CN116710483A

CN116710483A - Methods of treating cancer with Antibody Drug Conjugates (ADCs) that bind to 191P4D12 protein

Info

Publication number: CN116710483A
Application number: CN202180077229.3A
Authority: CN
Inventors: 伊莱娜·玛丽·加特纳
Original assignee: Sijin Co; Agensys Inc
Current assignee: Sijin Co; Agensys Inc
Priority date: 2020-09-17
Filing date: 2021-09-16
Publication date: 2023-09-05
Also published as: CN117224698A

Abstract

Provided herein are methods of treating cancer with Antibody Drug Conjugates (ADCs) that bind to 191P4D12 protein.

Description

Methods of treating cancer with Antibody Drug Conjugates (ADCs) that bind to 191P4D12 protein

Cross Reference to Related Applications

The present application claims the benefit of U.S. application Ser. No. 63/080,013, U.S. application Ser. No. 63/196,641, and U.S. application Ser. No. 63/240,794, both filed on 9 and 17, 2021, and 3, 2021, each of which is incorporated herein by reference in its entirety. Electronically submitted references to sequence listings

The present application contains a sequence listing submitted electronically via EFS-Web as an ASCII format sequence listing, with a file name of "14369-274-228_SEQ_LISTING. Txt" and a creation date of 2021, 9, 13 and a size of 39,755 bytes. The sequence listing submitted via EFS-Web is part of this specification and is incorporated by reference herein in its entirety.

1. Technical field

Provided herein are methods of treating cancer with Antibody Drug Conjugates (ADCs) that bind to 191P4D12 protein (connexin-4).

2. Background art

Cancer is the leading cause of death in the 35 to 65 year old population in the united states and it is the second leading cause of death in the worldwide population. It is estimated that there will be about 170 tens of thousands of new Cancer cases in the united states in 2019 and about 610000 cases die from Cancer (National Cancer institute.2019.cancer Stat pictures: cancer of Any Site.seer.cancer/status/html/all.html.2019, 6 months 5 acquisition). There were estimated 1810 ten thousand new cancer cases worldwide in 2018 and approximately 960 deaths from cancer in 2018 (World Health organization.press release.2018, 9 month.who.int/cancer/prglobochannel.pdf.2019, 6 month 5 day acquisition). Most deaths occur in patients with metastatic cancer. Indeed, advances in therapy, including surgery, radiation therapy, and adjuvant chemotherapy, have healed most patients with localized cancers over the past 20 years. The appropriate benefit of cancer as a metastatic disease occurrence or recurrence in patients from conventional therapy is only manifested in terms of total survival (OS) and little cure.

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

191P4D12 (also known as connexin-4) is a type I transmembrane protein and is a member of the family of related immunoglobulin-like adhesion molecules involved in intercellular adhesion. 191P4D12 belongs to the connexin family of adhesion molecules. 191P4D12 is composed of an extracellular domain (ECD) containing 3 Ig-like domains, a transmembrane helix and an intracellular domain (Takai Y et al Annu Rev Cell Dev Biol 2008; 24:309-42). It is thought that connexins mediate Ca via both homophilic and homophilic trans-interactions at the adhesion junction ²⁺ Independent Cell-Cell adhesion, where it recruits cadherins and regulates cytoskeletal rearrangement (Rikitake and Takai, cell Mol Life Sci.2008;65 (2): 253-63). 191P4D12 has very low sequence identity with other connexin family members and ranges between 25% and 30% in ECD (Reymond N et al J Biol Chem2001; 43205-15). The adhesion promoted by connexins supports several biological processes such as immunomodulation, host-pathogen interactions and immune evasion (Sakisaka T et al Current Opinion in Cell Biology2007; 19:593-602).

Urothelial carcinoma

According to the international cancer research institute (International Agency for Research on Cancer; IARC), urothelial cancer kills over 165000 patients annually and is the ninth most common cancer worldwide. Approximately 151000 new cases of urothelial cancer are diagnosed annually in europe, with 52000 deaths annually. Over 22000 new cases are diagnosed annually in japan, with 7600 deaths annually. Cancer Fact Sheets All cancers excluding Non-Melanoma skin.International Agency for Research on cancer2017 retrieved from gco.iarc.fr/today/face-pieces-cancer=29 & type=0 & sex= 0.2017, 12 months, 19 days. According to the national cancer institute (National Cancer Institute), it was estimated that over 79000 newly increased cases of urothelial cancer were diagnosed at 2017 and over 16000 people died from the disease in the united states (the United States/US). SEER Cancer Stat Facts Blader Cancer national Cancer Institute.Bethesda, MD, seer Cancer govstatfarads/html/url. Html.2017, 12 months 19.

First line therapy for metastatic urothelial cancer in patients with adequate renal function consists of cisplatin (cisplatin) based combinations, such as: methotrexate (methotrexa), vinblastine (vinblastine), doxorubicin (doxorubicin) and cisplatin (MVAC); or gemcitabine plus cisplatin, which indicates a total reaction rate of up to 50%, including about 10-15% complete reaction (complete response; CR). Bellmutt J et al, N Engl J med.2017;376:1015-26.DOI:10.1056/NEJMoa1613683. Regardless of the initial chemosensitivity, patients were incured and the outcome of metastatic urothelial cancer was poor following these regimens: median time to progression is only 7 months and median total survival (OS) is 14 months. Approximately 15% of patients survive for at least 5 years and are particularly poor prognosis in patients with visceral metastasis, with a 5 year OS rate of 7%. von der Maase H et al, J Clin Oncol.2005;23:4602-8.

For two-line therapy, the small molecule tubulin inhibitor vinflunine (vinflunine) is approved only in europeThe median OS was 6.9 months compared to the median OS of 4.6 months for optimal supportive care. Bellmunt J et al, J Clin Oncol.2009;27:4454-61. For decades, the therapeutic prospects of only available cytotoxic chemotherapy have not changed significantly until recently an immune checkpoint inhibitor (check point inhibitor; CPI) targeting programmed death 1/programmed death-ligand 1 (PD-1/PD-L1) was approved. Starting with the PD-L1 inhibitor atezolizumab, 5 months in 2016, several CPIs have been FDA approved in the united states for urothelial cancer in platinum-pretreated patients. Most approvals have been based on single set phase II data (single arm phase II data). See Tecentriq Prescribing Information, genentech, month 4 2017, opdivo Prescribing Information, bristol-Myers Squibb, month 9 2017, imfinzi Prescri bing Information, astraZeneca, 5 months 2017 and Bavencio Prescribing Information, EMD Serono, 3 months 2017. However, in 2017, the results of phase III trial KEYNOTE-045 demonstrated significantly longer survival in patients treated with pamglizumab (pembrolizumab) when compared to standard two-wire chemotherapy. Bellmutt J et al, N Engl jmed.2017;376:1015-26.DOI:10.1056/NEJMoa1613683. This resulted in the conventional approval of pamphlet Li Zhushan as a second line treatment for patients with locally advanced or metastatic urothelial cancer (mUC; keytruda Prescribing Information, merck, month 9 2017). The approval was based on a median OS of 10.3 months for palbociclizumab compared to a median OS of 7.4 months for taxane (taxane) chemotherapy or vinflunine. Bellmutt J et al, N Engl J med.2017;376:1015-26.DOI:10.1056/NEJMoa1613683. CPI is then approved for sale in europe and is expected to be approved in asia. Other PD-1 and PD-L1 inhibitors are currently evaluated as first and second line therapies in clinical trials for urothelial cancer. Mullane SA and Bellmunt J.Curr Opin Urol.2016;26:556-63.

Currently, no therapy is approved for patients with locally advanced or mUC who were previously treated with CPI.

Bladder cancer

In all new cancer cases in the united states, bladder cancer accounts for approximately 5% in men (fifth most common neoplasm) and 3% in women (eighth most common neoplasm). As the elderly population increases, the incidence is slowly increasing. The american cancer society (cancer. Org) estimates an annual increase in 81,400 cases, including male 62,100 and female 19,300, accounting for 4.5% of all cancer cases. In the United states, the incidence of age-adjusted male and female is 20/100,000. It is estimated that 17,980 deaths per year are due to bladder cancer (male 13,050 and female 4,930), accounting for 3% of cancer-related deaths. Bladder cancer morbidity and mortality increases dramatically with age and becomes an increasingly serious problem as the population becomes more aged.

Most bladder cancers recur in the bladder. Bladder cancer is treated with transurethral resection of the bladder (TUR) in combination with intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer indicates the limitations of TUR. TUR alone does not cure most muscle invasive cancers. Radical cystectomy and uroflow surgery are the most effective means of eliminating cancer, but have undeniable effects on urinary and sexual function. There continues to be a considerable need for therapeutic modalities that are beneficial to patients with bladder cancer.

There is a considerable need for additional treatments for urothelial and bladder cancers. These include the use of antibodies and antibody drug conjugates as therapeutic modalities.

3. Summary of the invention

Provided herein are methods of treating various cancers in a subject, including a subject having a previously treated locally advanced or metastatic urothelial cancer, using an Antibody Drug Conjugate (ADC) that binds 191P4D 12.

In some embodiments, the prior treatment comprises platinum-based chemotherapy. In certain embodiments, the prior treatment comprises an immune checkpoint inhibitor (CPI). In other embodiments, the prior treatment includes both platinum-based chemotherapy and CPI.

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

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

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

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

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

wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or More Monomethyl Auristatin E (MMAE) units, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a Complementarity Determining Region (CDR) comprising the amino acid sequence of a CDR of the heavy chain variable region shown in SEQ ID NO. 22; and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region shown in SEQ ID NO. 23.

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

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

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

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

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

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

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

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

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

Embodiment 10. The method of embodiment 1, wherein the subject has a total survival of at least or about 9 months after the treatment.

Embodiment 11. The method of embodiment 2, wherein the subject has a total survival of at least or about 12 months after the treatment.

Embodiment 12. The method of embodiment 3, wherein the subject has a total survival of at least or about 11 months after the treatment.

Embodiment 13. The method of any one of embodiments 1 to 3, wherein the subject has a total survival in the range of 9 to 19 months after the treatment.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

(ii) Platelet count is not less than 100X 109/L;

(iii) Hemoglobin is not less than 9g/dL;

(iv) Serum bilirubin does not exceed an upper normal limit of 1.5 times (upper limit of normal; ULN) or 3 times ULN for patients with Gilbert's disease;

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

(vi) Alanine aminotransferase and aspartate aminotransferase are no more than 3 times ULN.

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

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

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

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

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

Embodiment 53 the method of embodiment 52, wherein the uncontrolled diabetes is determined by hemoglobin A1c (HbA 1 c) of no less than 8% or HbA1c between 7% and 8% with associated diabetes symptoms not otherwise explained.

Embodiment 54 the method of embodiment 53, wherein the associated diabetic symptom comprises or consists of: polyuria, polydipsia, or both polyuria and polydipsia.

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

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

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

Embodiment 58 the method of embodiment 56, wherein the PD-L1 inhibitor is selected from the group consisting of: alemtuzumab (atezolizumab), avistuzumab (avelumab), and devaluzumab (durvalumab).

Embodiment 59. The method of any one of embodiments 1 to 58, wherein the antibody or antigen-binding fragment thereof comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO. 9, CDR-H2 comprising the amino acid sequence of SEQ ID NO. 10, CDR-H3 comprising the amino acid sequence of SEQ ID NO. 11; CDR-L1 comprising the amino acid sequence of SEQ ID NO. 12, CDR-L2 comprising the amino acid sequence of SEQ ID NO. 13 and CDR-L3 comprising the amino acid sequence of SEQ ID NO. 14, or

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

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

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

Embodiment 61 the method of any one of embodiments 1 to 60, wherein the antibody or antigen binding fragment thereof comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 23.

Embodiment 62 the method of any one of embodiments 1 to 61, wherein the antibody comprises: a heavy chain comprising an amino acid sequence in the range of amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID No. 7; and a light chain comprising an amino acid sequence in the range of amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO. 8.

Embodiment 63. The method of any one of embodiments 1 to 61, wherein the antigen binding fragment is a Fab, F (ab') 2, fv, or scFv.

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

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

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

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

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

Embodiment 69 the method of embodiment 67 or 68, wherein the linker has the formula: -Aa-Ww-Yy-; wherein-A-is an extension subunit, a is 0 or 1; -W-is an amino acid unit, W is an integer ranging from 0 to 12; and-Y-is a spacer unit, Y is 0, 1 or 2.

Embodiment 70. The method of embodiment 69 wherein the extension subunit has the structure of formula (1); the amino acid unit is valine-citrulline; and the spacer unit is a PAB group comprising the structure of formula (2):

embodiment 71 the method of embodiment 69 or 70 wherein the extension subunit forms a bond with a sulfur atom of the antibody or antigen binding fragment thereof; and wherein the spacer unit is linked to MMAE via a carbamate group.

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

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

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

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

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

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

Embodiment 77. The method of embodiment 76 wherein p is 2 to 8.

Embodiment 78. The method of embodiment 76 or 77, wherein p is 3 to 5.

Embodiment 79 the method of any one of embodiments 76 to 78 wherein p is 3 to 4.

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

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

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

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

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

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

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

Embodiment 87 the method of any one of embodiments 1 to 86, wherein said ADC is administered by IV injection or infusion three times every four week period.

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

Embodiment 89 the method of any one of embodiments 1-88, wherein the ADC is administered by IV injection or infusion for about 30 minutes three times every four week period.

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

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

Embodiment 92 the method of any one of embodiments 1 to 91, wherein the ADC is formulated in a pharmaceutical composition comprising about 20mM L-histidine, about 0.02% (w/v) TWEEN-20, about 5.5% (w/v) trehalose dihydrate and hydrochloride, and wherein the pH of the pharmaceutical composition at 25 ℃ is about 6.0.

Embodiment 93 the method of any one of embodiments 1-91, wherein the ADC is formulated in a pharmaceutical composition comprising about 9mM histidine, about 11mM histidine monohydrochloride, about 0.02% (w/v) TWEEN-20 and about 5.5% (w/v) trehalose dihydrate, and wherein the pH of the pharmaceutical composition at 25 ℃ is about 6.0.

Embodiment 94 the method of any one of embodiments 1 to 93 wherein the ADC is enrolment Shan Kangwei statin (enfortumab vedotin; EV) or a biological analogue thereof, wherein the EV is administered at a dose of about 1.25mg/kg of body weight of the subject, and wherein the dose is administered by IV injection or infusion for about 30 minutes on days 1, 8 and 15 of each four week cycle.

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

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

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

Embodiment 98 the method of any one of embodiments 1 to 94, wherein the population of subjects has stable disease after the treatment.

4. Description of the drawings

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

Fig. 2A depicts the overall study design of the clinical study described in section 6.1. Fig. 2B depicts a study protocol of the clinical study described in section 6.1. Fig. 2C depicts the EuroQOL 5 dimension (EQ-5D-5L) described in section 6.1.

Figure 3 depicts the analytical efficacy boundaries of the clinical study described in section 6.1. Note #: the predetermined efficacy boundary IDMC for IA (= 0.00661) of OS is adjusted using 299 deaths observed by the initial data snapshot up to each initial cut-off value. Two deaths were reported after the initial data snapshot, and thus 301 deaths were shown in the primary analysis (primary analysis) table of OS in TLR.

Fig. 4 depicts the overall survival Kaplan Meier curve-FAS for the clinical study described in section 6.1.

Figure 5 depicts overall survival subgroup results for the clinical study described in section 6.1.

FIG. 6 depicts the PFS Kaplan Meier Curve-FAS of the clinical study described in section 6.1.

Fig. 7 depicts the results of subgroup analysis, which shows that there is a progression free survival benefit of enrolment Shan Kangwei Statins (EVs) in multiple subgroups.

Fig. 8 depicts the results of subgroup analysis of total reaction rates.

Figure 9 depicts Kaplan-Meier estimates of response duration assessed by researchers in all patients with confirmed complete or partial responses based on treatment groups.

Fig. 10A-10D depict Kaplan-Meier estimates based on total survival of subgroups. FIG. 10A depicts subgroups aged 65 years or older. Fig. 10B depicts a subgroup in which liver metastasis is present. Fig. 10C depicts a subgroup with primary upper urinary tract disease. FIG. 10D depicts no response to a previous subgroup of PD-1/L1 inhibitors. Abbreviations: CI, confidence interval; HR, risk ratio; PD-1/L1, apoptosis protein-1 or apoptosis-ligand 1.

Fig. 11A-11D depict Kaplan-Meier estimates of non-progressive survival based on subgroups. FIG. 11A depicts subgroups aged 65 years or older. Fig. 11B depicts a subgroup in which liver metastasis is present. Fig. 11C depicts a subgroup with primary upper urinary tract disease. FIG. 11D depicts no response to a previous subgroup of PD-1/L1 inhibitors. Abbreviations: CI, confidence interval; HR, risk ratio; PD-1/L1, apoptosis protein-1 or apoptosis-ligand 1.

Fig. 12 depicts treatment-related adverse events (safety populations). Abbreviations: EV, enrolment Shan Kangwei statin; PD-1/L1, apoptosis protein-1 or apoptosis-ligand 1; SC, standard chemotherapy.

FIG. 13 depicts treatment-related adverse events of ≡3 with adjusted exposures in refractory subgroups. Abbreviations: EV, enrolment Shan Kangwei statin; PD-1/L1, apoptosis protein-1 or apoptosis-ligand 1; SC, standard chemotherapy.

5. Detailed description of the preferred embodiments

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

5.1 definition

Techniques and procedures described or referred to herein include those used by those skilled in the art using conventional methods, such as Sambrook et al, molecular Cloning: A Laboratory Manual (3 rd edition 2001); current Protocols in Molecular Biology (Ausubel et al, 2003); therapeutic Monoclonal Antibodies: from Bench to Clinic (An et al 2009); monoclonal Antibodies: methods and Protocols (Albitar 2010); and Antibody Engineering volumes 1 and 2 (Kontermann and Dubel, 2 nd edition 2010) are well understood and/or commonly employed in general.

Unless defined otherwise herein, technical and scientific terms used in this specification shall have the meanings commonly understood by one of ordinary skill in the art. For the purposes of explaining the present specification, the following description of terms will be applied and terms used in the singular will also include the plural and vice versa, as appropriate. To the extent that any description of the terms set forth conflicts with any document incorporated herein by reference, the terms set forth below shall govern.

