV contrast and CT urography of the abdomen and pelvis; mixed cell types with urothelial carcinoma as the predominant (>50%); an ECOG performance status of 0, 1, or 2; a life expectancy of ≥3 months; and are deemed suitable for RC+PLND by their urologist and / or oncologist.

[0246] In some embodiments, the human subjects targeted by the methods provided herein may have received prior intravesical injections of Bacillus Calmette-Guerin (BCG) or intravesical chemotherapy for NMIBC.

[0247] In other embodiments of the methods provided herein, including the methods of the preceding paragraphs, the human subjects to whom the methods provided herein may be used are human subjects with various other conditions. In one embodiment, the human subjects to whom the methods provided herein may be used also have the condition that the absolute neutrophil count (ANC) is not less than 1500 / μL. In other embodiments, the human subjects to whom the methods provided herein may be used also have the condition that the platelet count is not less than 100,000 / μL. In other embodiments, the human subjects to whom the methods provided herein may be used also have the condition that the hemoglobin is not less than 9.0 g / dL or 5.6 mmol / L. In other embodiments, the human subjects to whom the methods provided herein may be used also have the condition that the CrCl is not more than 1.5 times the upper limit of normal (ULN). In other embodiments, the human subjects to whom the methods provided herein may be used also have the condition that the creatinine is not more than 1.5 times the upper limit of normal (ULN). In other embodiments, the human subjects to whom the methods provided herein may be used also have the condition that the CrCl is not less than 30 mL / min. In other embodiments, human subjects to whom the methods provided herein can be used also have the following conditions: (i) serum total bilirubin does not exceed 1.5 times the upper limit of normal (ULN), (ii) for patients with total bilirubin levels exceeding 1.5 times the ULN, direct bilirubin does not exceed ULN, or (iii) for patients with Gilbert's disease, serum total bilirubin does not exceed 3 times the ULN. In other embodiments, human subjects to whom the methods provided herein can be used also have AST (SGOT) and ALT (SGPT) do not exceed 3 times the ULN. In other embodiments, human subjects to whom the methods provided herein can be used also have INR or PT do not exceed 1.5 times the upper limit of normal (ULN). In other embodiments, human subjects to whom the methods provided herein can be used also have aPTT or PTT do not exceed 1.5 times the upper limit of normal (ULN).In some embodiments, the human subjects for whom the methods provided herein can be used also have any two of the following: an absolute neutrophil count (ANC) of not less than 1500 / μL; a platelet count of not less than 100,000 / μL; a hemoglobin of not less than 9.0 g / dL or 5.6 mmol / L; a CrCl of not more than 1.5 times the upper limit of normal (ULN); a creatinine of not more than 1.5 times the upper limit of normal (ULN); a CrCl of not less than 30 mL / min; (i) serum total bilirubin of not more than 1.5 times the upper limit of normal (ULN), (ii) for patients with total bilirubin levels exceeding 1.5 times the ULN, direct bilirubin of not more than ULN, or (iii) for patients with Gilbert's disease, serum total bilirubin of not more than 3 times the ULN; AST (SGOT) and ALT (SGPT) of not more than 3 times the ULN; an INR or PT of not more than 1.5 times the upper limit of normal (ULN); and an aPTT or PTT of not more than 1.5 times the upper limit of normal (ULN). In some embodiments, the human subjects for whom the methods provided herein can be used also have any three of the following: absolute neutrophil count (ANC) not less than 1500 / μL; platelet count not less than 100,000 / μL; hemoglobin not less than 9.0 g / dL or 5.6 mmol / L; CrCl not more than 1.5 times the upper limit of normal (ULN); creatinine not more than 1.5 times the upper limit of normal (ULN); CrCl not less than 30 mL / min; (i) serum total cholesterol; (ii) for patients with total bilirubin levels exceeding 1.5 times the ULN, direct bilirubin not exceeding ULN, or (iii) for patients with Gilbert's disease, total serum bilirubin not exceeding 3 times the ULN; AST (SGOT) and ALT (SGPT) not exceeding 3 times the ULN; INR or PT not exceeding 1.5 times the ULN; and aPTT or PTT not exceeding 1.5 times the ULN.In some embodiments, the human subjects for whom the methods provided herein can be used also have any four of the following: absolute neutrophil count (ANC) not less than 1500 / μL; platelet count not less than 100,000 / μL; hemoglobin not less than 9.0 g / dL or 5.6 mmol / L; CrCl not more than 1.5 times the upper limit of normal (ULN); creatinine not more than 1.5 times the upper limit of normal (ULN); CrCl not less than 30 mL / min; (i) serum total cholesterol; (ii) for patients with total bilirubin levels exceeding 1.5 times the ULN, direct bilirubin not exceeding ULN, or (iii) for patients with Gilbert's disease, total serum bilirubin not exceeding 3 times the ULN; AST (SGOT) and ALT (SGPT) not exceeding 3 times the ULN; INR or PT not exceeding 1.5 times the ULN; and aPTT or PTT not exceeding 1.5 times the ULN. In some embodiments, the human subjects for whom the methods provided herein can be used also have any five of the following: absolute neutrophil count (ANC) not less than 1500 / μL; platelet count not less than 100,000 / μL; hemoglobin not less than 9.0 g / dL or 5.6 mmol / L; CrCl not more than 1.5 times the upper limit of normal (ULN); creatinine not more than 1.5 times the upper limit of normal (ULN); CrCl not less than 30 mL / min; (i) serum total cholesterol; (ii) for patients with total bilirubin levels exceeding 1.5 times the ULN, direct bilirubin not exceeding ULN, or (iii) for patients with Gilbert's disease, total serum bilirubin not exceeding 3 times the ULN; AST (SGOT) and ALT (SGPT) not exceeding 3 times the ULN; INR or PT not exceeding 1.5 times the ULN; and aPTT or PTT not exceeding 1.5 times the ULN.In some embodiments, the human subjects for whom the methods provided herein can be used also have any six of the following: absolute neutrophil count (ANC) not less than 1500 / μL; platelet count not less than 100,000 / μL; hemoglobin not less than 9.0 g / dL or 5.6 mmol / L; CrCl not more than 1.5 times the upper limit of normal (ULN); creatinine not more than 1.5 times the upper limit of normal (ULN); CrCl not less than 30 mL / min; (i) serum total cholesterol; (ii) for patients with total bilirubin levels exceeding 1.5 times the ULN, direct bilirubin not exceeding ULN, or (iii) for patients with Gilbert's disease, total serum bilirubin not exceeding 3 times the ULN; AST (SGOT) and ALT (SGPT) not exceeding 3 times the ULN; INR or PT not exceeding 1.5 times the ULN; and aPTT or PTT not exceeding 1.5 times the ULN. In some embodiments, the human subjects for whom the methods provided herein can be used also have any seven of the following: absolute neutrophil count (ANC) not less than 1500 / μL; platelet count not less than 100,000 / μL; hemoglobin not less than 9.0 g / dL or 5.6 mmol / L; CrCl not more than 1.5 times the upper limit of normal (ULN); creatinine not more than 1.5 times the upper limit of normal (ULN); CrCl not less than 30 mL / min; (i) serum total cholesterol; (ii) for patients with total bilirubin levels exceeding 1.5 times the ULN, direct bilirubin not exceeding ULN, or (iii) for patients with Gilbert's disease, total serum bilirubin not exceeding 3 times the ULN; AST (SGOT) and ALT (SGPT) not exceeding 3 times the ULN; INR or PT not exceeding 1.5 times the ULN; and aPTT or PTT not exceeding 1.5 times the ULN.In some embodiments, the human subjects for whom the methods provided herein can be used also have any eight of the following: absolute neutrophil count (ANC) not less than 1500 / μL; platelet count not less than 100,000 / μL; hemoglobin not less than 9.0 g / dL or 5.6 mmol / L; CrCl not more than 1.5 times the upper limit of normal (ULN); creatinine not more than 1.5 times the upper limit of normal (ULN); CrCl not less than 30 mL / min; (i) serum total cholesterol; (ii) for patients with total bilirubin levels exceeding 1.5 times the ULN, direct bilirubin not exceeding ULN, or (iii) for patients with Gilbert's disease, total serum bilirubin not exceeding 3 times the ULN; AST (SGOT) and ALT (SGPT) not exceeding 3 times the ULN; INR or PT not exceeding 1.5 times the ULN; and aPTT or PTT not exceeding 1.5 times the ULN. In some embodiments, the human subjects for whom the methods provided herein can be used also have all nine of the following: an absolute neutrophil count (ANC) of no less than 1500 / μL; a platelet count of no less than 100,000 / μL; hemoglobin of no less than 9.0 g / dL or 5.6 mmol / L; a CrCl of no more than 1.5 times the upper limit of normal (ULN); a creatinine of no more than 1.5 times the upper limit of normal (ULN); a CrCl of no less than 30 mL / min; (i) serum total bilirubin of no more than 1.5 times the upper limit of normal (ULN), (ii) for patients with total bilirubin levels exceeding 1.5 times the ULN, direct bilirubin of no more than ULN, or (iii) for patients with Gilbert's disease, serum total bilirubin of no more than 3 times the ULN; AST (SGOT) and ALT (SGPT) of no more than 3 times the ULN; INR or PT of no more than 1.5 times the upper limit of normal (ULN); and aPTT or PTT of no more than 1.5 times the upper limit of normal (ULN).In some embodiments, the human subjects for whom the methods provided herein can be used also have one or more of the following: absolute neutrophil count (ANC) not less than 1500 / μL; platelet count not less than 100,000 / μL; hemoglobin not less than 9.0 g / dL or 5.6 mmol / L; CrCl not more than 1.5 times the upper limit of normal (ULN); creatinine not more than 1.5 times the upper limit of normal (ULN); CrCl not less than 30 mL / min; (i) serum total Bilirubin not exceeding 1.5 times the upper limit of normal (ULN), (ii) for patients with total bilirubin levels exceeding 1.5 times the ULN, direct bilirubin not exceeding ULN, or (iii) for patients with Gilbert's disease, serum total bilirubin not exceeding 3 times the ULN; AST (SGOT) and ALT (SGPT) not exceeding 3 times the ULN; INR or PT not exceeding 1.5 times the upper limit of normal (ULN); and aPTT or PTT not exceeding 1.5 times the upper limit of normal (ULN).