The terms "antibody", "immunoglobulin" or "Ig" are used interchangeably herein and are used in the broadest sense and specifically cover, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions having multi-or mono-epitope specificity, polyclonal or monovalent antibodies, multivalent antibodies, multi-specific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single chain antibodies, and fragments thereof, as described below. The antibodies can be human antibodies, humanized antibodies, chimeric antibodies, and/or affinity matured antibodies, as well as antibodies from other species (e.g., mice and rabbits, etc.). The term "antibody" is intended to include the polypeptide product of a B cell within a polypeptide of the immunoglobulin class, which is capable of binding to a particular molecular antigen and is composed of two pairs of identical polypeptidesChains are constructed in which each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain includes a variable region having about 100 to about 130 or more amino acids, and each carboxy-terminal portion of each chain includes a constant region. See, e.g., antibody Engineering (Borrebaeck, 2 nd edition, 1995); and Kuby, immunology (3 rd edition, 1997). In particular embodiments, a particular molecular antigen may be bound by an antibody (including a polypeptide or epitope) provided herein. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments (e.g., antigen binding fragments) of any of the above, which refer to portions of an antibody heavy or light chain polypeptide 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 Fragments, F (ab') ₂ Fragments, disulfide-linked Fv (dsFv), fd fragments, fv fragments, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen binding domains or molecules (e.g., one or more CDRs of an antibody) that contain an antigen binding site that binds an antigen. Such antibody fragments can be found, for example, in Harlow and Lane, antibodies: A Laboratory Manual (1989); mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, 1995); huston et al, 1993,Cell Biophysics 22:189-224; pluckthun and Skerra,1989, meth. Enzymol.178:497-515; and Day, advanced Immunochemistry (2 nd edition 1990). 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 IgA 2) of immunoglobulin molecules. The antibody may be an agonistic or antagonistic antibody.

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

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

An "intact" antibody is an antibody comprising an antigen binding site, CL and at least heavy chain constant regions CH1, CH2 and CH 3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In certain embodiments, the intact antibody has one or more effector functions.

The terms "antigen binding fragment," "antigen binding domain," "antigen binding region," and similar terms refer to the portion of an antibody that comprises amino acid residues that interact with an antigen and confer upon the binding agent its specificity and affinity for the antigen (e.g., CDR). As used herein, "antigen binding fragment" includes "antibody fragment" that comprises a portion of an intact antibody, e.g., the antigen binding or variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, fab ', F (ab') ₂ And Fv fragments; bifunctional and di-bifunctional antibodies (see, e.g., holliger et al, 1993, proc. Natl. Acad. Sci.90:6444-48; lu et al, 2005, J. Biol. Chem.280:19665-72; hudson et al, 2003, nat. Med.9:129-34; WO 93/11161; and U.S. Pat. Nos. 5,837,242 and 6,492,123); single chain antibody molecules (see, e.g., U.S. Pat. Nos. 4,946,778; 5,260,203; 5,482,858; and 5,476,786); a double variable domain antibody (see, e.g., U.S. patent No. 7,612,181); single variable domain antibodies (sdabs) (see, e.g., woolven et al, 1999,Immunogenetics 50:98-101; and Streltsov et al, 2004,Proc Natl Acad Sci USA.101:12444-49); and multispecific antibodies formed from antibody fragments.

The term "binding" refers to interactions between molecules, including, for example, the formation of complexes. The interactions may be, for example, non-covalent interactions, including hydrogen bonding, ionic bonding, hydrophobic interactions, and/or van der Waals interactions (van der Waals interaction). A complex may also include the binding of two or more molecules that are 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., antigen) is the affinity of the antibody or functional fragment for that epitope. The ratio of the dissociation rate (koff) of the binding molecule (e.g., antibody) to the monovalent antigen (koff) to the binding rate (kon) is the dissociation constant K _D Which is inversely related to affinity. K (K) _D The lower the value, the higher the antibody affinity. K (K) _D The values vary depending on the different complexes of antibody and antigen and depend on k _on And k is equal to _off Both of which are located in the same plane. Dissociation constant K of the antibodies provided herein _D Any of the methods provided herein or any other method well known to those of skill in the art may be used. The affinity at one binding site does not always reflect the true strength of interaction between the antibody and antigen. When complex antigens containing multiple, repetitive epitopes (e.g., multivalent antigens) are contacted with antibodies containing multiple binding sites, the interaction of the antibodies with the antigen at one site will increase the probability of reaction at the second site. The strength of such multiple interactions between multivalent antibodies and antigens is referred to as avidity.

Terms that bind to an antibody or antigen-binding fragment thereof described herein, such as "bind to", "specifically bind to (that specifically bind to)", and the like, are also used interchangeably herein and refer to a binding molecule having an antigen-binding domain that specifically binds to an antigen (e.g., a polypeptide). Antibodies or antigen binding fragments that bind or specifically bind to an antigen may have cross-reactivity with the antigen of interest. In certain embodiments, an antibody or antigen binding fragment that binds or specifically binds to an antigen does not have cross-reactivity with other antigens. Bound to or specifically bound toAntibodies or antigen binding fragments of the antigen can be prepared, for example, by immunoassays,Or other techniques known to those skilled in the art. In some embodiments, an antibody or antigen-binding fragment binds to or specifically binds to an antigen 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, such as Radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). Typically, the specific or selective response will be at least twice the background signal or noise and may exceed 10 times the background. For a discussion of binding specificity see, e.g., fundamental Immunology 332-36 (Paul et al, 2 nd edition, 1989). In certain embodiments, the extent of binding of an antibody or antigen binding fragment to a "non-target" protein is less than about 10% of the binding molecule or antigen binding domain to its particular target antigen, e.g., as determined by fluorescence activated cell sorting (fluorescence activated cell sorting; FACS) analysis or RIA. Terms such as "specific binding," "specific binding to," or "specific for … …" mean binding measurably distinct from non-specific interactions. Specific binding can be measured, for example, by determining binding of a molecule as compared to binding of a control molecule, which is typically a molecule having a similar structure that is not binding active. For example, specific binding can be determined by competition with a control molecule similar to the target (e.g., an excess of unlabeled target). In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. Antibodies or antigen binding fragments that bind to an antigen include antibodies or antigen binding fragments that are capable of binding to an antigen with sufficient affinity to render the binding molecule useful as, for example, a diagnostic agent in targeting the antigen. In certain embodiments, the antibody or antigen binding fragment that binds to the antigen has a dissociation constant (K _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 among antigens from different species (e.g., between human and cynomolgus species).

"binding affinity" generally refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an inherent 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 determined by the dissociation constant (K _D ) And (3) representing. Affinity can be measured by common methods known in the art, including those described herein. Low affinity antibodies typically bind antigen slowly and tend to dissociate easily, while high affinity antibodies typically bind antigen faster and tend to remain bound for longer periods of time. A variety of methods of measuring binding affinity are known in the art, any of which may be used for the purposes of the present disclosure. Specific illustrative embodiments include the following. In one embodiment, "K _D "or" K _D The value "may be measured by assays known in the art, for example by binding assays. K (K) _D RIA measurements can be performed, for example, using the Fab form of the antibody of interest and its antigen (Chen et al, 1999,J.Mol Biol 293:865-81). K (K) _D Or K _D The values can also be determined by using biological layer interferometry (biolayer interferometry; BLI) or surface plasmon resonance (surface plasmon resonance; SPR), byUsing for example +.>QK384 system, or by->Using for example +.>TM-2000 or->TM-3000. "association rate" or "kon" may also be used with the same Biological Layer Interferometry (BLI) or Surface Plasmon Resonance (SPR) techniques described above using, for example>QK384、TM-2000 or->TM-3000 system.

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

In certain embodiments, an antibody or antigen binding fragment may comprise a portion of a "humanized" form of a non-human (e.g., murine) antibody, which is a chimeric antibody (e.g., recipient antibody) comprising a human immunoglobulin, in which the native CDR residues are replaced with residues from the corresponding CDRs of a non-human species (e.g., mouse, rat, rabbit, or non-human primate) having the desired specificity, affinity, and capacity (e.g., donor antibody). In some cases, one or more FR region residues of a human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or the donor antibody. These modifications were made to further optimize antibody performance. The humanized antibody heavy or light chain may comprise substantially all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to CDRs of a non-human immunoglobulin and all or substantially all of the FR are FR of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al, 1986,Nature 321:522-25; riechmann et al 1988,Nature 332:323-29; presta,1992, curr.op. Struct. Biol.2:593-96; carter et al, 1992, proc. Natl. Acad. Sci. USA89:4285-89; U.S. patent No. 6,800,738; 6,719,971; 6,639,055; 6,407,213; and No. 6,054,297.

In certain embodiments, an antibody or antigen binding fragment may comprise a "fully human antibody" or a portion of a "human antibody," wherein the terms are used interchangeably herein and refer to an antibody comprising human variable regions and, for example, human constant regions. In particular embodiments, the term refers to antibodies comprising variable and constant regions of human origin. In certain embodiments, "fully human" antibodies may also encompass antibodies that bind to polypeptides and are encoded by nucleic acid sequences that are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequences. The term "fully human antibody" includes antibodies comprising variable and constant regions 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, U.S. Pat. No. of Health and Human Services, NIH publication No. 91-3242). A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human and/or has been made using any technique for making human antibodies. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phagocytosis Cell display libraries (Hoogenboom and Winter,1991, J.mol. Biol.227:381; marks et al, 1991, J.mol. Biol. 222:581) and yeast display libraries (Chao et al, 2006,Nature Protocols 1:755-68). Cole et al, monoclonal Antibodies and Cancer Therapy 77 (1985); boerner et al, 1991, J.Immunol.147 (1): 86-95; and van Dijk and van de Winkel,2001, curr. Opin. Pharmacol.5:368-74, 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 antigen stimulation, but whose endogenous locus has been disabled (see, e.g., jakobovits,1995, curr. Opin. Biotechnol.6 (5): 561-66; brucggemann and Taussing,1997, curr. Opin. Biotechnol.8 (4): 455-58; and U.S. Pat. Nos. 6,075,181 and 6,150,584, in relation to XENOMOUSE) ^TM Technology). See also, for example, li et al 2006, proc.Natl. Acad.Sci.USA 103:3557-62 for human antibodies produced via human B cell hybridoma technology.

In certain embodiments, an antibody or antigen-binding fragment may comprise a portion of a "recombinant human antibody," wherein the phrase includes human antibodies produced, expressed, produced, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell; an antibody isolated from a recombinant combinatorial human antibody library; antibodies isolated from transgenic and/or transchromosomal animals (e.g., mice or cattle) of human immunoglobulin genes (see, e.g., taylor, l.d. et al (1992) nucleic acids res.20:6287-6295) or antibodies produced, expressed, produced or isolated by any other means that involves splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, e.a. et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. device of Health and Human Services, NIH disclosure No. 91-3242). However, in certain embodiments, such recombinant human antibodies are subject to in vitro mutagenesis (or somatic mutation induction in vivo when transgenic animals of human Ig sequences are used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and associated with human germline VH and VL sequences, do not naturally occur in vivo within the human antibody germline antibody repertoire.

In certain embodiments, an antibody or antigen binding fragment may comprise a portion of a "monoclonal antibody," wherein the term, as used herein, 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 typically recognize a single epitope on an antigen. In particular 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 producing antibodies. For example, monoclonal antibodies suitable for use in the present disclosure may be prepared by the hybridoma method described for the first time by Kohler et al, 1975,Nature 256:495, or may be prepared using recombinant DNA methods in bacterial 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 described, for example, 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 thereby are well known in the art. See, e.g., short Protocols in Molecular Biology (Ausubel et al, 5 th edition 2002).

Typical 4-chain antibody units are hetero-tetranectin proteins, which consist of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H chain and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for each α and γ chain and four CH domains of μ and ε isoforms. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at its other end. VL is aligned with VH and CL is aligned with the first constant domain of the heavy chain (CH 1). It is believed that a particular amino acid residue forms an interface between the light chain variable domain and the heavy chain variable domain. VH and VL pair together to form a single antigen binding site. See, e.g., basic and Clinical Immunology (Stites et al, 8 th edition 1994) for the structure and properties of different classes of antibodies; and Immunobiology (Janeway et al, 5 th edition 2001).

The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. Conventional IgG typically comprises two Fab regions, each residing on one of the two arms of a Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region for each of the heavy and light chains. More specifically, the heavy chain variable and constant regions in the Fab region are VH and CH1 regions, and the light chain variable and constant regions in the Fab region are VL and CL regions. VH, CH1, VL and CL in the Fab region may be arranged in various ways to confer antigen binding ability according to the present disclosure. For example, the VH and CH1 regions may be located on one polypeptide, and the VL and CL regions may be located on separate polypeptides, similar to the Fab region of a conventional IgG. Alternatively, the VH, CH1, VL, and CL regions may all be on the same polypeptide and oriented in different orders as described in more detail in the following sections.

The terms "variable region," "variable domain," "V region," or "V domain" refer to a portion of an antibody's light or heavy chain that is typically located at the amino terminus of the light or heavy chain and is about 120 to 130 amino acids in length in the heavy chain and about 100 to 110 amino acids in length in the light chain, and is used for the binding and specificity of each particular antibody for its particular 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 differ widely in terms of the sequence in an antibody. The V region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is unevenly distributed over 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 (referred to as a Framework Region (FR)) separated by shorter regions of greater variability (e.g., extreme variability), referred to as "hypervariable regions", each about 9-12 amino acids in length. The variable regions of the heavy and light chains each comprise four FR largely in the beta sheet configuration, joined by three hypervariable regions that form loops connecting the beta sheet structure and in some cases form part of the beta sheet structure. The hypervariable regions in each chain are tightly bound together by the FR to hypervariable regions from other chains, facilitating the formation of the antigen binding site of the antibody (see, e.g., kabat et al, sequences of Proteins of Immunological Interest (5 th edition, 1991)). The constant region is not directly involved in binding of an antibody to an antigen, but exhibits a variety of effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (antibody dependent cellular cytotoxicity; ADCC) and complement-dependent cytotoxicity (complement dependent cytotoxicity; CDC). The variable regions vary widely in sequence between different antibodies. In a specific embodiment, the variable region is a human variable region.

The term "variable region residue number according to Kabat" or "amino acid position number as in Kabat" and variations thereof, refers 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 fewer or additional amino acids corresponding to or inserted into the shortening of the FRs or CDRs of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion following residue 52 (residue 52a, according to Kabat) and three insertion residues following residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). For a given antibody, the Kabat numbering of residues can be determined by aligning the homologous regions of the antibody sequences with a "standard" Kabat numbering sequence. The Kabat numbering system is generally used in reference to residues in the variable domain (about residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., kabat et al, supra). The "EU numbering system" or "EU index" is generally used in reference to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in the co-pending document to Kabat et al). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems have been described, for example, by AbM, chothia, contact, IMGT and AHon.

When used with reference to an antibody, the term "heavy chain" refers to a polypeptide chain of about 50-70kDa, wherein the amino-terminal portion includes a variable region having about 120 to 130 or more amino acids, and the carboxy-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., isoforms), referred to as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). The different heavy chains vary in size: alpha, delta and gamma contain about 450 amino acids, while mu and epsilon contain about 550 amino acids. When combined with light chains, these different types of heavy chains produce five well-known classes (e.g., isotypes) of antibodies, igA, igD, igE, igG and IgM, respectively, including the four subclasses of IgG, namely IgG1, igG2, igG3, and IgG4.

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

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

CDR regions are well known to those skilled in the art and have been defined by the well known numbering system. For example, kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are most commonly used (see, e.g., kabat et al, supra). Chothia actually refers to the position of the structural loop (see, e.g., chothia and Lesk,1987, J.mol. Biol. 196:901-17). When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the length of the loop (since the Kabat numbering scheme places insertions in H35A and H35B;if neither 35A nor 35B is present, then the loop ends are located at 32; if only 35A is present, the loop end is at 33; if 35A and 35B are both present, then the loop end is at 34). The AbM hypervariable region represents a compromise between Kabat CDRs and Chothia structural loops and is used by Oxford Molecular AbM antibody modeling software (see, e.g., volume Antibody Engineering, volume 2 (Kontermann and dubel, editions 2, 2010)). The "contact" hypervariable region is based on analysis of available complex crystal structures. Another common numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information (Lafranc et al, 2003, dev. Comp. Immunol.27 (1): 55-77). IMGT is an integrated information system specific for Immunoglobulins (IG), T-cell receptors (TCR) and major histocompatibility complexes (major histocompatibility complex; MHC) of humans and other vertebrates. Herein, CDRs are referred to with respect to amino acid sequences and positions within the light chain or heavy chain. Since the "positions" of CDRs within an immunoglobulin variable domain structure are conserved across species and exist in a structure called a loop, CDRs and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from immunoglobulins of a species into the acceptor framework typically from human antibodies. Another numbering system (AHon) has been developed by Honyger and Pluckthun, 2001, J.mol.biol.309:657-70. Correspondence between numbering systems (including, for example, kabat numbering and IMGT unique numbering systems) is well known to those skilled in the art (see, for example, kabat, supra; chothia and Lesk, supra; martin, supra; lefranc et al, supra). Residues from each of these hypervariable regions or CDRs are indicated in table 1 below.

TABLE 1

The boundaries of a given CDR may vary depending on the scheme used for authentication. Thus, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody, or regions thereof, e.g., variable regions, as well as the individual CDRs (e.g., CDR-H1, CDR-H2) of the antibody, or regions thereof, are to be understood as encompassing complementarity determining regions as defined by any one of the known schemes described above. In some cases, schemes are specified for identifying one or more specific CDRs, such as CDRs defined by the Kabat, chothia or Contact methods. In other cases, specific amino acid sequences of CDRs are given.

The hypervariable region may comprise the following "extended hypervariable region": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL; and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH.

The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains, which are not directly involved in binding an antibody to an antigen, but which exhibit multiple effector functions, such as interactions with Fc receptors. The term refers to that portion of an immunoglobulin molecule that comprises an amino acid sequence that is more conserved relative to other portions of the immunoglobulin (the variable region, which contains the antigen binding site). The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

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

In this context, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, the native sequence Fc region, the recombinant Fc region, and the variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, a human IgG heavy chain Fc region is generally defined as extending from amino acid residue position Cys226 or from Pro230 to its carboxy-terminus. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region may be removed, for example during preparation or purification of the antibody, or by engineering the nucleic acid encoding the heavy chain of the antibody in a recombinant manner. Thus, a composition of intact antibodies may comprise a population of antibodies with all K447 residues removed, a population of antibodies with no K447 residues removed, and a population of antibodies comprising a mixture of antibodies with and without K447 residues. The "functional Fc region" possesses the "effector function" of the native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions typically require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed using various assays known to those of skill in the art. A "variant Fc region" comprises an amino acid sequence that differs from the 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, e.g., about one to about ten amino acid substitutions or about one to about five amino acid substitutions, in the native sequence Fc region or in the Fc region of the parent polypeptide as compared to the native sequence Fc region or as compared to the Fc region of the parent polypeptide. The variant Fc-regions 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, e.g., at least about 95% homology therewith.

As used herein, an "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. The 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 contiguous amino acid of a polypeptide ("linear" epitope), or an epitope may comprise amino acids from two or more non-contiguous regions of a polypeptide ("conformational", "non-linear" or "discontinuous" epitope). Those skilled in the art will appreciate that in general, linear epitopes may or may not be dependent on secondary, tertiary or quaternary structures. For example, in some embodiments, the binding molecule binds to a group of amino acids, whether or not it is folded in the native three-dimensional protein structure. In other embodiments, the binding molecule requires that the amino acid residues comprising the epitope assume a particular conformation (e.g., bending, twisting, turning or folding) to recognize and bind the epitope.

The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be linear or branched, it may comprise 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 polypeptides, for example, that contain one or more amino acid analogs (including but not limited to unnatural amino acids) as well as other modifications known in the art. It will be appreciated that since the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the "polypeptides" may occur in single chain form or in two or more related chain forms.

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

By "excipient" is meant 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, flavorants, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizing agents, wetting agents and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., freunds' adjuvant) (complete or incomplete), or vehicle.

In one embodiment, each component is "pharmaceutically acceptable" in the following sense: is compatible with the other ingredients of the pharmaceutical formulation and is suitable for use in contact with tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications commensurate with a reasonable benefit/risk ratio. See, e.g., lippincott Williams & Wilkins: philiadelphia, PA,2005; handbook of Pharmaceutical Excipients, 6 th edition; rowe et al; the Pharmaceutical Press and the American Pharmaceutical Association:2009; handbook of Pharmaceutical Additives, 3 rd edition; ash and Ash braiding; gower Publishing Company:2007; pharmaceutical Preformulation and Formulation, version 2; gibson braiding; CRC Press LLC, boca Raton, FL,2009. In some embodiments, the pharmaceutically acceptable excipient is non-toxic to the cells or mammal to which it is exposed at the dosage and concentration used. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffer solution.

The abbreviation "MMAE" refers to monomethyl auristatin E.

The hyphen (-) indicates the point of attachment to the side molecule unless the context indicates otherwise.

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 (nitrogen mustard), ethyleneimine compounds and alkyl sulfonates; antimetabolites, such as folic acid, purine or pyrimidine antagonists; mitotic inhibitors, such as anti-tubulin agents, e.g. derivatives of vinca alkaloids (vinca alkaloids), auristatins (auristatin) and podophyllotoxins (podophyllotoxin); cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, such as DNA minor groove binders; and growth factor receptor antagonists. In addition, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biomolecules, and small molecules.

As used herein, the term "conservative substitution" refers to the substitution of an amino acid that is known to those skilled in the art and that can be made generally without altering the biological activity of the resulting molecule. Those skilled in The art recognize that in general, single amino acid substitutions in non-essential regions of polypeptides do not substantially alter biological activity (see, e.g., watson et al, MOLECULAR BIOLOGY OF THE GENE, the Benjamin/Cummings pub. Co., page 224 (4 th edition 1987)). Such exemplary substitutions are preferably made according to those substitutions set forth in tables 2 and 3. For example, such changes include substitution of any of isoleucine (I), valine (V) and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) replaces glutamic acid (E) and vice versa; glutamine (Q) replaces asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions may also be considered conservative, depending on the context of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (a) are often interchangeable, and alanine (a) and valine (V) may also be interchangeable. Methionine (M), which is relatively hydrophobic, is often interchangeable with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are often interchangeable in the position of the amino acid residues that are characterized significantly by their charge and the pK of these two amino acid residues is not significantly different. In certain circumstances, still other variations may be considered "conservative" (see, e.g., table 3 herein; biochemistry, 2 nd edition Lubert Stryer, eds. (Stanford University), pages 13-15; henikoff et al, PNAS 1992, volume 89, 10915-10919; lei et al, J Biol Chem, 1995, month 19; 270 (20): 11882-11886). Other substitutions are also permissible and may be determined empirically or based on known conservative substitutions.

TABLE 2 amino acid abbreviations

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

TABLE 3 amino acid substitutions or similarity matrices

The amino acid substitution matrix (block) was adapted from GCG software 9.0BLOSUM62. The higher the value, the greater the likelihood of substitution being found in the relevant native protein.

The term "homology" or "homology" is intended to refer to sequence similarity between two polynucleotides or between two polypeptides. Similarity can be determined by comparing the positions in each sequence aligned for comparison purposes. If two polypeptide sequences differ at a given position, the similarity or conservation of the position can be determined by assessing the similarity of the amino acids at the position, e.g., according to Table 3. The degree of similarity between sequences varies with the number of matched or homologous positions shared by the sequences. Alignment of the two sequences to determine their percent sequence similarity can be performed using software programs known in the art, such as those described in Ausubel et al, current Protocols in Molecular Biology, john Wiley and Sons, baltimore, MD (1999). Preferably, the comparison is made using default parameters, examples of which are set forth below. One alignment program that can be used is BLAST, which is well known in the art, set to default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; chain = two; cut-off value = 60; expected value = 10; matrix = BLOSUM62; description = 50 sequences; sorting mode = HIGH SCORE; database = non-redundant, genBank + EMBL + DDBJ + PDB + GenBank CDS translation + Swissprotein + spldate + PIR. Details of these procedures can be found in the national center for biotechnology information (National Center for Biotechnology Information).

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

Determination of the percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using mathematical algorithms. Preferred non-limiting examples of mathematical algorithms for comparing two sequences are the algorithms of Karlin and Altschul,1990,Proc.Natl.Acad.Sci.U.S.A.87:2264 2268, as modified in Karlin and Altschul,1993,Proc.Natl.Acad.Sci.U.S.A.90:5873 5877. Such algorithms are incorporated in 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., word length=12 for score=100) 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., word length=3 for a score of 50, to obtain amino acid sequences homologous to protein molecules described herein. For comparison purposes, gap 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 iterative searches that detect long-range relationships between molecules (supra). When utilizing BLAST, gapped BLAST, and PSI BLAST programs, default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., national Center for Biotechnology Information (NCBI) on the global information network, NCBI. Another non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller,1988,CABIOS 4:11 17. Such algorithms are incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When amino acid sequences are compared using the ALIGN program, PAM120 weight residue table, gap length penalty 12, and gap penalty 4 can be used.

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

The term "cytotoxic agent" refers to a substance that inhibits or prevents the activity of cellular expression, cellular function, and/or causes cellular destruction. By a means ofThe term is intended to include radioisotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to, auristatin (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureomycin (auromyins), maytansinoids, ricin (ricin), ricin A chain, combretastatin, duocarmycin (duocarmycin), dolastatin (dolastatin), dolastatin, daunomycin (daunorubicin), paclitaxel (taxols), cisplatin, cc1065, ethidium bromide, mitomycin (mitomycin), etoposide (etoposide), teniposide (teniposide), vincristine (vincristine), vinblastine (vinblastine), colchicine (colchicine), dactinomycin (dactinomycin), and the like dihydroxyanthrax-toxin diketone, actinomycin (actinomycin), diphtheria toxin (diphtheria toxin), pseudomonas Exotoxin (PE) A, PE, abrin (abrin), abrin A chain, mo Disu A chain (modeccin A chain), alpha-sarcin (sarcin), gelonin (gelonin), mitosin (mitogellin), restrictocin (retstricin), phenomycin (phenomycin), enomycin (enomycin), curculin (curcin), crotin (crotin), calicheamicin (calicheamicin), soapberry (Sapaonaria officinalis) inhibitors and glucocorticoids and other chemotherapeutic agents, radioisotopes such as At ²¹¹ 、I ¹³¹ 、I ¹²⁵ 、Y ⁹⁰ 、Re ¹⁸⁶ 、Re ¹⁸⁸ 、Sm ¹⁵³ 、Bi ²¹² Or Bi ²¹³ 、P ³² And radioactive isotopes of Lu, including Lu ¹⁷⁷ . The antibodies may also be conjugated to an anticancer prodrug-activating enzyme capable of converting the prodrug into its active form.

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

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

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

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

The term "preventing" refers to reducing the likelihood of onset (or recurrence) of a disease, disorder, condition, or associated symptom (e.g., cancer).

The term "cancer" or "cancer cells" is used herein to refer to tissues or cells found in neoplasms that possess characteristics that distinguish them from normal tissues or tissue cells. Among such features are but not limited to: degree of degenerative development, irregularities in shape, unclear cellular contours, changes in nuclear size, nuclear or cytoplasmic structures, other phenotypic changes, presence of cellular proteins indicative of cancerous or precancerous conditions, increased number of mitoses and metastatic capacity. The term "cancer" includes carcinoma, sarcoma, tumor, epithelial tumor, leukemia, lymphoma, polyp and hard cancer, transformation, neoplasm, and the like.

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

As used herein, "metastatic" cancer refers to cancer that has spread from its beginning to different parts of the body.

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

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

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

The term "and/or" as used in the phrase, e.g., "a and/or B," herein is intended to include a and B; a or B; a (alone); and B (alone). Also, the term "and/or" as used in a phrase such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

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

The "191P4D12 proteins" and/or "191P4D12 related proteins" of the present disclosure include those specifically identified herein (see fig. 1A), as well as allelic variants, conservative substitution variants, analogs, and homologs that can be isolated/produced and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins combining portions of different 191P4D12 proteins or fragments thereof, as well as fusion proteins of 191P4D12 proteins with heterologous polypeptides, are also included. Such 191P4D12 proteins are collectively referred to as 191P4D12 related proteins, proteins of the 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 than 25 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 191P4D12 protein sequence. The term "191P4D12" is used interchangeably with connexin-4.

5.2 methods of treating cancer

5.2.1General methods of treating cancer and methods of treating patients who have previously received cancer treatment

In one aspect, provided herein are methods of treating urothelial cancer in a subject using an Antibody Drug Conjugate (ADC) that binds 191P4D 12. In certain embodiments, urothelial cancer has been previously treated with platinum-based chemotherapy and CPI. In some embodiments, the urothelial cancer has been previously treated with platinum-based chemotherapy. In other embodiments, the urothelial cancer has been previously treated with CPI. In one embodiment, the subject has been previously treated with platinum-based chemotherapy and CPI. In another embodiment, the subject has been previously treated with platinum-based chemotherapy. In another embodiment, the subject has been previously treated with CPI.

In another aspect, provided herein are methods of treating locally advanced urothelial cancer in a subject using an Antibody Drug Conjugate (ADC) that binds 191P4D 12. In certain embodiments, locally advanced urothelial cancer has been previously treated with platinum-based chemotherapy and CPI. In some embodiments, locally advanced urothelial cancer has been previously treated with platinum-based chemotherapy. In other embodiments, locally advanced urothelial cancer has been previously treated with CPI. In one embodiment, the subject has been previously treated with platinum-based chemotherapy and CPI. In another embodiment, the subject has been previously treated with platinum-based chemotherapy. In another embodiment, the subject has been previously treated with CPI.

In another aspect, provided herein are methods of treating metastatic urothelial cancer in a subject using an Antibody Drug Conjugate (ADC) that binds 191P4D 12. In certain embodiments, metastatic urothelial cancer has been previously treated with platinum-based chemotherapy and CPI. In some embodiments, metastatic urothelial cancer has been previously treated with platinum-based chemotherapy. In other embodiments, metastatic urothelial cancer has been previously treated with CPI. In one embodiment, the subject has been previously treated with platinum-based chemotherapy and CPI. In another embodiment, the subject has been previously treated with platinum-based chemotherapy. In another embodiment, the subject has been previously treated with CPI.

In one aspect, provided herein are methods of treating urothelial cancer in a subject using an Antibody Drug Conjugate (ADC) that binds 191P4D 12. In certain embodiments, the urothelial cancer has been previously treated with platinum-based chemotherapy and a PD-1 inhibitor. In some embodiments, the urothelial cancer has been previously treated with platinum-based chemotherapy. In other embodiments, the urothelial cancer has been previously treated with a PD-1 inhibitor. In one embodiment, the subject has been previously treated with platinum-based chemotherapy and a PD-1 inhibitor. In another embodiment, the subject has been previously treated with platinum-based chemotherapy. In another embodiment, the subject has been previously treated with a PD-1 inhibitor.

In another aspect, provided herein are methods of treating locally advanced urothelial cancer in a subject using an Antibody Drug Conjugate (ADC) that binds 191P4D 12. In certain embodiments, locally advanced urothelial cancer has been previously treated with platinum-based chemotherapy and PD-1 inhibitors. In some embodiments, locally advanced urothelial cancer has been previously treated with platinum-based chemotherapy. In other embodiments, locally advanced urothelial cancer has been previously treated with a PD-1 inhibitor. In one embodiment, the subject has been previously treated with platinum-based chemotherapy and a PD-1 inhibitor. In another embodiment, the subject has been previously treated with platinum-based chemotherapy. In another embodiment, the subject has been previously treated with a PD-1 inhibitor.

In another aspect, provided herein are methods of treating metastatic urothelial cancer in a subject using an Antibody Drug Conjugate (ADC) that binds 191P4D 12. In certain embodiments, metastatic urothelial cancer has been previously treated with platinum-based chemotherapy and PD-1 inhibitors. In some embodiments, metastatic urothelial cancer has been previously treated with platinum-based chemotherapy. In other embodiments, metastatic urothelial cancer has been previously treated with a PD-1 inhibitor. In one embodiment, the subject has been previously treated with platinum-based chemotherapy and a PD-1 inhibitor. In another embodiment, the subject has been previously treated with platinum-based chemotherapy. In another embodiment, the subject has been previously treated with a PD-1 inhibitor.

In one aspect, provided herein are methods of treating urothelial cancer in a subject using an Antibody Drug Conjugate (ADC) that binds 191P4D 12. In certain embodiments, the urothelial cancer has been previously treated with platinum-based chemotherapy and a PD-L1 inhibitor. In some embodiments, the urothelial cancer has been previously treated with platinum-based chemotherapy. In other embodiments, the urothelial cancer has been previously treated with a PD-L1 inhibitor. In one embodiment, the subject has been previously treated with platinum-based chemotherapy and a PD-L1 inhibitor. In another embodiment, the subject has been previously treated with platinum-based chemotherapy. In another embodiment, the subject has been previously treated with a PD-L1 inhibitor.

In another aspect, provided herein are methods of treating locally advanced urothelial cancer in a subject using an Antibody Drug Conjugate (ADC) that binds 191P4D 12. In certain embodiments, locally advanced urothelial cancer has been previously treated with platinum-based chemotherapy and PD-L1 inhibitors. In some embodiments, locally advanced urothelial cancer has been previously treated with platinum-based chemotherapy. In other embodiments, locally advanced urothelial cancer has been previously treated with a PD-L1 inhibitor. In one embodiment, the subject has been previously treated with platinum-based chemotherapy and a PD-L1 inhibitor. In another embodiment, the subject has been previously treated with platinum-based chemotherapy. In another embodiment, the subject has been previously treated with a PD-L1 inhibitor.

In another aspect, provided herein are methods of treating metastatic urothelial cancer in a subject using an Antibody Drug Conjugate (ADC) that binds 191P4D 12. In certain embodiments, metastatic urothelial cancer has been previously treated with platinum-based chemotherapy and PD-L1 inhibitors. In some embodiments, metastatic urothelial cancer has been previously treated with platinum-based chemotherapy. In other embodiments, metastatic urothelial cancer has been previously treated with a PD-L1 inhibitor. In one embodiment, the subject has been previously treated with platinum-based chemotherapy and a PD-L1 inhibitor. In another embodiment, the subject has been previously treated with platinum-based chemotherapy. In another embodiment, the subject has been previously treated with a PD-L1 inhibitor.

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

In some embodiments, the CPI provided for the method may comprise or consist of any CPI described in such section (section 5.2.1).

In all methods provided herein and in particular the methods described in the six preceding paragraphs: ADCs that may be used are described in sections 3, 5.2, 5.3, 5.4, 5.5, and 6; the selection of patients to be treated is described herein and exemplified in this section (section 5.2) and sections 3 and 6; the dosing regimen and pharmaceutical compositions for administration of the therapeutic agents are described in this section (section 5.2), sections 5.6, 5.7 and 6 below; biomarkers that can be used to identify therapeutic agents, select patients, determine the results of these methods, and/or serve in any way as criteria for these methods are described herein and exemplified in this section (section 5.2, including 5.2.1 and 5.2.2) and section 6; biomarkers can be determined as described in section 5.8 or as known in the art; the therapeutic results of the methods provided herein can be improvements to the biomarkers described herein, such as those described and exemplified in this section (section 5.2, including 5.2.2) and section 6. Thus, those of skill in the art will appreciate that the methods provided herein include all permutations and combinations of patients, therapeutic agents, dosing regimens, biomarkers, and therapeutic results as described above and below.

In certain embodiments, a subject having a urothelial cancer that expresses 1914d12 RNA, that expresses 191P4D12 protein, or that expresses both 191P4D12 RNA and 191P4D12 protein is treated using the methods provided herein. In some embodiments, the methods provided herein are used to treat subjects with urothelial cancer that express 191P4D12 RNA, that express 191P4D12 protein, or that express both 191P4D12 RNA and 191P4D12 protein, and subjects that have previously been treated with platinum-based chemotherapy and CPI. In one embodiment, the methods provided herein are used to treat subjects with urothelial cancer that express 191P4D12 RNA, 191P4D12 protein or both 191P4D12 RNA and 191P4D12 protein, and subjects that have previously been treated with platinum-based chemotherapy. In another embodiment, the methods provided herein are used to treat subjects with urothelial cancer that expresses 191P4D12 RNA, that expresses 191P4D12 protein, or that expresses both 191P4D12 RNA and 191P4D12 protein, and subjects that have been previously treated with CPI.

In certain embodiments, a subject having locally advanced urothelial cancer that expresses 191P4D12RNA, that expresses 191P4D12 protein, or that expresses both 191P4D12RNA and 191P4D12 protein is treated using the methods provided herein. In some embodiments, the methods provided herein are used to treat subjects with locally advanced urothelial cancer that expresses 191P4D12RNA, or that expresses both 191P4D12RNA and 191P4D12 protein, and subjects that have previously been treated with platinum-based chemotherapy and CPI. In one embodiment, the methods provided herein are used to treat subjects with locally advanced urothelial cancer that expresses 1914d12 RNA, that expresses 191P4D12 protein, or that expresses both 1914d12 RNA and 191P4D12 protein, and subjects that have been previously treated with platinum-based chemotherapy. In another embodiment, the methods provided herein are used to treat subjects with locally advanced urothelial cancer that expresses 191P4D12RNA, 191P4D12 protein or both 191P4D12RNA and 191P4D12 protein, and subjects that have been previously treated with CPI.

In certain embodiments, a subject having a metastatic cancer that expresses 1914D 12RNA, that expresses 191P4D12 protein, or that expresses both 191P4D12RNA and 191P4D12 protein is treated using the methods provided herein. In some embodiments, the methods provided herein are used to treat subjects having metastatic urothelial cancer that expresses 1914d12 RNA, that expresses 191P4D12RNA, or that expresses both 191P4D12RNA and 191P4D12 protein, and subjects that have been previously treated with platinum-based chemotherapy and CPI. In one embodiment, the methods provided herein are used to treat subjects having metastatic urothelial cancer that express 191P4D12RNA, that express 191P4D12 protein, or that express both 191P4D12RNA and 191P4D12 protein, and subjects that have been previously treated with platinum-based chemotherapy. In another embodiment, the methods provided herein are used to treat subjects having metastatic urothelial cancer that expresses 191P4D12RNA, 191P4D12 protein or both 191P4D12RNA and 191P4D12 protein, and subjects that have been previously treated with CPI.