[0248] In other embodiments of the methods provided herein, including the methods of the preceding paragraphs, the human subjects for whom the methods provided herein can be used are human subjects without certain conditions. In one embodiment, the human subjects for whom the methods provided herein are directed have not previously received systemic treatment, chemoradiotherapy, or radiotherapy for MIBC. In one embodiment, the human subjects for whom the methods provided herein are directed have not previously received any treatment with immune checkpoint inhibitors (CPIs) (e.g., PD-1 inhibitors, PD-L1 inhibitors, or PD-L2 inhibitors, such as atezolizumab, pembrolizumab, nivolumab, durvalumab, or avelumab). In one embodiment, the human subjects for whom the methods provided herein are directed have not previously received any treatment with agents for another stimulatory or co-inhibitory T cell receptor (e.g., CD137 agonists, CTLA-4 inhibitors, or OX-40 agonists). In one embodiment, the human subjects for whom the methods provided herein are directed have no evidence of lymph node disease on imaging. In one embodiment, the human subjects for whom the methods provided herein are directed have no evidence of metastatic disease on imaging. In one embodiment, the human subjects for whom the methods provided herein are directed have not yet undergone partial cystectomy to remove any NMIBC or MIBC. In one embodiment, the human subjects for whom the methods provided herein are directed do not suffer from persistent grade 2 or higher sensory or motor neuropathy. In one embodiment, the human subjects for whom the methods provided herein are directed do not suffer from diseases requiring high doses of steroids (>10 mg / day of prednisone or equivalent) or other immunosuppressive drugs. In one embodiment, the human subjects for whom the methods provided herein are directed have not previously been treated with enroku monoclonal antibody vedotin or other ADCs based on MMAE for urothelial carcinoma. In one embodiment, the human subjects for whom the methods provided herein are directed have not had a history of another invasive malignancy within 3 years before the first dose of enroku monoclonal antibody vedotin (EV), or have no evidence of residual disease of any previously diagnosed malignancy. In one embodiment, the human subjects for whom the methods provided herein are directed. In one embodiment, the human subjects for whom the methods provided herein are directed are not currently receiving systemic antibacterial treatment for active infection (i.e., virus, bacteria, or fungus) at the time of the first dose of enroku monoclonal antibody vedotin. In one embodiment, the human subjects for whom the methods provided herein are directed are not positive for hepatitis B surface antigen and / or anti-hepatitis B core antibodies. In one embodiment, the methods provided herein are directed to a human subject who does not have active hepatitis C infection or known HIV infection. In one embodiment, the methods provided herein are directed to a human subject who does not have active tuberculosis.In one embodiment, the human subjects for whom the methods provided herein are directed have no documented history of cerebrovascular events (stroke or transient ischemic attack), unstable angina, myocardial infarction, or cardiac symptoms (including congestive heart failure) that meet NYHA class IV within 6 months prior to the first dose of enroku vedotin. In one embodiment, the human subjects for whom the methods provided herein are directed do not have active keratitis or corneal ulcers. In one embodiment, the human subjects for whom the methods provided herein are directed do not have active autoimmune diseases that require systemic treatment (i.e., use of disease-modifying agents, corticosteroids, or immunosuppressive drugs) within the past 2 years. In one embodiment, the human subjects for whom the methods provided herein are directed do not have a history of idiopathic pulmonary fibrosis, organizing pneumonia, drug-induced pneumonia, idiopathic pneumonia, or no evidence of active pneumonia in a screening chest CT scan. In one embodiment, the human subjects for whom the methods provided herein are directed have not previously undergone allogeneic stem cell or solid organ transplantation. In one embodiment, the human subjects for whom the methods provided herein are directed have not been vaccinated with a live attenuated vaccine within 30 days prior to the first dose of study drug. In one embodiment, the methods provided herein are directed to a human subject who does not have uncontrolled diabetes, wherein uncontrolled diabetes is defined as HbA1c ≥ 8% or HbA1c 7% to < 8% with associated diabetic symptoms (polyuria or polydipsia) that cannot be explained otherwise.