In some embodiments, 191P4D12 RNA expression in cancer is determined by polynucleotide hybridization, sequencing (assessing the relative abundance of the sequences) and/or PCR (including RT-PCR). In some embodiments, 191P4D12 protein expression in cancer is determined by IHC, analysis in Fluorescence Activated Cell Sorting (FACS), and/or western blotting. In some embodiments, 191P4D12 protein expression in cancer is determined by more than one method. In some embodiments, 191P4D12 protein expression in cancer is determined by two IHC methods.

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

In some embodiments, a subject treatable in the methods provided herein includes a subject that has received one or more other cancer treatments. In some embodiments, subjects treatable in the methods provided herein include subjects who have received one or more other cancer treatments and whose cancers progress or recur after one or more treatments. Such one or more treatments include, for example, first-line or multi-line immune checkpoint inhibitor therapy, chemotherapy, and both immune checkpoint inhibitor therapy and chemotherapy. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI. In some embodiments, the subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs following platinum-containing chemotherapy. In some embodiments, the subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs following platinum-containing chemotherapy in a neoadjuvant setting. In some embodiments, the subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs following platinum-containing chemotherapy in a secondary setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed following platinum-containing chemotherapy in a neoadjuvant, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed following platinum-containing chemotherapy in a neoadjuvant, metastatic setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed following platinum-containing chemotherapy in a adjunctive, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed following platinum-containing chemotherapy in a adjunctive, metastatic setting. In some embodiments, the subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs following platinum-containing chemotherapy in a metastatic background. In some embodiments, the subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs following platinum-containing chemotherapy in a locally advanced setting.

In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI and platinum-containing chemotherapy. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI and platinum-containing chemotherapy in a neoadjuvant setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI and platinum-containing chemotherapy in a secondary setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI and platinum-containing chemotherapy in a locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI and platinum-containing chemotherapy in a metastatic background. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI and platinum-containing chemotherapy in a neoadjuvant, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI and platinum-containing chemotherapy in a neoadjuvant, metastatic setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI and platinum-containing chemotherapy in a adjunctive, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed following treatment with CPI and platinum-containing chemotherapy in a adjuvant, metastatic setting.

In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with CPI. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI in a neoadjuvant setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI in a secondary setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI in a locally advanced setting. In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer has progressed or relapsed following treatment with CPI in a metastatic background. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI in a neoadjuvant, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed following treatment with CPI in a neoadjuvant, metastatic setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with CPI in a adjunctive, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed following treatment with CPI in a secondary, metastatic background.

In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-1 inhibitor and platinum-containing chemotherapy. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with PD-1 inhibitors and platinum-containing chemotherapy in a neoadjuvant setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with PD-1 inhibitors and platinum-containing chemotherapies in a adjunctive setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with PD-1 inhibitors and platinum-containing chemotherapy in a locally advanced setting. In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-1 inhibitor and platinum-containing chemotherapy in a metastatic setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs after treatment with PD-1 inhibitors and platinum-containing chemotherapy in a neoadjuvant, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs following treatment with PD-1 inhibitors and platinum-containing chemotherapy in a neoadjuvant, metastatic setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs after treatment with PD-1 inhibitors and platinum-containing chemotherapy in a adjunctive, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs following treatment with PD-1 inhibitors and platinum-containing chemotherapy in a adjunctive, metastatic setting.

In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-1 inhibitor. In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-1 inhibitor in a neoadjuvant setting. In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-1 inhibitor in a secondary setting. In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-1 inhibitor in a locally advanced setting. In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-1 inhibitor in a metastatic setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with PD-1 inhibitors in a neoadjuvant, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs following treatment with PD-1 inhibitors in a neoadjuvant, metastatic setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs after treatment with PD-1 inhibitors in a helper, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs following treatment with PD-1 inhibitors in a adjunctive, metastatic setting.

In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-L1 inhibitor and platinum-containing chemotherapy. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with PD-L1 inhibitors and platinum-containing chemotherapy in a neoadjuvant setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with PD-L1 inhibitors and platinum-containing chemotherapy in a adjunctive setting. In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-L1 inhibitor and platinum-containing chemotherapy in a locally advanced setting. In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-L1 inhibitor and platinum-containing chemotherapy in a metastatic background. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs after treatment with PD-L1 inhibitors and platinum-containing chemotherapy in a neoadjuvant, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs following treatment with PD-L1 inhibitors and platinum-containing chemotherapy in a neoadjuvant, metastatic setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs after treatment with PD-L1 inhibitors and platinum-containing chemotherapy in a adjunctive, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs after treatment with PD-L1 inhibitors and platinum-containing chemotherapy in a adjunctive, metastatic setting.

In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-L1 inhibitor. In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-L1 inhibitor in a neoadjuvant setting. In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-L1 inhibitor in a secondary setting. In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-L1 inhibitor in a locally advanced setting. In some embodiments, a subject treatable in the methods provided herein includes a subject whose cancer progresses or recurs after treatment with a PD-L1 inhibitor in a metastatic setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progressed or relapsed after treatment with PD-L1 inhibitors in a neoadjuvant, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs following treatment with PD-L1 inhibitors in a neoadjuvant, metastatic setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs after treatment with PD-L1 inhibitors in a helper, locally advanced setting. In some embodiments, subjects treatable in the methods provided herein include subjects whose cancer progresses or recurs following treatment with PD-L1 inhibitors in a adjunctive, metastatic setting.

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

In some embodiments, a subject treatable in the methods provided herein has certain phenotypic or genotypic characteristics. In some embodiments, the subject has any permutation and combination of the phenotypic or genotypic characteristics described herein.

In some embodiments, the phenotypic or genotypic characteristic is determined histologically, cytologically, or both histologically and cytologically. In some embodiments of the methods provided herein, the histological and/or cytological determination of the phenotypic and/or genotypic characteristics is performed based on recently analyzed tissue as described in the american society of clinical oncology/american society of pathology (American Society of Clinical Oncology/College of American Pathologists; ASCO/CAP) guidelines, which are incorporated herein by reference in their entirety. In some embodiments, the phenotypic or genotypic characteristic is determined by sequencing, DNA hybridization, and/or RNA hybridization including next generation sequencing (e.g., NGS from Illumina, inc).

In various aspects or embodiments of the methods provided herein, including the methods provided in this section (section 5.2), e.g., the methods provided in this paragraph and the preceding paragraphs, the methods involve prior treatment with an immune checkpoint inhibitor as provided in the methods. As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that reduces, inhibits, interferes with, or modulates one or more checkpoint proteins, in whole or in part. Various checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 with its ligands PD-L1 and PD-L2 (Pardoll, nature Reviews Cancer,2012,12,252-264). Other exemplary checkpoint proteins include LAG-3, B7, TIM3 (HAVCR 2), OX40 (CD 134), GITR, CD137, CD40, VTCN1, IDO1, CD276, PVRIG, TIGIT, CD25 (IL 2 RA), IFNAR2, IFNAR1, CSF1R, VSIR (VISTA), or HLA. These proteins appear to be responsible for co-stimulatory or inhibitory interactions of the T cell response. Immune checkpoint proteins appear to regulate and maintain the duration and magnitude of self-tolerance and physiological immune responses. Immune checkpoint inhibitors include antibodies or are derived from antibodies.

In certain embodiments, the checkpoint inhibitor of the methods provided herein can be an inhibitor or activator of a checkpoint protein that is up-regulated in cancer. In some embodiments, the checkpoint inhibitor of the methods provided herein can be an inhibitor or activator for a checkpoint protein comprising: LAG-3, B7, TIM3 (HAVCR 2), OX40 (CD 134), GITR, CD137, CD40, VTCN1, IDO1, CD276, PVRIG, TIGIT, CD (IL 2 RA), IFNAR2, IFNAR1, CSF1R, VSIR (VISTA), or HLA. In some embodiments, the checkpoint inhibitor of the methods provided herein can be an inhibitor or activator selected from the group consisting of: PD-1 inhibitor, PD-L2 inhibitor, CTLA-4 inhibitor, LAG-3 inhibitor, B7 inhibitor, TIM3 (HAVCR 2) inhibitor, OX40 (CD 134) inhibitor, GITR agonist, CD137 agonist, or CD40 agonist, VTCN1 inhibitor, IDO1 inhibitor, CD276 inhibitor, PVRIG inhibitor, TIGIT inhibitor, CD25 (IL 2 RA) inhibitor, IFNAR2 inhibitor, IFNAR1 inhibitor, CSF1R inhibitor, VSIR (VISTA) inhibitor, or HLA-targeted therapeutic agent. Such inhibitors, activators, or therapeutic agents are further provided below.

In some embodiments, the checkpoint inhibitor is a CTLA-4 inhibitor. In one embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. Examples of anti-CTLA-4 antibodies include, but are not limited to, those described in: U.S. Pat. nos. 5,811,097, 5,811,097, 5,855,887, 6,051,227, 6,207,157, 6,682,736, 6,984,720 and 7,605,238, all of which are incorporated herein in their entirety. In one embodiment, the anti-CTLA-4 antibody is tremelimumab (also known as tibetamab (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. Ipimab under the trade name Yervoy ^TM And (5) selling.

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

In some embodiments, the checkpoint inhibitor is a PD-1 inhibitor. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody. In one embodiment, the anti-PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558 or MDX 1106), or pamglizumab (also known as MK-3475, SCH 900475 or Rankine-mab (lambrolizumab)). In one embodiment, the anti-PD-1 antibody is nivolumab. Nawuzumab is a human IgG4 anti-PD-1 monoclonal antibody and is under the trade name Opdivo ^TM And (5) selling. In another embodiment, the anti-PD-1 antibody is a pamphlet Li Zhushan antibody. Pamo Li Zhushan is a humanized monoclonal IgG4 antibody and is known under the trade name Keytruda ^TM And (5) selling. In another embodiment, the anti-PD-1 antibody is the humanized antibody CT-011. CT-011 alone failed to show a response in the treatment of acute myelogenous leukemia (acute myeloid leukemia; AML) in the recurrent cases. In another embodiment, the anti-PD-1 antibody is a fusion protein AMP-224. In another embodiment, the PD-1 antibody is BGB-A317.BGB-a317 is a monoclonal antibody in which the ability to bind fcγ receptor I is specifically engineered and which has the characteristic of unique binding to PD-1 with high affinity and excellent target specificity. In one embodiment, the PD-1 antibody is a cemipramiab Li Shan antibody (cemiplimab). In another embodiment, the PD-1 antibody is caprilizumab (camrelizumab). In another embodiment, the PD-1 antibody is a Xindi Li Shan antibody (sintillimab). In some embodiments, the PD-1 antibody is tirelizumab (tisrelizumab). 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 a terlipressimab Li Shan antibody (toripalimab).

In certain embodiments, the checkpoint inhibitor is a PD-L1 inhibitor. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one embodimentIn the present case, the anti-PD-L1 antibody is MEDI4736 (Dewaruzumab). In another embodiment, the anti-PD-L1 antibody is BMS-936559 (also referred to 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 avermectin.

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 rthigm 12B7A.

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

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

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

In one embodiment, the checkpoint inhibitor is an OX40 (CD 134) agonist. In one embodiment, the checkpoint inhibitor is an anti-OX 40 antibody. In one embodiment, the anti-OX 40 antibody is anti-OX-40. In another embodiment, the anti-OX 40 antibody is MEDI6469.

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

In one embodiment, the checkpoint inhibitor is a CD137 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD 137 antibody. In one embodiment, the anti-CD 137 antibody is Wu Ruilu mab (urelumab). In another embodiment, the anti-CD 137 antibody is PF-05082566.

In one embodiment, the checkpoint inhibitor is a CD40 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD 40 antibody. In one embodiment, the anti-CD 40 antibody is CF-870,893.

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

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

In one embodiment, the checkpoint inhibitor is an IDO inhibitor. In one embodiment, the IDO inhibitor is INCB024360. In another embodiment, the IDO inhibitor is indoximod (indoximod). In one embodiment, the IDO inhibitor is Ai Kaduo stat (epacoadostat). In another embodiment, the IDO inhibitor is BMS986205. In another embodiment, the IDO inhibitor is natamod (Navoximod). In one embodiment, the IDO inhibitor is PF-06840003. In another embodiment, the IDO inhibitor is KHK2455. In another embodiment, the IDO inhibitor is RG70099. In one embodiment, the IDO inhibitor is IOM-E. In another embodiment, the IDO inhibitor is IOM-D.

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

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

For some embodiments, the checkpoint inhibitor is a TIM3 inhibitor. For certain embodiments, the TIM3 inhibitor is an anti-TIM 3 antibody. For one embodiment, the TIM3 inhibitor is AJUD009.

In some embodiments, the checkpoint inhibitor is a CD25 (IL 2 RA) inhibitor. In certain embodiments, the CD25 (IL 2 RA) inhibitor is an anti-CD 25 (IL 2 RA) antibody. In one embodiment, the CD25 (IL 2 RA) inhibitor is daclizumab (daclizumab). In another embodiment, the CD25 (IL 2 RA) inhibitor is basiliximab.

In some embodiments, the checkpoint inhibitor is an IFNAR1 inhibitor. In certain embodiments, the IFNAR1 inhibitor is an anti-IFNAR 1 antibody. In one embodiment, the IFNAR1 inhibitor is anistuzumab (aniflolumab). In another embodiment, the IFNAR1 inhibitor is sibirimumab (sibalimumab).

In some embodiments, the checkpoint inhibitor is a CSF1R inhibitor. In certain embodiments, the CSF1R inhibitor is an anti-CSF 1R antibody. In one embodiment, the CSF1R inhibitor is piroxicam (pexidatinib). In another embodiment, the CSF1R inhibitor is emituzumab-b. In another embodiment, the CSF1R inhibitor is cabiralizumab (cabiralizumab). In one embodiment, the CSF1R inhibitor is ARRY-382. In another embodiment, the CSF1R inhibitor is BLZ945. In another embodiment, the CSF1R inhibitor is AJUD010. In one embodiment, the CSF1R inhibitor is AMG820. In another embodiment, the CSF1R inhibitor is IMC-CS4. In another embodiment, the CSF1R inhibitor is JNJ-40346527. In one embodiment, the CSF1R inhibitor is PLX5622. In another embodiment, the CSF1R inhibitor is FPA008.

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

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

In some embodiments, after administration of the ADCs provided herein, a checkpoint inhibitor is administered. In other embodiments, the checkpoint inhibitor is administered concurrently (e.g., in the same dosing period) with the ADCs provided herein. In other embodiments, the checkpoint inhibitor is administered after administration of the ADCs provided herein.

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

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

5.2.1.1 other patient demographics

Furthermore, the human subjects that can use the methods provided herein are human subjects having various other conditions. In one embodiment, a human subject that may use the methods provided herein may have a primary tumor site in the lower urinary tract. In some embodiments, a human subject that may use the methods provided herein may have visceral metastasis. In certain embodiments, a human subject that can use the methods provided herein can have liver metastasis. In other embodiments, a human subject that can use the methods provided herein can have at least 1 bellmut risk factor. In other embodiments, a human subject that may use the methods provided herein may have an ECOG behavioral state score of 0. In one embodiment, a human subject that may use the methods provided herein may have a primary tumor site and visceral metastasis in the lower urinary tract. In some embodiments, a human subject that may use the methods provided herein may have a primary tumor site and liver metastasis in the lower urinary tract. In certain embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the lower urinary tract and at least 1 bellmut risk factor. In other embodiments, a human subject that may use the methods provided herein may have a primary tumor site in the lower urinary tract and an ECOG behavioral state score of 0. In other embodiments, a human subject that may use the methods provided herein may have visceral and hepatic metastasis. In one embodiment, a human subject that can use the methods provided herein can have visceral metastasis and at least 1 bellmut risk factor. In some embodiments, a human subject that may use the methods provided herein may have visceral metastasis and an ECOG behavioral state score of 0. In other embodiments, a human subject that can use the methods provided herein can have liver metastasis and at least 1 bellmut risk factor. In other embodiments, a human subject that may use the methods provided herein may have liver metastasis and ECOG behavioral state scores of 0. In one embodiment, a human subject that can use the methods provided herein can have an ECOG behavioral state score of at least 1 bellmut risk factor and 0. In other embodiments, a human subject that may use the methods provided herein may have a primary tumor site, visceral metastasis, and hepatic metastasis in the lower urinary tract. In other embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the lower urinary tract, visceral metastasis, and at least 1 bellmut risk factor. In other embodiments, a human subject that may use the methods provided herein may have a primary tumor site in the lower urinary tract, visceral metastasis, and an ECOG behavioral state score of 0. In some embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the lower urinary tract, liver metastasis, and at least 1 bellmut risk factor. In certain embodiments, a human subject that may use the methods provided herein may have a primary tumor site in the lower urinary tract, liver metastasis, and ECOG behavioral state score of 0. In other embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the lower urinary tract, an ECOG behavioral state score of at least 1 bellmut risk factor, and 0. In some embodiments, a human subject that can use the methods provided herein can have visceral metastasis, hepatic metastasis, and at least 1 bellmut risk factor. In certain embodiments, a human subject that may use the methods provided herein may have visceral metastasis, hepatic metastasis, and ECOG behavioral state scores of 0. In other embodiments, a human subject that can use the methods provided herein can have visceral metastasis, an ECOG behavioral state score of at least 1 bellmut risk factor, and 0. In other embodiments, a human subject that can use the methods provided herein can have liver metastasis, an ECOG behavioral state score of at least 1 bellmut risk factor, and 0. In other embodiments, a human subject that can use the methods provided herein can have a primary tumor site, visceral metastasis, hepatic metastasis, and at least 1 bellmut risk factor in the lower urinary tract. In other embodiments, a human subject that may use the methods provided herein may have a primary tumor site in the lower urinary tract, visceral metastasis, hepatic metastasis, and ECOG behavioral state score of 0. In some embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the lower urinary tract, visceral metastasis, an ECOG behavioral state score of at least 1 bellmut risk factor, and 0. In certain embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the lower urinary tract, liver metastasis, an ECOG behavioral state score of at least 1 bellmut risk factor, and 0. In other embodiments, a human subject that can use the methods provided herein can have visceral metastasis, hepatic metastasis, an ECOG behavioral state score of at least 1 bellmut risk factor, and 0. In certain embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the lower urinary tract, visceral metastasis, hepatic metastasis, an ECOG behavioral state score of at least 1 bellmut risk factor, and 0. In some embodiments, a human subject that can use the methods provided herein can have any one of the following: primary tumor sites in the lower urinary tract, visceral metastasis, hepatic metastasis, at least 1 bellmut risk factor, and ECOG behavioral status scores of 0. In some embodiments, a human subject that can use the methods provided herein can have any two of the following: primary tumor sites in the lower urinary tract, visceral metastases, hepatic metastases, ECOG behavioral state scores of at least 1 bellmut risk factor and 0, in any combination or permutation. In some embodiments, a human subject that can use the methods provided herein can have any of the following: primary tumor sites in the lower urinary tract, visceral metastases, hepatic metastases, ECOG behavioral state scores of at least 1 bellmut risk factor and 0, in any combination or permutation. In some embodiments, a human subject that may use the methods provided herein may have any four of the following: primary tumor sites in the lower urinary tract, visceral metastases, hepatic metastases, ECOG behavioral state scores of at least 1 bellmut risk factor and 0, in any combination or permutation. In some embodiments, a human subject that can use the methods provided herein can have all five of the following: primary tumor sites in the lower urinary tract, visceral metastasis, hepatic metastasis, at least 1 bellmut risk factor, and ECOG behavioral status scores of 0.

In other embodiments, a human subject that may use the methods provided herein may have a primary tumor site in the upper urinary tract. In one embodiment, a human subject that may use the methods provided herein may have a primary tumor site and visceral metastasis in the upper urinary tract. In some embodiments, a human subject that may use the methods provided herein may have a primary tumor site and liver metastasis in the upper urinary tract. In certain embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the upper urinary tract and at least 1 bellmut risk factor. In other embodiments, a human subject that may use the methods provided herein may have a primary tumor site in the upper urinary tract and an ECOG behavioral state score of 0. In other embodiments, a human subject that may use the methods provided herein may have a primary tumor site, visceral metastasis, and hepatic metastasis in the upper urinary tract. In other embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the upper urinary tract, visceral metastasis, and at least 1 bellmut risk factor. In other embodiments, a human subject that may use the methods provided herein may have a primary tumor site in the upper urinary tract, visceral metastasis, and an ECOG behavioral state score of 0. In some embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the upper urinary tract, liver metastasis, and at least 1 bellmut risk factor. In certain embodiments, a human subject that may use the methods provided herein may have a primary tumor site in the upper urinary tract, liver metastasis, and ECOG behavioral state score of 0. In other embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the upper urinary tract, an ECOG behavioral state score of at least 1 bellmut risk factor, and 0. In other embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the upper urinary tract, visceral metastasis, hepatic metastasis, and at least 1 bellmut risk factor. In other embodiments, a human subject that may use the methods provided herein may have a primary tumor site in the upper urinary tract, visceral metastasis, hepatic metastasis, and ECOG behavioral state score of 0. In some embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the upper urinary tract, visceral metastasis, an ECOG behavioral state score of at least 1 bellmut risk factor, and 0. In certain embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the upper urinary tract, liver metastasis, an ECOG behavioral state score of at least 1 bellmut risk factor, and 0. In certain embodiments, a human subject that can use the methods provided herein can have a primary tumor site in the upper urinary tract, visceral metastasis, hepatic metastasis, an ECOG behavioral state score of at least 1 bellmut risk factor, and 0. In some embodiments, a human subject that can use the methods provided herein can have any one of the following: primary tumor sites in the upper urinary tract, visceral metastasis, hepatic metastasis, at least 1 bellmut risk factor, and ECOG behavioral status scores of 0. In some embodiments, a human subject that can use the methods provided herein can have any two of the following: primary tumor sites in the upper urinary tract, visceral metastases, hepatic metastases, ECOG behavioral state scores of at least 1 bellmut risk factor and 0, in any combination or permutation. In some embodiments, a human subject that can use the methods provided herein can have any of the following: primary tumor sites in the upper urinary tract, visceral metastases, hepatic metastases, ECOG behavioral state scores of at least 1 bellmut risk factor and 0, in any combination or permutation. In some embodiments, a human subject that may use the methods provided herein may have any four of the following: primary tumor sites in the upper urinary tract, visceral metastases, hepatic metastases, ECOG behavioral state scores of at least 1 bellmut risk factor and 0, in any combination or permutation. In some embodiments, a human subject that can use the methods provided herein can have all five of the following: primary tumor sites in the upper urinary tract, visceral metastasis, hepatic metastasis, at least 1 bellmut risk factor, and ECOG behavioral status scores of 0.

In other embodiments of the methods provided herein, including the methods of the preceding paragraphs, the human subject to which the methods provided herein may be applied is a subject havingHuman subjects under other conditions. In one embodiment, a human subject that can use the methods provided herein further has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ and/L. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ and/L. In certain embodiments, a human subject that can use the methods provided herein also has the following conditions: hemoglobin is not less than 9g/dL. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: serum bilirubin does not exceed an Upper Limit of Normal (ULN) of 1.5 times or 3 times ULN for patients with gilbert disease. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: crCl is not less than 30mL/min. In another embodiment, a human subject that can use the methods provided herein further has the following conditions: alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) are no more than 3 times ULN. In one embodiment, a human subject that can use the methods provided herein further has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet is not less than 100×10 ⁹ and/L. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The ratio of the hemoglobin to the total hemoglobin is not less than 9g/dL. In certain embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ personal/L and serum bilirubin does not exceed 1.5 times ULN or 3 times ULN for patients with gilbert disease. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The individual/L and CrCl are not less than 30mL/min. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ personal/L and ALT and AST are not more than3 times ULN. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ The ratio of the hemoglobin to the total hemoglobin is not less than 9g/dL. In one embodiment, a human subject that can use the methods provided herein further has the following conditions: platelet count is not less than 100X 10 ⁹ personal/L and serum bilirubin does not exceed 1.5 times ULN or 3 times ULN for patients with gilbert disease. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ The individual/L and CrCl are not less than 30mL/min. In certain embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ The number of units/L and ALT and AST is not more than 3 times ULN. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: hemoglobin is no less than 9g/dL and serum bilirubin is no more than 1.5 times ULN or no more than 3 times ULN for patients with Gilbert's disease. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: hemoglobin is not less than 9g/dL and CrCl is not less than 30mL/min. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: hemoglobin is not less than 9g/dL and ALT and AST are not more than 3 times ULN. In one embodiment, a human subject that can use the methods provided herein further has the following conditions: serum bilirubin is no more than 1.5 times ULN or no more than 3 times ULN and CrCl is no less than 30mL/min for patients with gilbert disease. In another embodiment, a human subject that can use the methods provided herein further has the following conditions: serum bilirubin is no more than 1.5 times ULN or no more than 3 times ULN and ALT and AST are no more than 3 times ULN for patients with gilbert disease. In another embodiment, a human subject that can use the methods provided herein further has the following conditions: crCl is not less than 30mL/min and ALT and AST are not more than 3 times ULN. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophils Cell count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The ratio of the hemoglobin to the total hemoglobin is not less than 9g/dL. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ personal/L and serum bilirubin does not exceed 1.5 times ULN or 3 times ULN for patients with gilbert disease. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The individual/L and CrCl are not less than 30mL/min. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The number of units/L and ALT and AST is not more than 3 times ULN. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of units/L, the hemoglobin is not less than 9g/dL and the serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease. In certain embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The ratio of the hemoglobin to the CrCl is not less than 9g/dL and not less than 30mL/min. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The ratio of hemoglobin to ALT and AST is not less than 9g/dL and not more than 3 times ULN. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN and CrCl is not less than 30mL/min for patients with Gilbert's disease. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: insulation typeCount neutrophil at not less than 1.0X10 ⁹ No more than 1.5 times ULN per liter, serum bilirubin, or no more than 3 times ULN for patients with gilbert disease and no more than 3 times ULN for ALT and AST. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ Each/L, crCl is not less than 30mL/min and ALT and AST are not more than 3 times ULN. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ The number of units/L, the hemoglobin is not less than 9g/dL and the serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease. In certain embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ The ratio of the hemoglobin to the CrCl is not less than 9g/dL and not less than 30mL/min. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ The ratio of hemoglobin to ALT and AST is not less than 9g/dL and not more than 3 times ULN. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ The serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN and CrCl is not less than 30mL/min for patients with Gilbert's disease. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ No more than 1.5 times ULN per liter, serum bilirubin, or no more than 3 times ULN for patients with gilbert disease and no more than 3 times ULN for ALT and AST. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ Each/L, crCl is not less than 30mL/min and ALT and AST are not more than 3 times ULN. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: hemoglobin is not less than 9g/dL, serum bilirubin is not more than 1.5 times ULN or not more than for patients with Gilbert's disease3 times ULN and CrCl is not lower than 30mL/min. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: hemoglobin is no less than 9g/dL, serum bilirubin is no more than 1.5 times ULN or no more than 3 times ULN for patients with Gilbert's disease and ALT and AST are no more than 3 times ULN. In certain embodiments, a human subject that can use the methods provided herein also has the following conditions: hemoglobin is not less than 9g/dL, crCl is not less than 30mL/min, and ALT and AST are not more than 3 times ULN. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: serum bilirubin is no more than 1.5 times ULN or no more than 3 times ULN for patients with gilbert disease, crCl is no less than 30mL/min and ALT and AST are no more than 3 times ULN. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The number of units/L, the hemoglobin is not less than 9g/dL and the serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The ratio of the hemoglobin to the CrCl is not less than 9g/dL and not less than 30mL/min. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The ratio of hemoglobin to ALT and AST is not less than 9g/dL and not more than 3 times ULN. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN and CrCl is not less than 30mL/min for patients with Gilbert's disease. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrality Granulocyte count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ No more than 1.5 times ULN per liter, serum bilirubin, or no more than 3 times ULN for patients with gilbert disease and no more than 3 times ULN for ALT and AST. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ Each/L, crCl is not less than 30mL/min and ALT and AST are not more than 3 times ULN. In certain embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The ratio of serum bilirubin to ULN is not less than 1.5 times ULN, not less than 9g/dL, not more than 3 times ULN and not less than 30mL/min for patients with Gilbert disease. In certain embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The amount of hemoglobin is not less than 9g/dL, serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN and ALT and AST are not more than 3 times ULN for patients with Gilbert's disease. In certain embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The ratio of the total hemoglobin to the total hemoglobin is not less than 9g/dL, the ratio of the total hemoglobin to the total hemoglobin is not less than 30mL/min, and the ratio of ALT to AST is not more than 3 times ULN. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ No more than 1.5 times ULN per liter, serum bilirubin, or no more than 3 times ULN for patients with gilbert disease, no less than 30mL/min CrCl, and no more than 3 times ULN for ALT and AST. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ The ratio of serum bilirubin to ULN is not less than 1.5 times ULN, not less than 9g/dL, not more than 3 times ULN and not less than 30mL/min for patients with Gilbert disease. In some embodiments, human subjects who may use the methods provided hereinThe test subjects also had the following conditions: platelet count is not less than 100X 10 ⁹ The amount of hemoglobin is not less than 9g/dL, serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN and ALT and AST are not more than 3 times ULN for patients with Gilbert's disease. In other embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ The ratio of the total hemoglobin to the total hemoglobin is not less than 9g/dL, the ratio of the total hemoglobin to the total hemoglobin is not less than 30mL/min, and the ratio of ALT to AST is not more than 3 times ULN. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ No more than 1.5 times ULN per liter, serum bilirubin, or no more than 3 times ULN for patients with gilbert disease, no less than 30mL/min CrCl, and no more than 3 times ULN for ALT and AST. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: hemoglobin no less than 9g/dL, serum bilirubin no more than 1.5 times ULN or no more than 3 times ULN for patients with Gilbert's disease, crCl no less than 30mL/min, and ALT and AST no more than 3 times ULN. In certain embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The ratio of serum bilirubin to ULN is not less than 1.5 times ULN, not less than 9g/dL, not more than 3 times ULN and not less than 30mL/min for patients with Gilbert disease. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The amount of hemoglobin is not less than 9g/dL, serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN and ALT and AST are not more than 3 times ULN for patients with Gilbert's disease. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The ratio of the hemoglobin to the hemoglobin is not lower than 9g/dL, the CrCl is not lower than 30mL/min, and ALT and AST are not exceeding3 times ULN. In certain embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ No more than 1.5 times ULN per liter, serum bilirubin, or no more than 3 times ULN for patients with gilbert disease, no less than 30mL/min CrCl, and no more than 3 times ULN for ALT and AST. In certain embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The amount of hemoglobin is not less than 9g/dL, serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, crCl is not less than 30mL/min and ALT and AST are not more than 3 times ULN. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: platelet count is not less than 100X 10 ⁹ The amount of hemoglobin is not less than 9g/dL, serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, crCl is not less than 30mL/min and ALT and AST are not more than 3 times ULN. In some embodiments, a human subject that can use the methods provided herein also has the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The amount of hemoglobin is not less than 9g/dL, serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, crCl is not less than 30mL/min and ALT and AST are not more than 3 times ULN. In some embodiments, a human subject that can use the methods provided herein also has any of the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The amount of hemoglobin is not less than 9g/dL, serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, crCl is not less than 30mL/min and ALT and AST are not more than 3 times ULN. In some embodiments, a human subject that can use the methods provided herein also has any two of the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of platelets per liter is not less than 100×10 ⁹ The serum bilirubin is no more than 1.5 times ULN, no more than 3 times ULN, no more than 30mL/min CrCl and no more than 3 times ULN for ALT and AST in any combination or permutation. In some embodiments, a human subject that can use the methods provided herein also has any of the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The serum bilirubin is no more than 1.5 times ULN, no more than 3 times ULN, no more than 30mL/min CrCl and no more than 3 times ULN for ALT and AST in any combination or permutation. In some embodiments, a human subject that can use the methods provided herein also has any four of the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The serum bilirubin is no more than 1.5 times ULN, no more than 3 times ULN, no more than 30mL/min CrCl and no more than 3 times ULN for ALT and AST in any combination or permutation. In some embodiments, a human subject that can use the methods provided herein also has any five of the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The serum bilirubin is no more than 1.5 times ULN, no more than 3 times ULN, no more than 30mL/min CrCl and no more than 3 times ULN for ALT and AST in any combination or permutation. In some embodiments, the human subject who can use the methods provided herein also has all six of the following conditions: absolute neutrophil count is not less than 1.0X10 ⁹ The number of individual/L and platelet count is not less than 100×10 ⁹ The amount of hemoglobin is not less than 9g/dL, serum bilirubin is not more than 1.5 times ULN or not more than 3 times ULN for patients with Gilbert's disease, crCl is not less than 30mL/min and ALT and AST are not more than 3 times ULN.

In other embodiments of the methods provided herein, including the methods of the preceding paragraphs, the human subjects to which the methods provided herein can be applied are human subjects that do not contain certain conditions. In one embodiment, a human subject that may use the methods provided herein may have no more than grade 2 sensory or motor neuropathy. In some embodiments, a human subject who may use the methods provided herein may not have active central nervous system metastasis. In certain embodiments, a human subject that may use the methods provided herein may not have uncontrolled diabetes. In one embodiment, a human subject that may use the methods provided herein may have no more than grade 2 sensory or motor neuropathy and no active central nervous system metastasis. In some embodiments, a human subject that may use the methods provided herein may have no more than grade 2 sensory or motor neuropathy and no uncontrolled diabetes. In other embodiments, a human subject that may use the methods provided herein may not have active central nervous system metastasis and uncontrolled diabetes. In other embodiments, a human subject that may use the methods provided herein may have no more than grade 2 sensory or motor neuropathy, no active central nervous system metastasis, and uncontrolled diabetes. In some embodiments, a human subject that can use the methods provided herein can have any one of the following: no more than grade 2 sensory or motor neuropathy, no active central nervous system metastasis and uncontrolled diabetes. In some embodiments, a human subject that can use the methods provided herein can have any two of the following: no more than level 2 sensory or motor neuropathy, no active central nervous system metastasis and uncontrolled diabetes, in any combination or permutation. In some embodiments, a human subject that can use the methods provided herein can have all three of the following: no more than grade 2 sensory or motor neuropathy, no active central nervous system metastasis and uncontrolled diabetes. In one embodiment of the method provided in this paragraph, uncontrolled diabetes is determined by hemoglobin A1c (HbA 1 c) being no less than 8%. In some embodiments of the methods provided in this paragraph, uncontrolled diabetes is determined by HbA1c being between 7% and 8% and accompanied by associated diabetes symptoms that are not otherwise explained. In other embodiments of the methods provided in this paragraph, the associated diabetic condition comprises or consists of polyuria. In some other embodiments of the methods provided in this paragraph, the associated diabetic condition comprises or consists of polydipsia. In other embodiments of the methods provided in this paragraph, the associated diabetic condition comprises or consists of both polyuria and polydipsia.

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

In some embodiments, the CPI provided for the methods may comprise or consist of any of the CPIs described in such section (section 5.2.1.1).

5.2.1.2 therapeutic results of the methods provided herein

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

In some embodiments, the response (complete or partial response) is determined by assessing the tumor or cancer site (lesion). Criteria for determining Complete Response (CR), partial Response (PR), progressive Disease (PD) and Stable Disease (SD) are described in section 6 (e.g., in section 6.1.6.3).

Thus, the therapeutic results of the methods provided herein can be assessed based on any one or more of the reaction criteria described above. In one embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 30%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 35%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 40%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 45%, using the baseline sum diameter as a reference. In one embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 50%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 55%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 60%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 65%, using the baseline sum diameter as a reference. In one embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 70%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 75%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 80%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 85%, using the baseline sum diameter as a reference. In one embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 90%, using the baseline sum diameter as a reference. In another embodiment, the human subject has a partial response after treatment by the methods provided herein, wherein the partial response is defined by a reduction in the sum of diameters of the target lesions of at least or about 95%, using the baseline sum diameter as a reference. In some embodiments, the diameter is determined based on the longest diameter of the lesion. In certain embodiments, the diameter is determined from the longest diameter of the lesion in the measurement plane. In some embodiments, the diameter is determined from the longest diameter of the lesion in the measurement plane, wherein the smallest dimension is 10mm by CT scanning. In certain embodiments, the diameter is determined from the longest diameter of the lesion in the measurement plane, wherein the smallest dimension is 10mm and the CT slice thickness is no more than 5mm by CT scanning.

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

Alternatively, a therapeutic outcome based on a complete response, partial response, or stable disease may be assessed with respect to a population of human subjects treated by the methods provided herein by assessing the percentage of subjects in the treated population that have a complete response, partial response, or stable disease. Thus, in some embodiments, the therapeutic outcome or efficacy measurement is applicable to the outcome achieved by actually treating the population of subjects. In other embodiments, a therapeutic outcome or efficacy measurement refers to an outcome or efficacy that can be achieved when a population of human subjects is treated in a method as disclosed herein. While the following section discusses treatment of an actual population of human subjects, it is to be understood that corresponding methods in which results or efficacy measurements can be achieved in a patient population are also contemplated herein. Briefly, the two cases described above apply to the following sections; for simplicity and to avoid redundancy, only one case is described below.

In some embodiments of the methods provided herein, including sections 3, 5.3 and 6 and this section (section 5.2), the ADC is enrolment Shan Kangwei statin. In certain embodiments of the methods provided herein, including sections 3, 5.3 and 6 and this section (section 5.2), the ADC is a biological analog of enrolment Shan Kangwei statin.

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

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

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

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

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

Furthermore, the therapeutic outcome of the methods provided herein may be assessed based on the duration of the response as set forth in section 6.1.8.4. In one embodiment, the human subject has a duration of response of at least or about 4 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 5 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 6 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 7 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 8 months after treatment. In one embodiment, the human subject has a duration of response of at least or about 9 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 10 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 11 months after treatment. In one embodiment, the human subject has a duration of response of at least or about 12 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 13 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 14 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 15 months after treatment. In one embodiment, the human subject has a duration of response of at least or about 16 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 17 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 18 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 19 months after treatment. In another embodiment, the human subject has a duration of response of at least or about 20 months after treatment.