[0249] In all methods provided herein, particularly the methods described in the preceding 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 in Sections 3 and 6; dosing regimens and pharmaceutical compositions for administering therapeutic agents are described in this section (Section 5.2), the following Section 5.4, Section 5.7, and Section 6; methods that can be used to identify therapeutic agents, select patients, determine The results of these methods and / or biomarkers that serve as criteria for these methods in any way, the biomarkers can be determined as described in Section 5.8 or as known in the art; the treatment results of the methods provided herein are described in this section (Section 5.2, including Section 5.2.1.4) and Sections 3 and 6, and additional treatment results can be improvements in biomarkers described herein, such as those described and exemplified in this section (Section 5.2, including Section 5.2.2) and Sections 3 and 6; and combination therapies including ADC and other therapeutic agents are described in this section (Section 5.2) and Section 5.5. Therefore, those skilled in the art will appreciate that the methods provided herein include all permutations and combinations of patients, therapeutic agents, dosing regimens, biomarkers, and treatment results as described above and below.

[0250] In certain embodiments, the methods provided herein are used to treat subjects with urothelial carcinoma that 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 carcinoma that expresses 191P4D12 RNA or 191P4D12 protein.

[0251] In certain embodiments, the methods provided herein are used to treat subjects with urothelial carcinoma that 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 carcinoma that expresses 191P4D12 RNA or 191P4D12 protein.

[0252] 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 IHC, fluorescence activated cell sorting (FACS) analysis and / or Western blot. 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.

[0253] In some embodiments, the subject that can be treated by 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.

[0254] In some embodiments, the phenotypic or genotypic characteristics are determined histologically, cytologically, or histologically and cytologically. In some embodiments of the methods provided herein, the phenotypic and / or genotypic characteristics are determined histologically and / or cytologically as described in the American Society of Clinical Oncology / College of American Pathologists (ASCO / CAP) guidelines based on the most recently analyzed tissue, and the standards are incorporated herein by reference in their entirety. In some embodiments, the phenotypic or genotypic characteristics are determined by sequencing, DNA hybridization, and / or RNA hybridization including next-generation sequencing (e.g., NGS by Illumina).

[0255] 5.2.1.3 Optional exclusion of patients previously treated with checkpoint inhibitors (CPIs)

[0256] 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 can refer to subjects who have not previously received any immune checkpoint inhibitor treatment (e.g., human subjects for whom the methods provided herein are directed have not previously received any immune checkpoint inhibitor (CPI) (e.g., PD-1 inhibitor, PD-L1 inhibitor or PD-L2 inhibitor, such as atezolizumab, pembrolizumab, nivolumab, durvalumab or avelumab)).

[0257] As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with or regulates one or more checkpoint proteins. 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, IDOl, CD276, PVRIG, TIGIT, CD25 (IL2RA), IFNAR2, IFNAR1, CSF1R, VSIR (VISTA) or HLA. These proteins appear to be responsible for the costimulatory 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.

[0258] In certain embodiments, the checkpoint inhibitor may be an inhibitor or activator for a checkpoint protein that is upregulated in cancer. In some specific embodiments, the checkpoint inhibitor may be an inhibitor or activator for a checkpoint protein including: 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 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.

[0259] 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 U.S. Patents 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 by reference 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 marketed under the trade name Yervoy TM Commercially available.

[0260] In certain embodiments, the checkpoint inhibitor is a PD-1 / PD-L1 inhibitor. Examples of PD-1 / PD-L1 inhibitors include, but are not limited to, 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, which are incorporated herein by reference in their entirety.

[0261] 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 marketed under the trade name Opdivo. TMIn another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 antibody and is marketed under the trade name Keytruda TM Commercially available. In another embodiment, the anti-PD-1 antibody is the humanized antibody CT-011. CT-011 alone was administered without response in the treatment of relapsed 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 whose ability to bind to Fcγ receptor I is explicitly engineered, and it has a unique binding tag, high affinity, and excellent target specificity for PD-1. 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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).

[0266] 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).

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] In one embodiment, the checkpoint inhibitor is recombinant human leukocyte factor-15 (rhIL-15).

[0272] In one embodiment, the checkpoint inhibitor is a VTCN inhibitor. In one embodiment, the VTCN inhibitor is FPA150.

[0273] 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 or IOM-D.

[0274] 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 M TIG7192A. 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] In certain embodiments, the immune checkpoint inhibitors provided herein include two or more checkpoint inhibitors described herein (including checkpoint inhibitors of the same or different classes).

[0282] In some embodiments, the subject treatable with the methods provided herein is a mammal. In some embodiments, the subject treatable with the methods provided herein is a human.

[0283] 5.2.1.4 Treatment results of the methods provided herein

[0284] Although there is a poor prognosis for cisplatin-ineligible human subjects who are frail, have multiple comorbidities in addition to urothelial cancer / bladder cancer (e.g., muscle-invasive urothelial carcinoma (MIUC) and muscle-invasive bladder cancer (MIBC)) and are unable to tolerate other treatments other than immunotherapy as described above, the methods provided herein, including the methods described in this section (Section 5.2) and Sections 3 and 6, can provide beneficial treatment results to these cisplatin-ineligible human subjects. In one embodiment, a human subject has a complete response after treatment by the methods provided herein. In another embodiment, a human subject has a partial response after treatment by the methods provided herein.

[0285] In some embodiments, the response (complete or partial response) is determined by evaluating 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 Table 15.

[0286] In some embodiments of the methods provided herein, the pathological complete remission rate (pCRR) of the subject is at least 30%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 35%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 40%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 45%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 50%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 55%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 60%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 65%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 70%. In some embodiments, the pathological complete remission rate (pCRR) of the subject is at least 75%.

[0287] In some embodiments of the methods provided herein, the pathological complete remission rate (pCRR) is at least 30%. In some embodiments, the pathological complete remission rate (pCRR) is at least 35%. In some embodiments, the pathological complete remission rate (pCRR) is at least 40%. In some embodiments, the pathological complete remission rate (pCRR) is at least 45%. In some embodiments, the pathological complete remission rate (pCRR) is at least 50%. In some embodiments, the pathological complete remission rate (pCRR) is at least 55%. In some embodiments, the pathological complete remission rate (pCRR) is at least 60%. In some embodiments, the pathological complete remission rate (pCRR) is at least 65%. In some embodiments, the pathological complete remission rate (pCRR) is at least 70%. In some embodiments, the pathological complete remission rate (pCRR) is at least 75%.

[0288] In some embodiments of the methods provided herein, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 30%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 35%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 40%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 45%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 50%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 55%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 60%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 65%. In some embodiments, the pathological complete remission rate (pCRR) of the subject population treated with ADC is at least 70%. In some embodiments, the pathological complete response rate (pCRR) in the population of subjects treated with the ADC is at least 75%.

[0289] In some embodiments of the methods provided herein, the human subject has a disease-free survival period of at least or about 2 months after treatment. In another embodiment, the human subject has a disease-free survival period of at least or about 3 months after treatment. In another embodiment, the human subject has a disease-free survival period of at least or about 4 months after treatment. In another embodiment, the human subject has a disease-free survival period of at least or about 5 months after treatment. In another embodiment, the human subject has a disease-free survival period of at least or about 6 months after treatment. In another embodiment, the human subject has a disease-free survival period of at least or about 7 months after treatment. In another embodiment, the human subject has a disease-free survival period of at least or about 8 months after treatment. In one embodiment, the human subject has a disease-free survival period of at least or about 9 months after treatment. In another embodiment, the human subject has a disease-free survival period of at least or about 10 months after treatment. In another embodiment, the human subject has a disease-free survival period of at least or about 11 months after treatment. In another embodiment, the human subject has a disease-free survival period of at least or about 12 months after treatment. In one embodiment, the human subject has a disease-free survival of at least or about 13 months after treatment. In another embodiment, the human subject has a disease-free survival of at least or about 14 months after treatment. In another embodiment, the human subject has a disease-free survival of at least or about 15 months after treatment. In some embodiments, the overall survival of the subject is extended by at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, or at least 12 months.

[0290] In one embodiment, the human subject has a disease-free survival period in the range of 2 to 15 months after treatment. In another embodiment, the human subject has a disease-free survival period in the range of 2 to 14 months after treatment. In another embodiment, the human subject has a disease-free survival period in the range of 2 to 13 months after treatment. In one embodiment, the human subject has a disease-free survival period in the range of 2 to 12 months after treatment. In another embodiment, the human subject has a disease-free survival period in the range of 3 to 12 months after treatment. In another embodiment, the human subject has a disease-free survival period in the range of 3 to 11 months after treatment. In another embodiment, the human subject has a disease-free survival period in the range of 4 to 11 months after treatment. In one embodiment, the human subject has a disease-free survival period in the range of 4 to 10 months after treatment. In another embodiment, the human subject has a disease-free survival period in the range of 5 to 10 months after treatment. In another embodiment, the human subject has a disease-free survival period in the range of 5 to 9 months after treatment. In another embodiment, the human subject has a disease-free survival period in the range of 5 to 8 months after treatment. In one embodiment, the human subjects have a disease-free survival period in the range of 5 to 7 months after treatment. In one embodiment, the human subjects have a disease-free survival period in the range of 5 to 6 months after treatment. In one embodiment, the human subjects have a disease-free survival period in the range of 6 to 7 months after treatment. In another embodiment, the human subjects have a disease-free survival period in the range of 6 to 8 months after treatment.

[0291] In addition, in some embodiments, the disease-free survival of a human subject population treated by the methods provided herein is evaluated by evaluating the median or average disease-free survival of the treated population. In one embodiment, a human subject population is treated by the methods provided herein, wherein the median or average disease-free survival of the treated population is at least or about 2 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average disease-free survival of the treated population is at least or about 3 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average disease-free survival of the treated population is at least or about 4 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average disease-free survival of the treated 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 disease-free survival of the treated 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 disease-free survival of the treated population is at least or about 7 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average disease-free survival of the treated population is at least or about 8 months. In one embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average disease-free survival of the treated population is at least or about 9 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average disease-free survival of the treated population is at least or about 10 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average disease-free survival of the treated population is at least or about 11 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average disease-free survival of the treated population is at least or about 12 months. In one embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average disease-free survival of the treated population is at least or about 13 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average disease-free survival of the treated population is at least or about 14 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or mean disease-free survival of the treated population is at least or about 15 months.

[0292] In one embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 2 to 15 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 2 to 14 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 2 to 13 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 2 to 12.32 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 2 to 12 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 3 to 12 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 3 to 11 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 4 to 11 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 4 to 10 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 5 to 10 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 5 to 9 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 5 to 8 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 5 to 7 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 5 to 6 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 6 to 7 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the disease-free survival of the treated population is in the range of 6 to 8 months.

[0293] In some embodiments of the methods provided herein, the human subject has an event-free survival of at least or about 2 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 2.6 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 3 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 4 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 5 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 6 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 6.54 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 7 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 8 months after treatment. In one embodiment, the human subject has an event-free survival of at least or about 9 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 10 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 11 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 12 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 12.32 months after treatment. In one embodiment, the human subject has an event-free survival of at least or about 13 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 14 months after treatment. In another embodiment, the human subject has an event-free survival of at least or about 15 months after treatment.