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

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

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

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

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

In addition, in some embodiments, the progression-free survival of a population of human subjects treated by the methods provided herein is assessed by assessing the median or average progression-free survival in the treatment population. In one embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average progression-free survival in the treated population is at least or about 2 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average progression-free survival in the treated population is at least or about 3 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average progression-free survival in the treated population is at least or about 4 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average progression-free survival in the treated population is at least or about 5 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average progression-free survival in the treated population is at least or about 6 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average progression-free survival in 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 progression-free survival in 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 progression-free survival in 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 progression-free survival in 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 progression-free survival in 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 progression-free survival in 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 progression-free survival in 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 progression-free survival in 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 average progression-free survival in the treated population is at least or about 15 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average progression-free survival in the treated population is at least or about 16 months. In one embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average progression-free survival in the treated population is at least or about 17 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average progression-free survival in the treated population is at least or about 18 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average progression-free survival in the treated population is at least or about 19 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average progression-free survival in the treated population is at least or about 20 months.

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

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

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

Furthermore, in some embodiments, the total survival of a population of human subjects treated by the methods provided herein is assessed by assessing the median or average total survival in the treatment population. In one embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 5 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 6 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in 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 total survival in 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 total survival in 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 total survival in 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 total survival in 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 total survival in 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 total survival in 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 total survival in 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 average total survival in the treated population is at least or about 15 months. In one embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 16 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 17 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 18 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 19 months. In one embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 20 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 21 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 22 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 23 months. In one embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 24 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 25 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 26 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 27 months. In one embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 28 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 29 months. In another embodiment, a population of human subjects is treated by the methods provided herein, wherein the median or average total survival in the treated population is at least or about 30 months.

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

5.2.2Methods of treating cancer in selected patient populations

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

In one aspect, provided herein is a method of preventing or treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or More Monomethyl Auristatin E (MMAE) units, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a Complementarity Determining Region (CDR) comprising the amino acid sequence of a CDR of the heavy chain variable region shown in SEQ ID NO. 22; and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region shown in SEQ ID NO. 23; and wherein the subject has any of the suitable features as provided in section 6.

In some aspects, provided herein is a method of preventing or treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or More Monomethyl Auristatin E (MMAE) units, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a Complementarity Determining Region (CDR) comprising the amino acid sequence of a CDR of the heavy chain variable region shown in SEQ ID NO. 22; and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region shown in SEQ ID NO. 23; and wherein the cancer has any of the suitable markers and/or features as provided in section 6.

In some aspects, provided herein is a method of treating urothelial cancer or bladder cancer in a human subject having liver metastasis, the method comprising administering to the subject having liver metastasis an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or More Monomethyl Auristatin E (MMAE) units, wherein the subject has received immune checkpoint inhibitor (CPI) therapy, and wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or More Monomethyl Auristatin E (MMAE) units, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a Complementarity Determining Region (CDR) comprising the amino acid sequence of a CDR of the heavy chain variable region shown in SEQ ID NO. 22; and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region shown in SEQ ID NO. 23; and wherein the subject has any of the suitable features as provided in section 6.

In some aspects, provided herein is a method of treating urothelial cancer or bladder cancer in a human subject having liver metastasis, the method comprising administering to the subject having liver metastasis an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or More Monomethyl Auristatin E (MMAE) units, wherein the subject has received immune checkpoint inhibitor (CPI) therapy, and wherein the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 that has been conjugated to one or More Monomethyl Auristatin E (MMAE) units, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a Complementarity Determining Region (CDR) comprising the amino acid sequence of a CDR of the heavy chain variable region shown in SEQ ID NO. 22; and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region shown in SEQ ID NO. 23; and wherein the urothelial cancer or bladder cancer has any of the suitable markers and/or features as provided in section 6.

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

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

In some aspects, provided herein is a method of treating urothelial cancer or bladder cancer in a human subject, the method comprising administering to the subject an effective amount of an antibody drug conjugate, wherein the antibody drug conjugate comprises an antibody or antigen binding fragment thereof that binds to 191P4D12 that has been conjugated to one or More Monomethyl Auristatin E (MMAE) units, wherein the subject has received immune checkpoint inhibitor (CPI) therapy, wherein the subject has progression or recurrence of the cancer during or after the CPI therapy, and wherein the antibody drug conjugate comprises an antibody or antigen binding fragment thereof that binds to 191P4D12 that has been conjugated to one or More Monomethyl Auristatin E (MMAE) units, wherein the antibody or antigen binding fragment thereof comprises: a heavy chain variable region comprising a Complementarity Determining Region (CDR) comprising the amino acid sequence of a CDR of the heavy chain variable region shown in SEQ ID NO. 22; and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region shown in SEQ ID NO. 23; and wherein the subject has any of the suitable features as provided in section 6.

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

In all methods provided herein and specifically described in sections 5.2.1 and 5.2.2: therapeutic agents that can be used are described in chapter 5.3 and chapter 6; the selection of patients to be treated is described herein and exemplified in chapter 5.2 (including chapter 5.2.2) and chapter 6; dosage regimens and pharmaceutical compositions for administering therapeutic agents are described in the following articles section 5.4, 5.6, 5.7 and section 6; biomarkers that can be used to identify therapeutic agents, select patients, determine the results of these methods, and/or serve in any way as criteria for these methods are described herein and exemplified in section 5.2 (including section 5.2.2) and section 6; the therapeutic results of the methods provided herein can be improvements of the biomarkers described herein, such as those described and exemplified in chapter 5.2 (including chapter 5.2.2) and chapter 6. Thus, those of skill in the art will appreciate that the methods provided herein include all permutations and combinations of patients, therapeutic agents, dosing regimens, biomarkers, and therapeutic results as described above and below.

5.3 antibody drug conjugates for use in methods

In various embodiments of the methods provided herein, including the methods provided in section 5.2, the ADC used in the methods comprise or are anti-1914d12 ADC described herein and/or in U.S. patent No. 8,637,642, which is incorporated herein by reference in its entirety. In some embodiments, an anti-191P 4D12 antibody drug conjugate provided in the methods herein comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 as provided herein (including subsection 5.3.1), the 191P4D12 being conjugated to one or more cytotoxic agent (drug unit or D) units as provided herein (including this subsection (subsection 5.3), further disclosed in subsection 5.3.2). In certain embodiments, the cytotoxic agent (drug unit or D) may be directly covalently linked or linked via a Linker Unit (LU).

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

L-(LU-D) _p (I)

or a pharmaceutically acceptable salt or solvate thereof; wherein:

l is an antibody unit, e.g., an anti-connexin-4 antibody or antigen-binding fragment thereof, as provided in subsection 5.3.1 below, and

(LU-D) is a linker unit-drug unit moiety, wherein:

LU-is a linker unit, and

d is a drug unit having cytostatic or cytotoxic activity against the target cell; and is also provided with

p is an integer of 1 to 20.

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

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

L-(A _a -W _w -Y _y -D) _p (II)

or a pharmaceutically acceptable salt or solvate thereof, wherein:

l is an antibody unit, such as an anti-connexin-4 antibody or antigen-binding fragment thereof as provided in subsection 5.3.1 below; and is also provided with

-A _a -W _w -Y _y -a joint unit (LU), wherein:

-a-is an extension subunit,

a is 0 or 1, and the number of the components is 1,

each-W-is independently an amino acid unit,

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

y-is a self-decomposing spacer unit,

y is 0, 1 or 2;

p is an integer of 1 to 20.

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

In some specific embodiments of the methods provided herein (including the methods provided in section 5.2), the cytotoxic agent that is part of any ADC provided herein to the methods comprises, consists of, or is MMAE.

For compositions comprising multiple antibodies or antigen binding fragments thereof, drug loading is represented by p (average number of drug molecules per antibody unit). Drug loading can range from 1 to 20 drugs (D)/antibody. The average drug number/antibody in the preparation of the coupling reaction can be characterized by conventional means such as mass spectrometry, ELISA assay and HPLC. Quantitative distribution of antibody drug conjugates was also determined according to p. In some cases, isolation, purification and characterization of homogeneous antibody drug conjugates, where p is a particular value of the antibody drug conjugate with other drug loading, can be accomplished by means such as reverse phase HPLC or electrophoresis. In an exemplary embodiment, p is 2 to 8.

Additional embodiments of ADCs for use in the methods provided herein have been described in U.S. patent No. 8,637,642 and international application No. PCT/US2019/056214 (publication No. WO 2020/117373), both of which are incorporated herein by reference in their entirety.

5.3.1anti-191P 4D12 antibodies or antigen binding fragments

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

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

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

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

In some embodiments, the anti-connexin-4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a Complementarity Determining Region (CDR) comprising the amino acid sequence of a heavy chain variable region shown as SEQ ID NO. 22, which is an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 136 (serine) of SEQ ID NO. 7; and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region shown as SEQ ID NO. 23, which is an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 130 (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 comprising the amino acid sequence corresponding to complementarity determining region 1 (CDR-H1), CDR-H2 and CDR-H3 in the heavy chain variable region sequence shown as SEQ ID NO. 22, which is an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 136 (serine) of SEQ ID NO. 7; and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences corresponding to CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region sequence shown as SEQ ID NO:23, which is an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 130 (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 as SEQ ID No. 22, which is the amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 136 (serine) of SEQ ID No. 7; and a light chain variable region comprising a CDR consisting of the amino acid sequence of the CDR of the light chain variable region shown as SEQ ID NO. 23, which is an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 130 (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 corresponding to complementarity determining region 1 (CDR-H1), CDR-H2 and CDR-H3 in the heavy chain variable region sequence shown as SEQ ID NO. 22, which is an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 136 (serine) of SEQ ID NO. 7; and a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 consisting of the amino acid sequences corresponding to CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region sequence shown as SEQ ID NO:23, which is an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 130 (arginine) of SEQ ID NO: 8. SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 7 and SEQ ID NO. 8 are shown in FIG. 1D and FIG. 1E and listed below:

SEO ID NO：22

SEQ ID NO23

SEQ ID NO7

SEQ ID NO：8

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, kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are most commonly used (see, e.g., kabat et al, supra). Chothia actually refers to the position of the structural loop (see, e.g., chothia and Lesk,1987, J.mol. Biol. 196:901-17). The ends of the Chothia CDR-H1 loop vary between H32 and H34 when numbered using the Kabat numbering convention, depending on the length of the loop (since the Kabat numbering convention will place insertions in H35A and H35B; if neither 35A nor 35B is present, the loop ends are at 32; if only 35A is present, the loop ends are at 33; if both 35A and 35B are present, the loop ends are at 34). The AbM hypervariable region represents a compromise between Kabat CDR and Chothia structural loops, and is made by Oxford Molecular AbM antibody modeling softwareUsing (see e.g.Antibody EngineerinVolume g 2 (Kontermann and Dubel, 2 nd edition, 2010)). The "contact" hypervariable region is based on analysis of available complex crystal structures. Another common numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information (Lafranc et al, 2003, dev. Comp. Immunol.27 (1): 55-77). IMGT is an integrated information system specific for Immunoglobulins (IG), T Cell Receptors (TCR) and Major Histocompatibility Complex (MHC) in humans and other vertebrates. Herein, CDRs are referred to with respect to amino acid sequences and positions within the light chain or heavy chain. Since the "positions" of CDRs within an immunoglobulin variable domain structure are conserved across species and exist in a structure called a loop, CDRs and framework residues are readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used to graft and replace CDR residues from immunoglobulins of a species into the acceptor framework typically from human antibodies. Another numbering system (AHon) has been developed by Honyger and Pluckthun, 2001, J.mol.biol.309:657-70. Correspondence between numbering systems (including, for example, kabat numbering and IMGT unique numbering systems) is well known to those skilled in the art (see, for example, kabat, supra; chothia and Lesk, supra; martin, supra; lefranc et al, supra). Residues from each of these hypervariable regions or CDRs are indicated in table 1 above.

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

In some embodiments, the anti-connexin-4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a CDR (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 AbM numbering; 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 according to AbM numbering.

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

In other embodiments, the anti-connexin-4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a CDR (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 the Contact numbering; 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 according to the Contact number.

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

In some 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 Kabat numbering; 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 according to Kabat numbering.

In some embodiments, the anti-connexin-4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a CDR (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 AbM numbering; 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 according to AbM numbering.

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

In other embodiments, the anti-connexin-4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a CDR (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 the Contact numbering; 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 according to the Contact number.

In other embodiments, the anti-connexin-4 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a CDR (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 IMGT numbering; 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 according to IMGT numbering.

As described above, CDR sequences according to different numbering systems can be readily determined, for example using online tools such as those provided by antigen receptor numbering and receptor classification (Antigen receptor Numbering And Receptor ClassificatIon; ANARCI). For example, 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, as determined by ANARCI.

TABLE 4 Table 4

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

TABLE 5

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

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

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

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

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.

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

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

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

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 in the range of amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO. 8.

In some embodiments, amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to optimize the binding affinity and/or other biological properties of an antibody, including (but not limited to) specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity or solubility. Thus, it is contemplated that antibody variants may be made in addition to the antibodies described herein. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and/or by synthesizing the desired antibody or polypeptide. Those skilled in the art having knowledge of amino acid changes can alter the post-translational processes of the antibody, for example, alter the number or position of glycosylation sites or alter membrane anchoring characteristics.

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

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

Amino acid sequence insertions include amino and/or carboxy-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.

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

Amino acids can be grouped according to their similarity of side chain properties (see, e.g., lehninger, biochemistry73-75 (2 nd edition, 1975)): (1) nonpolar: ala (A), val (V), leu (L), ile (I), pro (P), phe (F), trp (W), met (M); (2) uncharged polarity: gly (G), ser (S), thr (T), cys (C), tyr (Y), asn (N), gln (Q); (3) acidity: asp (D), glu (E); and (4) alkaline: lys (K), arg (R), his (H). Alternatively, naturally occurring residues can be grouped based on common side chain characteristics: (1) hydrophobicity: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilicity: cys, ser, thr, asn, gln; (3) acidity: asp, glu; (4) alkaline: his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

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

Variations can be generated using methods known in the art, such as oligonucleotide-mediated (site-directed) mutation induction, alanine scanning, and PCR mutation induction. The cloned DNA may be subjected to site-directed mutagenesis (see, e.g., carter,1986,Biochem J.237:1-7; and Zoller et al, 1982,Nucl.Acids Res.10:6487-500), cassette mutagenesis (see, e.g., wells et al, 1985, gene 34:315-23), or other known techniques to generate anti-MSLN antibody variant DNA.

Covalent modifications of antibodies are included within the scope of the present disclosure. Covalent modification includes reacting the amino acid residues of interest of the antibody with an organic derivatizing agent 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 the corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; hydroxy phosphorylation of seryl or threonyl residues; methylation of alpha-amino groups of lysine, arginine and histidine side chains (see, e.g., cright on, proteins: structure and Molecular Properties-86 (1983)); acetylation of the N-terminal amine; and amidation of any C-terminal carboxyl groups.

Other types of covalent modification 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 non-proteinaceous polymers (e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene), in a manner such as set forth in the following documents: U.S. Pat. nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337.

In certain embodiments, the antibodies or antigen binding fragments provided herein comprise a heavy chain having a degree of homology or identity to the heavy chain as set forth in SEQ ID NO. 7 and a light chain having a degree of homology or identity to the light chain as set forth in SEQ ID NO. 8. Such embodiments of heavy/light chains having homology or identity are further provided below. 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 as 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 75% homology or identity to the heavy chain as 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 80% homology or identity to the heavy chain as 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 85% homology or identity to the heavy chain as set forth in SEQ ID NO. 7. In some embodiments, the antibodies or antigen binding fragments provided herein comprise a heavy chain having greater than 90% homology or identity to the heavy chain as 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 as 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 provided homologies or identities to the heavy chain as 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 as 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 as set forth in SEQ ID NO. 8. In some embodiments, the antibodies or antigen binding fragments provided herein comprise a light chain having greater than 75% homology or identity to the light chain as set forth in SEQ ID NO. 8. In some embodiments, the antibodies or antigen binding fragments provided herein comprise a light chain having greater than 80% homology or identity to the light chain as set forth in SEQ ID NO. 8. In some embodiments, the antibodies or antigen binding fragments provided herein comprise a light chain having greater than 85% homology or identity to the light chain as set forth in SEQ ID NO. 8. In some embodiments, the antibodies or antigen binding fragments provided herein comprise a light chain having greater than 90% homology or identity to the light chain as set forth in SEQ ID NO. 8. In some embodiments, the antibodies or antigen binding fragments provided herein comprise a light chain having greater than 95% homology or identity to the light chain as set forth in SEQ ID NO. 8. In certain embodiments, the antibodies or antigen binding fragments provided herein comprise a light chain having any one of the provided homologies or identities to the light chain as set forth in SEQ ID NO. 8, wherein the CDRs (CDR-L1, CDR-L2 and CDR-L3) are identical to the CDRs in the light chain as set forth in SEQ ID NO. 8. In certain embodiments, an antibody or antigen binding fragment provided herein comprises any combination or permutation of any of the cognate light chains provided in such a paragraph with any of the cognate heavy chains.

In certain embodiments, an antibody or antigen binding fragment provided herein comprises a heavy chain variable region having a homology or identity to the heavy chain variable region as set forth in SEQ ID NO. 22 and a light chain variable region having a homology or identity to the light chain variable region as 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 below. In some embodiments, the antibodies or antigen binding fragments provided herein comprise a heavy chain variable region having greater than 70% homology or identity to the heavy chain variable region as 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 as 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 as 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 as 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 as 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 as 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 one of the provided homologies or identities to the heavy chain variable region as 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 as set forth in SEQ ID NO. 22. In some embodiments, an antibody or antigen binding fragment provided herein comprises a light chain variable region having greater than 70% homology or identity to the light chain variable region as set forth in SEQ ID NO. 23. In some embodiments, an antibody or antigen binding fragment provided herein comprises a light chain variable region having greater than 75% homology or identity to a light chain variable region as set forth in SEQ ID NO. 23. In some embodiments, an antibody or antigen binding fragment provided herein comprises a light chain variable region having greater than 80% homology or identity to the light chain variable region as set forth in SEQ ID NO. 23. In some embodiments, an antibody or antigen binding fragment provided herein comprises a light chain variable region having greater than 85% homology or identity to the light chain variable region as set forth in SEQ ID NO. 23. In some embodiments, an antibody or antigen binding fragment provided herein comprises a light chain variable region having greater than 90% homology or identity to the light chain variable region as set forth in SEQ ID NO. 23. In some embodiments, an antibody or antigen binding fragment provided herein comprises a light chain variable region having greater than 95% homology or identity to the light chain variable region as 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 one of the provided homologies or identities to the light chain variable region as 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 as set forth in SEQ ID NO. 23. In certain embodiments, an antibody or antigen binding fragment provided herein comprises any combination or permutation of any of the homologous light chain variable regions provided in such paragraph with any of the homologous heavy chain variable regions.