[0294] In one embodiment, the human subject has an event-free survival in the range of 2 to 15 months after treatment. In another embodiment, the human subject has an event-free survival in the range of 2 to 14 months after treatment. In another embodiment, the human subject has an event-free survival in the range of 2 to 13 months after treatment. In another embodiment, the human subject has an event-free survival in the range of 2 to 12.32 months after treatment. In one embodiment, the human subject has an event-free survival in the range of 2 to 12 months after treatment. In another embodiment, the human subject has an event-free survival in the range of 3 to 12 months after treatment. In another embodiment, the human subject has an event-free survival in the range of 3 to 11 months after treatment. In another embodiment, the human subject has an event-free survival in the range of 4 to 11 months after treatment. In one embodiment, the human subject has an event-free survival in the range of 4 to 10 months after treatment. In another embodiment, the human subject has an event-free survival in the range of 5 to 10 months after treatment. In another embodiment, the human subject has an event-free survival in the range of 5 to 9 months after treatment. In another embodiment, the human subject has an event-free survival in the range of 5 to 8 months following treatment. In one embodiment, the human subject has an event-free survival in the range of 5 to 7 months following treatment. In one embodiment, the human subject has an event-free survival in the range of 5 to 6 months following treatment. In one embodiment, the human subject has an event-free survival in the range of 6 to 7 months following treatment. In another embodiment, the human subject has an event-free survival in the range of 6 to 8 months following treatment.

[0295] In addition, in some embodiments, the event-free survival of a human subject population treated by the methods provided herein is evaluated by evaluating the median or average event-free survival of the treated population. In one embodiment, a human subject population is treated by the methods provided herein, wherein the median or average event-free survival of the treated population is at least or about 2 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average event-free survival of the treated population is at least or about 2.6 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average event-free survival of the treated population is at least or about 3 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average event-free survival of the treated population is at least or about 4 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average event-free survival of the treated 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 event-free survival of the treated 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 event-free survival of the treated 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 event-free survival of the treated 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 event-free survival of the treated 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 event-free survival of the treated 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 event-free survival of the treated population is at least or about 11 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average event-free survival of the treated population is at least or about 12 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average event-free survival of the treated population is at least or about 12.32 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the median or average event-free survival of the treated population is at least or about 13 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average event-free survival of the treated population is at least or about 14 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average event-free survival of the treated population is at least or about 15 months.

[0296] In one embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is in the range of 2 to 15 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is in the range of 2 to 14 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is in the range of 2 to 13 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is in the range of 2 to 12.32 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is in the range of 2 to 12 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is in the range of 3 to 12 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is in the range of 3 to 11 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is in the range of 4 to 11 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is within the range of 4 to 10 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is within the range of 5 to 10 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is within the range of 5 to 9 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is within the range of 5 to 8 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is within the range of 5 to 7 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is within the range of 5 to 6 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is within the range of 6 to 7 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the event-free survival of the treated population is within the range of 6 to 8 months.

[0297] In some embodiments of the methods provided herein, the human subject has an overall survival of at least or about 5 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 6 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 7 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 8 months after treatment. In one embodiment, the human subject has an overall survival of at least or about 9 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 10 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 11 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 12 months after treatment. In one embodiment, the human subject has an overall survival of at least or about 13 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 14 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 15 months after treatment. In one embodiment, the human subject has an overall survival of at least or about 16 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 17 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 18 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 19 months after treatment. In one embodiment, the human subject has an overall survival of at least or about 20 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 21 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 22 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 23 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 24 months after treatment. In one embodiment, the human subject has an overall survival of at least or about 25 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 26 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 27 months after treatment. In one embodiment, the human subject has an overall survival of at least or about 28 months after treatment. In another embodiment, the human subject has an overall survival of at least or about 29 months following treatment. In another embodiment, the human subject has an overall survival of at least or about 30 months following treatment.

[0298] In one embodiment, the human subject has an overall survival in the range of 10 to 19 months after treatment. In another embodiment, the human subject has an overall survival in the range of 10 to 18 months after treatment. In another embodiment, the human subject has an overall survival in the range of 10 to 17 months after treatment. In another embodiment, the human subject has an overall survival in the range of 10 to 16 months after treatment. In one embodiment, the human subject has an overall survival in the range of 10 to 15 months after treatment. In another embodiment, the human subject has an overall survival in the range of 10 to 14 months after treatment. In another embodiment, the human subject has an overall survival in the range of 10 to 13 months after treatment. In another embodiment, the human subject has an overall survival in the range of 10 to 12 months after treatment. In one embodiment, the human subject has an overall survival in the range of 10 to 11 months after treatment. In another embodiment, the human subject has an overall survival in the range of 11 to 19 months after treatment. In another embodiment, the human subject has an overall survival in the range of 12 to 19 months after treatment. In another embodiment, the human subject has an overall survival in the range of 13 to 19 months after treatment. In one embodiment, the human subject has an overall survival in the range of 14 to 18 months after treatment. In one embodiment, the human subject has an overall survival in the range of 14 to 19 months after treatment. In one embodiment, the human subject has an overall survival in the range of 15 to 18 months after treatment. In another embodiment, the human subject has an overall survival in the range of 15 to 19 months after treatment. In another embodiment, the human subject has an overall survival in the range of 16 to 19 months after treatment. In another embodiment, the human subject has an overall survival in the range of 17 to 19 months after treatment. In one embodiment, the human subject has an overall survival in the range of 18 to 19 months after treatment. In another embodiment, the human subject has an overall survival in the range of 11 to 18 months after treatment. In another embodiment, the human subject has an overall survival in the range of 12 to 17 months after treatment. In another embodiment, the human subject has an overall survival in the range of 13 to 16 months after treatment. In another embodiment, the human subjects have an overall survival in the range of 14 to 15 months following treatment. In one embodiment, the human subjects have an overall survival in the range of 10 to 20 months following treatment. In another embodiment, the human subjects have an overall survival in the range of 11 to 20 months following treatment. In one embodiment, the human subjects have an overall survival in the range of 11 to 24 months following treatment.In one embodiment, the human subject has an overall survival in the range of 11 to 25 months after treatment. In one embodiment, the human subject has an overall survival in the range of 12 to 24 months after treatment. In one embodiment, the human subject has an overall survival in the range of 12 to 25 months after treatment. In another embodiment, the human subject has an overall survival in the range of 12 to 20 months after treatment. In one embodiment, the human subject has an overall survival in the range of 13 to 20 months after treatment. In another embodiment, the human subject has an overall survival in the range of 14 to 20 months after treatment. In another embodiment, the human subject has an overall survival in the range of 15 to 20 months after treatment. In another embodiment, the human subject has an overall survival in the range of 16 to 20 months after treatment. In one embodiment, the human subject has an overall survival in the range of 17 to 20 months after treatment. In another embodiment, the human subject has an overall survival in the range of 18 to 20 months after treatment. In another embodiment, the human subject has an overall survival in the range of 19 to 20 months after treatment. In one embodiment, the human subject has an overall survival in the range of 9 to 20 months after treatment. In another embodiment, the human subject has an overall survival in the range of 9 to 19 months after treatment. In another embodiment, the human subject has an overall survival in the range of 9 to 18 months after treatment. In one embodiment, the human subject has an overall survival in the range of 9 to 17 months after treatment. In another embodiment, the human subject has an overall survival in the range of 9 to 16 months after treatment. In another embodiment, the human subject has an overall survival in the range of 9 to 15 months after treatment. In one embodiment, the human subject has an overall survival in the range of 9 to 14 months after treatment. In another embodiment, the human subject has an overall survival in the range of 9 to 13 months after treatment. In another embodiment, the human subject has an overall survival in the range of 9 to 12 months after treatment. In one embodiment, the human subject has an overall survival in the range of 9 to 11 months after treatment. In another embodiment, the human subject has an overall survival in the range of 9 to 10 months after treatment.

[0299] In addition, in some embodiments, the overall survival of a human subject population treated by the methods provided herein is evaluated by evaluating the median or average overall survival of the treated population. In one embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated 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 overall survival of the treated 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 overall survival of the treated 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 overall survival of the treated 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 overall survival of the treated 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 overall survival of the treated 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 overall survival of the treated population is at least or about 11 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 12 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 13 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 14 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 15 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 16 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 17 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 18 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 19 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 20 months.In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 21 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 22 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 23 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 24 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 25 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 26 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 27 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 28 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 29 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the median or average overall survival of the treated population is at least or about 30 months.

[0300] In one embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 10 to 19 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 10 to 18 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 10 to 17 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 10 to 16 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 10 to 15 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 10 to 14 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 10 to 13 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 10 to 12 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 10 to 11 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 10 to 19 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 11 to 19 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 11 to 24 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 11 to 25 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 12 to 24 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 12 to 25 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 12 to 19 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is in the range of 13 to 19 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is in the range of 14 to 19 months.In another embodiment, the human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 15 to 19 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 16 to 19 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 17 to 19 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 18 to 19 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 11 to 18 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 12 to 17 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 13 to 16 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 14 to 15 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total survival of the treated population is within the range of 10 to 20 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total survival of the treated population is within the range of 11 to 20 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total survival of the treated population is within the range of 12 to 20 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total survival of the treated population is within the range of 13 to 20 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total survival of the treated population is within the range of 14 to 20 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total survival of the treated population is within the range of 15 to 20 months. In one embodiment, the human subject population is treated by the methods provided herein, wherein the total survival of the treated population is within the range of 16 to 20 months. In another embodiment, the human subject population is treated by the methods provided herein, wherein the total survival of the treated population is within the range of 17 to 20 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is in the range of 18 to 20 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is in the range of 19 to 20 months.In one embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 9 to 20 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 9 to 19 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 9 to 18 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 9 to 17 months. In one embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 9 to 16 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 9 to 15 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated population is within the range of 9 to 14 months. In another embodiment, a human subject population is treated by the methods provided herein, wherein the overall survival of the treated 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 overall survival of the treated 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 overall survival of the treated 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 overall survival of the treated population is within the range of 9 to 10 months.

[0301] In some embodiments of the methods provided herein, the pathological downstaging rate (pDSR) of the subject is at least 50%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 55%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 60%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 65%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 70%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 75%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 80%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 85%. In some embodiments, the pathological downstaging rate (pDSR) of the subject is at least 90%.

[0302] In some embodiments of the methods provided herein, the pathological downstaging rate (pDSR) is at least 50%. In some embodiments, the pathological downstaging rate (pDSR) is at least 55%. In some embodiments, the pathological downstaging rate (pDSR) is at least 60%. In some embodiments, the pathological downstaging rate (pDSR) is at least 65%. In some embodiments, the pathological downstaging rate (pDSR) is at least 70%. In some embodiments, the pathological downstaging rate (pDSR) is at least 75%. In some embodiments, the pathological downstaging rate (pDSR) is at least 80%. In some embodiments, the pathological downstaging rate (pDSR) is at least 85%. In some embodiments, the pathological downstaging rate (pDSR) is at least 90%.

[0303] In some embodiments of the methods provided herein, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 50%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 55%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 60%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 65%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 70%. In some embodiments, the pathological down-stage rate (pDSR) of the subject population treated with ADC is at least 75%.

[0304] In some embodiments, human subjects and patients are used interchangeably. Thus, it will be appreciated by those skilled in the art that in any of the methods provided herein, human subjects and patients are interchangeable.

[0305] 5.2.2 Methods of treating cancer in a patient population based on additional selection criteria

[0306] Provided herein are methods for treating a variety of cancers in a subject, wherein the cancer has any one of the suitable markers and / or characteristics provided in Section 6. Also provided herein are methods for treating a variety of cancers in a subject, wherein the subject has any one of the suitable characteristics provided in Section 6.

[0307] 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 a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 and one or more units of monomethyl auristatin E (MMAE), wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the 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.

[0308] 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 a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 and one or more units of monomethyl auristatin E (MMAE), wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the 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.

[0309] In another 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 a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 and one or more units of monomethyl auristatin E (MMAE), and wherein the subject has any one of the suitable characteristics provided in Section 6. In another 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 a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 and one or more units of monomethyl auristatin E (MMAE), and wherein the cancer has any one of the suitable markers and / or characteristics provided in Section 6.

[0310] In some embodiments of the methods provided herein (including Section 5.2, including Sections 5.2.1 and 5.2.2 and Sections 3 and 6), the subject is a human subject.

[0311] Of all methods provided herein and methods specifically described in Sections 5.2.1 and 5.2.2: therapeutic agents (including ADCs) that can be used are described in Sections 3, 5.2, 5.3, 5.4, 5.5, and 6, selecting patients for treatment is described herein and exemplified in Section 5.2 (including Sections 5.2.1 and 5.2.2) and Sections 3 and 6, dosing regimens and pharmaceutical compositions for administering therapeutic agents are described in Sections 5.4, 5.6, 5.7, and 6 below, and methods that can be used to identify therapeutic agents, select patients, and treat the disease are described herein and exemplified in Sections 5.2 (including Sections 5.2.1 and 5.2.2) and Sections 3 and 6. , determining the results of these methods and / or serving as a biomarker of criteria for these methods in any way, the biomarker can be determined as described in Section 5.8 or as known in the art, the treatment results of the methods provided herein are described in this section (Section 5.2, including Section 5.2.1.4) and Sections 3 and 6, other treatment results of the methods provided herein can be improvements of the biomarkers described herein, such as those described and exemplified in Sections 5.2 (including Sections 5.2.1 and 5.2.2) and Sections 3 and 6, and the combination therapy comprising ADC and other therapeutic agents is described in this section (Section 5.2) and Section 5.5. 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 results as described above and below.