In some embodiments, an anti-connexin-4 antibody provided herein comprises: the heavy and light chain CDR regions of an antibody known as Ha22-2 (2, 4) 6.1 produced by the hybridoma (accession number: PTA-11267) which is maintained in the American type culture Collection (American Type Culture Collection; ATCC), or heavy and light chain CDR regions comprising an amino acid sequence homologous to the amino acid sequence of the heavy and light chain CDR regions of Ha22-2 (2, 4) 6.1, and wherein the antibody retains the desired functional properties of an anti-connexin-4 antibody known as Ha22-2 (2, 4) 6.1 produced by the hybridoma (accession number: PTA-11267) which is maintained in the American Type Culture Collection (ATCC).

In some embodiments, an anti-connexin-4 antibody provided herein comprises: the heavy and light chain CDR regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) of the antibody known as Ha22-2 (2, 4) 6.1 produced by the hybridoma (accession number: PTA-11267) maintained in the American Type Culture Collection (ATCC); or heavy and light chain CDR regions consisting of amino acid sequences homologous to those of the heavy and light chain CDR regions of Ha22-2 (2, 4) 6.1, and wherein the antibody retains the desired functional properties of an anti-connexin-4 antibody known as Ha22-2 (2, 4) 6.1 produced by the hybridoma (accession number: PTA-11267) deposited with the American Type Culture Collection (ATCC).

In some embodiments, an antibody or antigen binding fragment thereof provided herein comprises a humanized heavy chain variable region and a humanized light chain variable region, wherein:

(a) The heavy chain variable region is contained in the polypeptide and is stored in the polypeptide American Type Culture Collection (ATCC) accession number: the CDRs (CDR-H1, CDR-H2 and CDR-H3) of the amino acid sequences of the heavy chain variable region CDRs set forth in the antibody produced by the hybridoma under PTA-11267;

(b) The light chain variable region comprises a polypeptide sequence that is encoded by a polypeptide sequence deposited in the American Type Culture Collection (ATCC) accession number: the hybridomas under PTA-11267 produce the CDRs (CDR-L1, CDR-L2 and CDR-L3) of the amino acid sequences of the CDRs of the light chain variable region described in the antibody.

(a) The heavy chain variable region is contained in the heavy chain American Type Culture Collection (ATCC) accession number: CDRs (CDR-H1, CDR-H2 and CDR-H3) consisting of the amino acid sequences of the CDRs of the heavy chain variable region set forth in the antibody produced by the hybridoma under PTA-11267;

(b) The light chain variable region is contained in the polypeptide by preservation American Type Culture Collection (ATCC) accession number: the hybridoma under PTA-11267 produces CDRs (CDR-L1, CDR-L2 and CDR-L3) consisting of the amino acid sequences of the CDRs of the light chain variable region set forth in the antibody.

In some embodiments, an anti-connexin-4 antibody provided herein comprises: the heavy and light chain variable regions of an antibody designated Ha22-2 (2, 4) 6.1 produced by the hybridoma maintained under American Type Culture Collection (ATCC) accession number PTA-11267, or heavy and light chain variable regions comprising an amino acid sequence homologous to the amino acid sequences of the heavy and light chain variable regions of Ha22-2 (2, 4) 6.1, and wherein the antibody retains the desired functional properties of the anti-connexin-4 antibodies provided herein. In some embodiments, a connexin-4 antibody provided herein comprises: the heavy and light chain variable regions of an antibody designated Ha22-2 (2, 4) 6.1 produced by the hybridoma maintained under American Type Culture Collection (ATCC) accession number PTA-11267, or 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 antibody retains the desired functional properties of the anti-connexin-4 antibodies provided herein. Any subclass of constant region can be selected as the constant region of the antibodies of the present disclosure. In one embodiment, a human IgG1 constant region may be used as the heavy chain constant region and a human igκ constant region may be used as the light chain constant region.

In some embodiments, an anti-connexin-4 antibody provided herein comprises: the heavy and light chains of an antibody designated Ha22-2 (2, 4) 6.1 (produced by a hybridoma maintained under American Type Culture Collection (ATCC) accession number PTA-11267), or heavy and light chains comprising an amino acid sequence homologous to the amino acid sequences of Ha22-2 (2, 4) 6.1, and wherein the antibody retains the desired functional properties of the anti-connexin-4 antibody provided herein. In some embodiments, the anti-connexin-4 antibodies provided herein comprise the heavy and light chains of an antibody known as Ha22-2 (2, 4) 6.1 (produced by a hybridoma maintained at American Type Culture Collection (ATCC) accession number: PTA-11267), or 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 antibody retains the desired functional properties of the anti-connexin-4 antibodies provided herein.

In some embodiments, an antibody or antigen binding fragment thereof provided herein comprises a heavy chain variable region and a light chain variable region, wherein:

(a) The heavy chain variable region comprises and is preserved by accession number in the American Type Culture Collection (ATCC): the heavy chain variable region amino acid sequence of the antibody produced by the hybridoma under PTA-11267 is at least 80% homologous or identical; and is also provided with

(b) The light chain variable region comprises a sequence as set forth in the American Type Culture Collection (ATCC) accession number: the light chain variable region amino acid sequence of the antibody produced by the hybridoma under PTA-11267 is at least 80% homologous or identical.

In certain embodiments, an antibody or antigen binding fragment provided herein comprises a polypeptide that is purified by a polypeptide that is deposited in the American Type Culture Collection (ATCC) accession No.: the heavy chain variable region of the antibody produced by the hybridoma under PTA-11267 has a degree of homology or identity to the heavy chain variable region amino acid sequence by the accession number of the American Type Culture Collection (ATCC): the light chain variable region amino acid sequence of the antibody produced by the hybridoma under PTA-11267 has a certain homology or identity. Such embodiments of heavy chain variable regions and light chain variable regions having homology or identity are further provided below. In some embodiments of the present invention, in some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in the American Type Culture Collection (ATCC) accession number: the heavy chain variable region amino acid sequence of the antibody produced by the hybridoma under PTA-11267 is at least 85% homologous or identical. In the case of a further embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence set forth in the American Type Culture Collection (ATCC) accession number: the heavy chain variable region amino acid sequence of the antibody produced by the hybridoma under PTA-11267 is at least 90% homologous or identical. In the case of a further embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence set forth in the American Type Culture Collection (ATCC) accession number: the heavy chain variable region amino acid sequence of the antibody produced by the hybridoma under PTA-11267 is at least 95% homologous or identical. In other embodiments, the heavy chain variable region may be identified by the accession number saved in the American Type Culture Collection (ATCC): the heavy chain variable region amino acid sequences of antibodies produced by the hybridomas under PTA-11267 are 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical. In some embodiments of the present invention, in some embodiments, the light chain variable region comprises the sequence set forth in accession number by accession number deposited in the American Type Culture Collection (ATCC): the light chain variable region amino acid sequence of the antibody produced by the hybridoma under PTA-11267 is at least 85% homologous or identical. In the case of a further embodiment of the present invention, the light chain variable region comprises the sequence set forth in accession number by accession number deposited in the American Type Culture Collection (ATCC): the light chain variable region amino acid sequence of the antibody produced by the hybridoma under PTA-11267 is at least 90% homologous or identical. In the case of a further embodiment of the present invention, the light chain variable region comprises the sequence set forth in accession number by accession number deposited in the American Type Culture Collection (ATCC): the light chain variable region amino acid sequence of the antibody produced by the hybridoma under PTA-11267 is at least 95% homologous or identical. In other embodiments, the light chain variable region may be identified by the accession number saved in the American Type Culture Collection (ATCC): the light chain variable region amino acid sequences of antibodies produced by the hybridomas under PTA-11267 are 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical. In certain embodiments, an antibody or antigen binding fragment provided herein comprises any combination or permutation of any of the homologous light chain variable regions provided in such paragraph with any of the homologous heavy chain variable regions.

In other embodiments, an antibody or antigen binding fragment thereof provided herein comprises a heavy chain and a light chain, wherein:

(a) The heavy chain comprises and is deposited by accession numbers in the American Type Culture Collection (ATCC): the heavy chain amino acid sequence of the antibody produced by the hybridoma under PTA-11267 is at least 80% homologous or identical; and

(b) The light chain comprises and is obtained by the method described in American Type Culture Collection (ATCC) accession number: the light chain amino acid sequence of the antibody produced by the hybridoma at PTA-11267 is at least 80% homologous or identical.

In certain embodiments, an antibody or antigen binding fragment provided herein comprises a polypeptide that is purified by a polypeptide that is deposited in the American Type Culture Collection (ATCC) accession No.: the heavy chain amino acid sequence of the antibody produced by the hybridoma under PTA-11267 has a degree of homology or identity to the heavy chain by accession numbers stored in the American Type Culture Collection (ATCC): the light chain amino acid sequence of the antibody produced by the hybridoma under PTA-11267 has a light chain with certain homology or identity. Such embodiments of heavy and light chains having homology or identity are further provided below. In some embodiments, the heavy chain comprises a heavy chain that is encoded by a heavy chain encoded by the American Type Culture Collection (ATCC) accession No.: the heavy chain amino acid sequence of the antibody produced by the hybridoma at PTA-11267 is at least 85% homologous or identical. In other embodiments, the heavy chain comprises and is deposited by the American Type Culture Collection (ATCC) accession No.: the heavy chain amino acid sequence of the antibody produced by the hybridoma at PTA-11267 is at least 90% homologous or identical. In other embodiments, the heavy chain comprises and is deposited by the American Type Culture Collection (ATCC) accession No.: the heavy chain amino acid sequence of the antibody produced by the hybridoma at PTA-11267 is at least 95% homologous or identical. In other embodiments, the heavy chain may be identified by the accession number saved in the American Type Culture Collection (ATCC): the heavy chain amino acid sequences of antibodies produced by the hybridomas under PTA-11267 are 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical. In some embodiments, the light chain comprises a heavy chain that is encoded by a heavy chain encoded by the American Type Culture Collection (ATCC) accession No.: the light chain amino acid sequence of the antibody produced by the hybridoma at PTA-11267 is at least 85% homologous or identical. In other embodiments, the light chain comprises a heavy chain that is encoded by a heavy chain encoded by the American Type Culture Collection (ATCC) accession No.: the light chain amino acid sequence of the antibody produced by the hybridoma at PTA-11267 is at least 90% homologous or identical. In other embodiments, the light chain comprises a heavy chain that is encoded by a heavy chain encoded by the American Type Culture Collection (ATCC) accession No.: the light chain amino acid sequence of the antibody produced by the hybridoma at PTA-11267 is at least 95% homologous or identical. In other embodiments, the light chain may be identified by the accession number saved in the American Type Culture Collection (ATCC): the light chain amino acid sequences of antibodies produced by the hybridomas under PTA-11267 are 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical. In certain embodiments, an antibody or antigen binding fragment provided herein comprises any combination or permutation of any of the cognate light chains provided in such a paragraph with any of the cognate heavy chains.

In some embodiments, the antibodies provided herein, or antigen binding fragments thereof, bind to a specific epitope in 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to the VC1 domain of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to the VC1 domain of 191P4D12 but not to the C1C2 domain. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 1-147 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to an epitope located in amino acid residues 1-147 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 1-10 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 11-20 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 21 to 30 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 31-40 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 41-50 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 51-60 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid 61 to 70 residues of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 71-80 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 81 to 90 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 91 to 100 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 101-110 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 111 to 120 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 121 to 130 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid 131 to 140 of 191P4D 12. In some embodiments, an antibody or antigen binding fragment thereof provided herein binds to amino acid residues 141 through 147 of 191P4D 12. Binding epitopes of certain embodiments of the antibodies or antigen binding fragments thereof provided herein have been determined and described in WO 2012/047724, which is incorporated herein by reference in its entirety.

In some embodiments, the antibodies or antigen binding fragments thereof provided herein bind to an epitope in 191P4D12 between 191P4D12 variants commonly observed in humans. In some embodiments, the antibodies or antigen binding fragments thereof provided herein bind to an epitope in 191P4D12 that is common between 191P4D12 polymorphisms observed in humans. In some embodiments, the antibodies or antigen binding fragments thereof provided herein bind to an epitope in 191P4D12 between 191P4D12 polymorphisms commonly observed in human cancers. In some embodiments, the antibodies provided herein, or antigen binding fragments thereof, bind to an epitope of 191P4D12 that will bind, internalize, disrupt, or modulate a biological function of 191P4D12 or 191P4D12 variant. In some embodiments, the antibodies provided herein, or antigen binding fragments thereof, bind to an epitope of 191P4D12 that would disrupt the interaction between 191P4D12 and the ligand, substrate, and binding partner.

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

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

In addition to or instead of modifications made within the framework or CDR regions, antibodies of the present disclosure may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, fc receptor binding, and/or antigen-dependent cytotoxicity. Furthermore, the anti-191P 4D12 antibodies provided herein may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation to again alter one or more functional properties of the antibody. Each of these implementations is described in further detail below.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. Such a process is further described in U.S. Pat. No. 5,677,425 to Bodmer et al. The number of cysteine residues in the CH1 hinge region is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the anti-191P 4D12 antibody.

In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the anti-191P 4D12 antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment such that the binding of the antibody to staphylococcal protein A (Staphylococcyl protein A; spA) is reduced relative to the native Fc hinge domain SpA binding. Such a method is described in further detail in U.S. Pat. No. 6,165,745 to Ward et al.

In another embodiment, the anti-191P 4D12 antibody is modified to increase its biological half-life. A variety of methods are possible. For example, mutations can be introduced as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively, to increase biological half-life, antibodies may be altered within the CH1 or CL region to contain two loops of the CH2 domain obtained from the Fc region of IgG to bind epitopes to the rescue receptor, as described in U.S. Pat. nos. 5,869,046 and 6,121,022 to presa et al.

In other embodiments, the Fc region is altered by substitution of at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from a particular amino acid residue may be substituted with a different amino acid residue such that the affinity of the antibody for the effector ligand is altered but the antigen binding capacity of the parent antibody is preserved. The affinity-altering effector ligand may be, for example, an Fc receptor or the C1 component of complement. Such a method is described in further detail in, for example, U.S. Pat. Nos. 5,624,821 and 5,648,260 to Winter et al.

The reactivity of the anti-191P 4D12 antibody with 191P4D 12-associated protein can be established by a variety of well-known means including western blot, immunoprecipitation, ELISA and FACS analysis using 191P4D 12-associated protein, 191P4D 12-expressing cells or extracts thereof, as appropriate. The 191P4D12 antibody or fragment thereof may be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymes. Furthermore, bispecific antibodies specific for two or more 191P4D12 epitopes are generated using methods generally known in the art. Homodimeric antibodies may also be produced by crosslinking techniques known in the art (e.g., wolff et al, cancer Res. 53:2560-2565).

In another specific embodiment, the anti-191P 4D12 antibodies provided herein are antibodies comprising heavy and light chains of an antibody known as Ha22-2 (2, 4) 6.1. The heavy chain of Ha22-2 (2, 4) 6.1 consists of an amino acid sequence in the range of residues 20 (E) to 466 (K) of SEQ ID NO. 7; and the light chain of Ha22-2 (2, 4) 6.1 consists of an amino acid sequence in the range of residues (D) to 236 (C) of the sequence of SEQ ID NO. 8.

Hybridomas producing the antibody designated Ha22-2 (2, 4) 6.1 were sent (via Federal Express) to the American Type Culture Collection (ATCC) of p.o. box 1549,Manassas,VA 20108 at month 8 and 18 2010 and assigned accession number PTA-11267.

Additional embodiments of anti-connexin-4 antibodies have been described in U.S. patent No. 8,637,642 and international application No. PCT/US2019/056214 (publication No. WO 2020/117373), both of which are incorporated herein by reference in their entirety.

5.3.2Cytotoxic agents (pharmaceutical units)

Since the ADCs used in the methods provided herein comprise antibodies or antigen binding fragments thereof conjugated to a cytotoxic agent, the present disclosure further provides various embodiments of the cytotoxic agent as part of the ADCs used in the methods. In various embodiments of the methods provided herein (including the methods provided in section 5.2), the cytotoxic agent that is part of any ADC provided herein to the methods comprises, consists of, or is a tubulin disrupting agent. In one embodiment, the cytotoxic agent is a tubulin disrupting agent. In some embodiments, the tubulin disrupting agent is selected from the group consisting of: dolastatin, auristatin, hamiltin, vinca alkaloids, maytansinoids, eribulin, colchicine, pribulin, phomopsin, epothilone, candidiasis, and taxanes. In a specific embodiment, the tubulin disrupting agent is auristatin. In another specific embodiment, the auristatin is monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), AFP, or auristatin T. In another specific embodiment, the auristatin is monomethyl auristatin E (MMAE).

In various embodiments of the methods provided herein (including the methods provided in section 5.2), the cytotoxic agent that is part of any ADC provided herein to the methods comprises, consists of, or is the following: any agent selected from the group consisting of the cytotoxic agents described in U.S. patent No. 8,637,642 and international application No. PCT/US2019/056214 (publication No. WO 2020/117373), both of which are incorporated herein by reference in their entirety.

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

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

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

In general, peptide-based drug units can be prepared by forming peptide bonds between two or more amino acids and/or peptide fragments. Such peptide bonds may for example be according to liquid phase synthesis methods well known in the art of peptide chemistry (see E.And K.L u bke, "The Peptides", volume 1, pages 76-136, 1965,Academic Press). The auristatin/dolastatin drug unit can be prepared according to the following literature: US 5635483; US 5780588; pettit et al (1989) J.am.chem.Soc.111:5463-5465; pettit et al (1998) Anti-Cancer Drug Design 13:243-277; pettit, g.r. et al, synthesis,1996,719-725; pettit et al (1996) J.chem. Soc. Perkin Trans.1:859-863; and Doronina (2003) Nat Biotechnol 21 (7): 778-784.

Additional embodiments of cytotoxic agents have been described in U.S. patent No. 8,637,642 and international application No. PCT/US2019/056214 (publication No. WO 2020/117373), both of which are incorporated herein by reference in their entirety.

5.3.3Joint

Typically, antibody drug conjugates comprise a linker unit between the drug unit (e.g., MMAE) and the antibody unit (e.g., anti-191P 4D12 antibody or antigen-binding fragment thereof). In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker in the intracellular environment releases the drug unit from the antibody. In other embodiments, the linker unit is not cleavable and releases the drug, e.g., by antibody degradation. In some embodiments, the linker is cleavable by a cleavage agent present in the intracellular environment (e.g., lysosomes or endosomes or within a cell membrane pit). The linker may be, for example, a peptidyl linker cleavable by an intracellular peptidase or protease, including but not limited to lysosomal or endosomal proteases. For example, a peptidyl linker (e.g., phe-Leu or Gly-Phe-Leu-Gly linker (SEQ ID NO: 15)) cleavable by thiol-dependent protease cathepsin B, which is highly expressed in cancerous tissue, may be used. In some embodiments, the peptide-based linker is at least two amino acids or at least three amino acids in length. In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH sensitive linker is hydrolyzable under acidic conditions. For example, acid labile linkers (e.g., hydrazone, semi-carbazone, thiosemi-carbazone, cis-aconitamide, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used. In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). Various disulfide linkers are known in the art, including, for example, those that may be formed using N-succinimidyl-S-acetylthioacetate (SATA), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), N-succinimidyl-3- (2-pyridyldithio) butyrate (SPDB), and N-succinimidyl-oxycarbonyl- α -methyl- α - (2-pyridyldithio) toluene (SMPT), SPDB, and SMPT.