[0312] 5.3 Antibody Drug Conjugates for Use in the Methods

[0313] In various embodiments of the methods provided herein (including the methods provided in Section 5.2), the ADC used in the method comprises or is an anti-191P4D12 ADC described herein and / or U.S. Patent No. 8,637,642, which is incorporated herein by reference in its entirety. In some embodiments, the anti-191P4D12 antibody drug conjugate provided herein to the method comprises an antibody or antigen-binding fragment thereof bound to 191P4D12 as provided herein (including Sections 3, 5.3.1 and 6) and a conjugate of one or more units (drug units, or D) of a cytotoxic agent as provided herein (including Sections 3 and 6, and this Section (Section 5.3)), and other contents are disclosed in Sections 5.3.2 and 5.3.4. In certain embodiments, a cytotoxic agent (drug unit, or D) may be covalently linked directly or via a linker unit (LU) as provided herein (including Sections 3 and 6, and this Section (Section 5.3)), and other contents are disclosed in Section 5.3.3.

[0314] In some embodiments, the antibody drug conjugate compound has the formula:

[0315] L-(LU-D) p (I)

[0316] or a pharmaceutically acceptable salt or solvate thereof; wherein:

[0317] L is an antibody unit, such as an anti-connexin-4 antibody or an antigen-binding fragment thereof, such as provided in Sections 3, 5.3.1 and 6, and

[0318] (LU-D) is a Linker Unit-Drug Unit moiety, wherein:

[0319] LU- is a linker unit, such as provided in Sections 3 and 6 and this section (Section 5.3), and otherwise disclosed in Section 5.3.3, and

[0320] D is a Drug unit having cytostatic or cytotoxic activity against target cells, such as provided in Sections 3 and 6 and this section (Section 5.3), with additional disclosure in Sections 5.3.2 and 5.3.4; and

[0321] p is an integer from 1 to 20, further examples are provided in Sections 3 and 6 and in this section (Section 5.3).

[0322] In some embodiments, p ranges from 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p ranges from 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.

[0323] In some embodiments, the antibody drug conjugate compound has the formula:

[0324] L-(A a -W w -Y y -D) p (II)

[0325] or a pharmaceutically acceptable salt or solvate thereof, wherein:

[0326] L is an antibody unit, e.g., an anti-Connexin-4 antibody or an antigen-binding fragment thereof, e.g., as provided in Sections 3, 5.3.1, and 6; and

[0327] -A a -W w -Y y - is a joint unit (LU), wherein:

[0328] -A- is the extension subunit,

[0329] a is 0 or 1,

[0330] Each -W- is independently an amino acid unit,

[0331] w is an integer in the range of 0 to 12,

[0332] -Y- is a self-degradable spacer unit,

[0333] y is 0, 1, or 2,

[0334] Each as provided, for example, in Sections 3 and 6 and this section (Section 5.3), with additional disclosure in Section 5.3.3;

[0335] D is a Drug unit having cytostatic or cytotoxic activity against target cells, such as provided in Sections 3 and 6 and this section (Section 5.3), with additional disclosure in Sections 5.3.2 and 5.3.4; and

[0336] p is an integer from 1 to 20, further examples are provided in Sections 3 and 6 and in this section (Section 5.3).

[0337] 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 ranges from 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p ranges from 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 zero, 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.

[0338] 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.

[0339] 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 per antibody during the preparation of the coupling reaction can be characterized by conventional methods such as mass spectrometry, ELISA assays and HPLC. The quantitative distribution of antibody drug conjugates can also be determined according to p. In some cases, separation, purification and characterization of homogeneous antibody drug conjugates (wherein p is a specific value of antibody drug conjugates with other drug loadings) can be achieved by means of, for example, reverse phase HPLC or electrophoresis. In certain exemplary embodiments, p is 2 to 8.

[0340] Other 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 incorporated herein by reference in their entirety.

[0341] In some embodiments of the methods provided herein (including Sections 3, 5.2, and 6 and this section (Section 5.3)), the ADC is enroku vedotin. In certain embodiments of the methods provided herein (including Sections 3, 5.2, and 6 and this section (Section 5.3)), the ADC is a biosimilar of enroku vedotin.

[0342] In some embodiments of the methods provided herein, the ADC is administered as a monotherapy.

[0343] 5.3.1 Anti-191P4D12 antibody or antigen-binding fragment

[0344] 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 the amino acid sequence of SEQ ID NO: 2 (see Figure 1A The corresponding cDNA encoding the 191P4D12 protein has the sequence of SEQ ID NO: 1 (see Figure 1A ).

[0345] 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.

[0346] In some embodiments, the anti-Connexin-4 antibodies provided herein are monoclonal antibodies.

[0347] In some embodiments, the antigen binding fragment is Fab, F(ab')2, Fv or scFv. In some embodiments, the antibody is a fully human antibody. In some embodiments, the antibody is IgG1 and the light chain is a kappa light chain. In some embodiments, the antibody or its antigen binding fragment is recombinantly produced.

[0348] 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), such as Figure 1B and Figure 1C as shown in .

[0349] In some embodiments, an anti-connexin-4 antibody or an 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 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 the corresponding complementary 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 the 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 as. Figure 1D and Figure 1E are shown in and listed below:

[0350] SEQ ID NO: 22

[0351]

[0352] SEQ ID NO: 23

[0353]

[0354]

[0355] SEQ ID NO: 7

[0356]

[0357] SEQ ID NO: 8

[0358]

[0359] CDR sequences can be determined according to well-known numbering systems. As described above, CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems. For example, the 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 loops (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 is present, the loop end is at 32; if only 35A is present, the loop end is at 33; if both 35A and 35B are present, 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 eds., 2nd edition, 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 specific to immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complex (MHC) of humans and other vertebrates. Herein, CDRs are referred to according to the amino acid sequence and the position within the light or heavy chain. Since the "position" of CDRs within the immunoglobulin variable domain structure is conserved between species and is present in structures called loops, CDR and framework residues are easily identified by using a numbering system that aligns the variable domain sequences according to structural features. This information can be used to transplant and replace CDR residues from an immunoglobulin from one species into an acceptor framework typically from a human antibody. 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 above.

[0360] 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.

[0361] 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.

[0362] 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 set forth 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 set forth in SEQ ID NO: 23 according to Chothia numbering.

[0363] In other embodiments, the anti-connexin-4 antibody or its antigen-binding fragment comprises: a heavy chain variable region, which includes CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) comprising 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, which includes CDRs comprising the amino acid sequence of the CDR of the light chain variable region shown in SEQ ID NO: 23 according to Contact numbering.

[0364] 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.

[0365] 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.

[0366] 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 sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO: 22 according to AbM numbering; and a light chain variable region comprising CDRs consisting of the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to AbM numbering.

[0367] 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 sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO: 22 according to Chothia numbering; and a light chain variable region comprising CDRs consisting of the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to Chothia numbering.

[0368] 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.

[0369] 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.

[0370] 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.

[0371] Table 4

[0372]

[0373] For another example, 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 as determined by ANARCI are listed in Table 5 below.

[0374] Table 5

[0375] 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: 1 8) QQANSFPPT (SEQ ID NO: 21)

[0376] In some embodiments, 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.

[0377] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 16; a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 17; a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 18; a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 19; a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 20; and a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 21.

[0378] In some embodiments, the antibody or antigen-binding fragment thereof 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.

[0379] In some embodiments, the antibody or antigen-binding fragment thereof comprises: CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 16; CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 17; CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 18; CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 19; CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 20; and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 21.

[0380] 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.

[0381] 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.

[0382] 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.

[0383] 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.

[0384] 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.

[0385] 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, being connected to cell ligands or other proteins, etc. Chemically modified antibodies. Any of a variety of chemical modifications can be carried out 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.

[0386] 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 a leucine replaced by a 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, the substitution is a conservative amino acid substitution produced at one or more predicted non-essential amino acid residues. The allowed variation 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.

[0387] 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.

[0388] 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, 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, such as 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.

[0389] Amino acids can be grouped according to the similarity of the properties of their side chains (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.

[0390] 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.

[0391] 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.

[0392] Covalent modifications of antibodies are included within the scope of the present disclosure. Covalent modifications include reacting 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; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the α-amino groups of lysine, arginine and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)); acetylation of the N-terminal amine; and amidation of any C-terminal carboxyl group.

[0393] 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.

[0394] 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 set forth as SEQ ID NO: 7 and a light chain having a certain homology or identity to the light chain set forth as 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 greater than 70% homology or identity to the heavy chain set forth as SEQ ID NO: 7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 75% homology or identity to the heavy chain set forth as SEQ ID NO: 7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 80% homology or identity to the heavy chain set forth as SEQ ID NO: 7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 85% homology or identity to the heavy chain set forth as 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 the light chain set forth in SEQ ID NO: 8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 85% homology or identity to the light chain set forth in SEQ ID NO: 8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 90% homology or identity to the light chain set forth in SEQ ID NO: 8. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 95% homology or identity to the light chain set forth in SEQ ID NO: 8.In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having any of the homologies or identities provided with the light chain 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 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.

[0395] 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 set forth in SEQ ID NO: 22 and a light chain variable region having a certain homology or identity to the light chain variable region set forth 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 greater than 70% homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having greater than 75% homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having greater than 80% homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having greater than 85% homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having greater than 90% homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having greater than 95% homology or identity to the heavy chain variable region set forth in SEQ ID NO: 22. In certain embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain variable region having any of the 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 to 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.

[0396] 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)).

[0397] 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)).

[0398] 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:

[0399] (a) the heavy chain variable region includes CDRs (CDR-H1, CDR-H2, and CDR-H3) comprising 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;

[0400] (b) The light chain variable region includes 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.

[0401] 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:

[0402] (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;

[0403] (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 under the American Type Culture Collection (ATCC) Accession No.: PTA-11267.

[0404] 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 known as 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 can be selected as the constant region of the antibodies of the present disclosure. In one embodiment, the human IgG1 constant region can be used as the heavy chain constant region and the human Igκ constant region can be used as the light chain constant region.

[0405] 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.

[0406] 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:

[0407] (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

[0408] (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.

[0409] 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 the antibody produced by the 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 variable region with any cognate heavy chain variable region as provided in this paragraph.

[0410] In other embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain and a light chain, wherein:

[0411] (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

[0412] (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 the antibody produced by the hybridoma deposited with the American Type Culture Collection (ATCC) under Accession No.: PTA-11267.

[0413] 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 amino acid sequence of the light chain 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 amino acid sequence of the light chain 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 amino acid sequence of the light chain 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 with any cognate heavy chain as provided in this paragraph.

[0414] 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 an epitope 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 embodi...

Claims

1. A method for treating muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) in a human subject, the method comprising (a) administering to the subject an effective amount of an antibody drug conjugate (ADC); wherein the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE); wherein the subject is not suitable for treatment with cisplatin (cisplatin-ineligible); and The pathological complete response rate (pCRR) was at least 30%.

2. A method of treating muscle-invasive urothelial carcinoma (MIUC) or muscle-invasive bladder cancer (MIBC) in a human subject, the method comprising (a) administering to the subject an effective amount of an antibody drug conjugate (ADC); wherein the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE); wherein the subject is not suitable for treatment with cisplatin (cisplatin-ineligible); and The pathological downstaging rate (pDSR) was at least 50%.

3. The method of claim 1 or claim 2, wherein the method comprises administering 3 cycles of the ADC to the subject.

4. The method of any one of claims 1 to 3, wherein the treatment of the cancer further comprises radical cystectomy and pelvic lymph node dissection (RC+PLND).

5. The method of claim 4, wherein the human subject receives the RC+PLND about four (4) weeks to about twelve (12) weeks after administration of the ADC to the human subject.

6. The method of claim 1 or 2, further comprising (b) performing radical cystectomy and pelvic lymph node dissection (RC+PLND) on the subject.

7. The method of claim 6, wherein (b) is performed from about four (4) weeks to about twelve (12) weeks after (a).

8. A method of treating muscle invasive urothelial carcinoma (MIUC) or muscle invasive bladder cancer (MIBC) in a human subject, the method comprising: administering to the subject an effective amount of an antibody drug conjugate (ADC) for multiple cycles, wherein (a) the ADC comprises a conjugate of an antibody or antigen-binding fragment thereof that binds to 191P4D12 (connexin-4) and one or more units of monomethyl auristatin E (MMAE); (b) the subject is not suitable for treatment with cisplatin (cisplatin-ineligible); and (c) the number of ADC treatment cycles administered to the cisplatin-ineligible subject is equal to or less than the number of cycles of standard of care (SOC) therapy used to treat cisplatin-eligible subjects with MIUC or MIBC.

9. The method of claim 8, wherein the SOC therapy for a cisplatin-appropriate subject comprises: i. Cisplatin; ii methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC); iii. Gemcitabine plus cisplatin; iv. administration of programmed cell death 1 (PD-1) inhibitors; or V. Administration of programmed cell death ligand 1 (PD--L1) inhibitors.

10. The method of claim 8 or 9, wherein the number of ADC treatment cycles administered to the cisplatin-ineligible subject is 3 or 4.

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