"linker units" (LU) are bifunctional compounds useful for linking a drug unit to an antibody unit to form an antibody drug conjugate. In some embodiments, the linker unit has the formula:

-A _a -W _w -Y _y -

wherein: -a-is an extension subunit,

a is 0 or 1, and the number of the components is 1,

each-W-is independently an amino acid unit,

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

y-is a self-decomposing spacer unit, and

y is 0, 1 or 2.

In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1, or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, when w is 1 to 12, y is 1 or 2. In some embodiments, w is 2 to 12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0. Each of the linker and extension subunits, amino acid units, and spacer subunits have been described in U.S. patent No. 8,637,642 and international application No. PCT/US2019/056214 (publication No. WO 2020/117373), both of which are incorporated herein by reference in their entirety.

Embodiments of the antibody-drug conjugate may include:

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

wherein w and y are each 0,

5.3.4drug loading

Drug loading is represented by p and is the average number of drug units per antibody in the molecule. Drug loading can range from 1 to 20 drug units (D)/antibody. The ADCs provided herein comprise a collection of antibodies or antigen binding fragments conjugated to a series of drug units (e.g., 1 to 20). In preparing ADCs from coupling reactions, the average number of drug units per antibody can be characterized by conventional means such as mass spectrometry and ELISA assays. Quantitative distribution of ADCs can also be determined from p. In some cases, the separation, purification and characterization of a homogeneous ADC, where p is a particular value, from an ADC with other drug loading may be achieved by means such as electrophoresis.

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

In certain embodiments, the drug loading of the ADCs provided herein is within the following range: 1 to about 8; about 2 to about 6; about 3 to about 5; about 3 to about 4; about 3.1 to about 3.9; about 3.2 to about 3.8; about 3.2 to about 3.7; about 3.2 to about 3.6; about 3.3 to about 3.8; or about 3.3 to about 3.7.

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

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

In certain embodiments, less than the theoretical maximum of drug units are conjugated to the antibody during the conjugation reaction. Antibodies may contain lysine residues that are, for example, not reactive with drug-linker intermediates or linker reagents. Generally, antibodies do not contain a number of free and reactive cysteine thiol groups that can be linked to a drug unit; in practice, most of the cysteine thiol residues in antibodies exist as disulfide bridges. In certain embodiments, the antibodies can be reduced with a reducing agent such as Dithiothreitol (DTT) or tricarbonyl ethyl phosphine (TCEP) under partial or complete reducing conditions to produce reactive cysteine thiol groups. In certain embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine. In some embodiments, the linker unit or the drug unit is coupled via a lysine residue on the antibody unit. In some embodiments, the linker unit or the drug unit is coupled via a cysteine residue on the antibody unit.

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

The load of the ADC (drug/antibody ratio) can be controlled in different ways, for example by: (i) limit the molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limit the coupling reaction time or temperature, (iii) use part of cysteine thiol modification or limiting reduction conditions, (iv) engineer the amino acid sequence of the antibody by recombinant techniques such that the number and position of cysteine residues are modified to control the number and/or position of linker-drug linkages (e.g., thioMab or thioFab prepared as disclosed herein and in WO2006/034488 (incorporated herein by reference in its entirety).

It will be appreciated that where more than one nucleophilic group is reacted with a drug-linker intermediate or linker reagent, followed by reaction with a drug unit reagent, then the resulting product is a distributed mixture of the ADC compound and one or more drug units linked to an antibody unit. The average drug number/antibody can be calculated from the mixture by a dual ELISA antibody assay specific for the antibody and specific for the drug. Individual ADC molecules can be identified in the mixture by mass spectrometry and isolated by HPLC (e.g., hydrophobic interaction chromatography) (see, e.g., hamburg, k.j. Et al, "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 anti-drug conjugate", abstract No. 624,American Association for Cancer Research,2004 annual meeting, 2004, 27-31 days, AACR meeting paper, 45, 2004, 3 months, alley, s.c. et al, "Controlling the location of drug attachment in antibody-drug conjugates", abstract No. 627,American Association for Cancer Research,2004 annual meeting, 2004, 3 months, 27-31 days, AACR meeting paper, 45, 2004, 3 months). In certain embodiments, a homogeneous ADC having a single loading value may be separated from the conjugate mixture by electrophoresis or chromatography.

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

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

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

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

In some embodiments, the ADC used in the methods provided herein is enrolment Shan Kangwei statin. Enrolment Shan Kangwei statins are fully human immunoglobulins G1 kappa (IgG 1) comprising a microtubule disrupting agent (MMAE) coupled via a protease-cleavable linker _K ) Antibody ADC (Challita-Eid PM et al, cancer Res.2016;76 (10):3003-13). Enrolment Shan Kangwei spines induce antitumor activity by binding to 191P4D12 protein on the cell surface, causing internalization of the ADC-191P4D12 complex, followed by transport to the lysosomal compartment and release of MMAE in the compartment via proteolytic cleavage linker. Intracellular release of MMAE subsequently interferes with tubulin polymerization, leading to G2/M phase cell cycle arrest and apoptotic cell death (Francisco JA et al, blood.2003, 8, 15; 102 (4): 1458-65).

AGS-22M6E is an ADC derived from a murine hybridoma cell line, as described above and in U.S. Pat. No. 8,637,642. Enrolment Shan Kangwei statins are equivalent of AGS-226 m6e ADC derived from Chinese Hamster Ovary (CHO) cell line and are exemplary products for human therapy. Enrolment Shan Kangwei statin has the same amino acid sequence, linker and cytotoxic drug as AGS-22M 6E. The comparability between enrolment Shan Kangwei statin and AGS-22M6E is determined via in-depth analysis and biological characterization studies, such as binding affinity to 191P4D12, cytotoxicity in vitro and antitumor activity in vivo.

In one embodiment, the ADC provided herein is enrolment Shan Kangwei statin, also known as EV, PADCEV, AGS-22M6E, AGS-22C3E, ASG-22C3E. Enrolment Shan Kangwei spines include anti-191P 4D12 antibodies, wherein the antibodies or antigen binding fragments thereof comprise a heavy chain comprising amino acid residues 20 to 466 of SEQ ID No. 7 and a light chain comprising amino acid residues 23 to 236 of SEQ ID No. 8.

Enrolment Shan Kangwei statin is an antibody-drug conjugate (ADC) to connexin-4 consisting of a fully human anti-connexin-4 IgG1 kappa monoclonal antibody (AGS-22C 3) conjugated to a small molecule microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease cleavable maleimidocaproyl valine-citrulline (vc) linker (SGD-1006). Coupling occurs at cysteine residues comprising interchain disulfide bonds of the antibody, yielding a drug to antibody ratio of about 3.8:1 product. The molecular weight is about 152kDa.

The enrolment Shan Kangwei statin has the following structural formula:

approximately 4 molecules of MMAE were attached to each antibody molecule. Enrolment Shan Kangwei statins are produced by chemically coupling antibodies and small molecule components. Antibodies are produced by mammalian (chinese hamster ovary) cells and small molecule components are produced by chemical synthesis.

The enrolment Shan Kangwei statin injections are provided in single dose vials as sterile, preservative-free, white to off-white lyophilized powders for intravenous use. Enrolment Shan Kangwei is supplied in 20 mg/vial and 30 mg/vial form and is required to be reconstituted with sterile water for injection (2.3 mL and 3.3mL respectively) USP to produce a clear to slightly milky, colorless to slightly yellow solution with a final concentration of 10 mg/mL. After reconstitution, each vial was allowed to draw 2mL (20 mg) and 3mL (30 mg). Each mL of reconstituted solution contained 10mg of enrolment Shan Kangwei statin, histidine (1.4 mg), histidine monohydrochloride (2.31 mg), polysorbate 20 (0.2 mg) and trehalose dihydrate (55 mg), pH 6.0.

5.4 pharmaceutical compositions

In certain embodiments of the methods provided herein, the ADC used in the methods is provided in the form of a "pharmaceutical composition". Such pharmaceutical compositions include an antibody drug conjugate provided herein, and one or more pharmaceutically or physiologically acceptable excipients. In certain embodiments, the antibody drug conjugate is provided in combination with or separate from one or more additional agents. Also provided is a composition comprising such one or more additional agents and one or more pharmaceutically or physiologically acceptable excipients. In certain embodiments, the antibody drug conjugate and the additional agent are present in therapeutically acceptable amounts. The pharmaceutical compositions may be used according to the methods and uses provided herein. Thus, for example, the pharmaceutical compositions can be administered to a subject ex vivo or in vivo in order to practice the methods of treatment and uses provided herein. The pharmaceutical compositions provided herein may be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are set forth herein.

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

In certain embodiments of the methods provided herein, the pharmaceutical compositions comprising ADCs may also comprise other therapeutically active agents or compounds disclosed herein or known to those of skill in the art that may be useful in the treatment or prevention of various diseases and disorders (e.g., cancer) as set forth herein. As set forth above, additional therapeutically active agents or compounds may be present in a separate pharmaceutical composition.

The pharmaceutical composition generally comprises a therapeutically effective amount of at least one of the antibody drug conjugates provided herein and one or more pharmaceutically acceptable formulations. In certain embodiments, the pharmaceutical composition further comprises one or more additional agents described herein.

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

In some embodiments, the antibody drug conjugate in the pharmaceutical compositions provided herein is selected from the antibody drug conjugates described in section 5.3 above.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In one embodiment, provided herein is an article comprising

(a) An antibody drug conjugate comprising the structure:

wherein L-represents an antibody or antigen-binding fragment thereof (e.g., an anti-connexin-4 antibody or antigen-binding fragment thereof) and p is 1 to 10; and

(b) A pharmaceutically acceptable excipient comprising about 20mM L-histidine, about 0.02% (w/v) TWEEN-20, about 5.5% (w/v) trehalose dihydrate, and HCl, wherein the concentration of the antibody drug conjugate is about 10mg/mL, and wherein the pH is about 6.0 at 25 ℃.

In another specific embodiment, the pharmaceutical compositions provided herein comprise:

(a) An antibody drug conjugate comprising the structure:

(b) Pharmaceutically acceptable excipients comprising about 20mM L-histidine, about 0.02% (w/v) TWEEN-20, about 5.5% (w/v) trehalose dihydrate and succinic acid,

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

(a) An antibody drug conjugate comprising the structure:

(b) Pharmaceutically acceptable excipients comprising about 20mM L-histidine, about 0.02% (w/v) TWEEN-20, about 5.0% (w/v) sucrose and HCl,

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

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

The pharmaceutical compositions provided herein may also contain other pharmaceutically acceptable formulations for modifying or maintaining the release rate of the antibody drug conjugate and/or additional agent as described herein. Such formulations include those known to the skilled artisan for preparing sustained release formulations. For other references regarding pharmaceutically and physiologically acceptable formulations, see, e.g., remington's Pharmaceutical Sciences, 18 th edition (1990,Mack Publishing Co., easton, pa.18042), pages 1435-1712, the Merck Index, 12 th edition (1996,Merck Publishing Group,Whitehouse,NJ); and Pharmaceutical Principles of Solid Dosage Forms (1993,Technonic Publishing Co., inc., lancaster, pa.). Additional pharmaceutical compositions suitable for administration are known in the art and may be employed in the methods and compositions provided herein.

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

The pharmaceutical compositions may be formulated in accordance with their intended route of administration. Thus, the pharmaceutical composition comprises excipients suitable for administration by a route comprising: parenteral (e.g., subcutaneous (s.c.), intravenous, intramuscular, or intraperitoneal), intradermal, oral (e.g., ingestion), inhalation, intracavity, intracranial, and transdermal (topical). Other exemplary routes of administration are set forth herein.

The pharmaceutical compositions may be in the form of sterile injectable aqueous or oleaginous suspensions. Such suspensions may be formulated using suitable dispersing or wetting agents and suspending agents as disclosed herein or as known to those skilled in the art. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable diluents, solvents and dispersion media which may be employed include water, ringer's solution, isotonic sodium chloride solution, cremophor EL ^TM (BASF, parippany, NJ) or Phosphate Buffered Saline (PBS), ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Prolonged absorption of a particular injectable formulation can be brought about by including an agent which delays absorption (for example, aluminum monostearate or gelatin).

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

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

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

In one embodiment, suitable aqueous vehicles include, but are not limited to, water, saline, physiological saline or Phosphate Buffered Saline (PBS), sodium chloride injection, ringer's injection, isotonic dextrose injection, sterile water injection, dextrose, and lactated ringer's injection. Non-aqueous vehicles include, but are not limited to, non-volatile oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, medium chain triglycerides of hydrogenated soybean oil and coconut oil, and palm seed oil. Water-miscible vehicles include, but are not limited to, ethanol, 1, 3-butanediol, liquid polyethylene glycols (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerol, N-methyl-2-pyrrolidone, N-dimethylacetamide, and dimethylsulfoxide.

In one embodiment, suitable antimicrobial agents or preservatives include, but are not limited to, phenol, cresol, mercuric agents, benzyl alcohol, chlorobutanol, methyl and propyl parahydroxybenzoates, thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl and propyl parahydroxybenzoates, and sorbic acid. Suitable isotonic agents include, but are not limited to, sodium chloride, glycerol, and dextrose. Suitable buffers include, but are not limited to, phosphates and citrates. Suitable antioxidants are those as described herein, including bisulfites and sodium metabisulfites. Suitable local anesthetics include, but are not limited to procaine hydrochloride (procaine hydrochloride). Suitable suspending and dispersing agents are those as described herein, including sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Suitable emulsifiers include the emulsifiers described herein, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate. Suitable chelating or chelating agents include, but are not limited to, EDTA. Suitable pH adjusting agents include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Suitable complexing agents include, but are not limited to, cyclodextrins including alpha-cyclodextrin, beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, sulfobutyl ether-beta-cyclodextrin and sulfobutyl ether 7-beta-cyclodextrin CyDex,Lenexa,KS)。

In one embodiment, the pharmaceutical compositions provided herein may be formulated for single or multiple dose administration. The single dose formulation is packaged in an ampoule, vial or syringe. Multiple doses of parenteral formulations may contain antimicrobial agents at antibacterial or antifungal concentrations. As known and practiced in the art, all parenteral formulations must be sterile.

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

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

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

The pharmaceutical compositions may also include excipients to protect the composition from rapid degradation or expulsion from the body, such as controlled release formulations, including implants, liposomes, hydrogels, prodrugs, and microencapsulated delivery systems. For example, time delay materials such as glyceryl monostearate or glyceryl stearate, alone or in combination with a wax, may be employed. Prolonged absorption of the injectable pharmaceutical composition can be brought about by including an agent which delays absorption, for example, aluminum monostearate or gelatin. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

The pharmaceutical compositions provided herein may be stored at-80 ℃, 4 ℃, 25 ℃, or 37 ℃.

The lyophilized compositions can be prepared by freeze-drying the liquid pharmaceutical compositions provided herein. In a specific embodiment, the pharmaceutical composition provided herein is a lyophilized pharmaceutical composition. In some embodiments, the pharmaceutical formulation is a lyophilized powder that can be reconstituted for administration in the form of solutions, emulsions, and other mixtures. They may also be reconstituted and formulated as solids or gels.

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

The lyophilizer can be used to prepare lyophilized formulations. For example, a VirTis Genesis Model EL test cell may be used. The unit incorporates a chamber with three working shelves (to a total usable shelf area of about 0.4 square meters), an external condenser and a mechanical vacuum pump system. The cascading mechanical refrigeration allows the shelves to cool to-70 ℃ or less and the external condenser to cool to-90 ℃ or less. The shelf temperature and chamber pressure were automatically controlled to +/-0.5 ℃ and +/-2 microns (millitorr), respectively. The cell was equipped with a vacuum capacitance manometer, pirani vacuum gauge, pressure transducer (measuring range: 0 to 1 atm) and relative humidity sensor.

Lyophilized powders may be prepared by dissolving the antibody drug conjugates provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solution is then sterile filtered and then lyophilized under standard conditions known to those skilled in the art to yield the desired formulation. In one embodiment, the resulting solution will be dispensed into vials for lyophilization. Each vial will contain a single dose or multiple doses of antibody drug conjugate. The lyophilized powder may be stored under suitable conditions, for example, at about 4 ℃ to room temperature.

The lyophilized powder is reconstituted with water for injection to give a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable excipients. This amount may be determined empirically and adjusted according to particular needs.

An exemplary reconstruction procedure is illustrated as follows: (1) A 5mL or 3mL syringe was fitted with an 18 or 20 gauge needle and the syringe was filled with water for injection (WFI) grade water; (2) Proper amount of WFI is measured using syringe scales, ensuring that the syringe is free of bubbles; (3) inserting the needle through the rubber stopper; (4) Dispensing the entire contents of the syringe down the wall of the bottle into the container, removing the syringe and needle and placing in a pointed container; (4) The vortex vial is continued to carefully dissolve the entire vial contents until complete reconstitution (e.g., about 20-40 seconds) and to minimize agitation of the excess protein solution that can cause foaming.

In some embodiments, the pharmaceutical compositions provided herein are supplied in a dry sterile lyophilized powder or anhydrous concentrate form in a hermetically sealed container, and can be reconstituted with, for example, water or saline to an appropriate concentration for administration to a subject. In certain embodiments, the antibody drug conjugate is supplied in dry sterile lyophilized powder form in hermetically sealed containers in unit doses of: at least 0.1mg, at least 0.5mg, at least 1mg, at least 2mg, at least 3mg, at least 5mg, at least 10mg, at least 15mg, at least 25mg, at least 30mg, at least 35mg, at least 45mg, at least 50mg, at least 60mg, at least 75mg, at least 80mg, at least 85mg, at least 90mg, at least 95mg, or at least 100mg. The lyophilized antibody drug conjugate may be stored in its original container at between 2 and 8 ℃, and the antibody drug conjugate may be administered within 12 hours, e.g., within 6 hours, within 5 hours, within 3 hours, or within 1 hour after reconstitution. In an alternative embodiment, a pharmaceutical composition comprising an antibody drug conjugate provided herein is supplied in liquid form in a hermetically sealed container that indicates the quantity and concentration of the antibody drug conjugate. In certain embodiments, the antibody drug conjugate in liquid form is supplied in a hermetically sealed container at the following doses: at least 0.1mg/ml, at least 0.5mg/ml, at least 1mg/ml, at least 5mg/ml, at least 10mg/ml, at least 15mg/ml, at least 25mg/ml, at least 30mg/ml, at least 40mg/ml, at least 50mg/ml, at least 60mg/ml, at least 70mg/ml, at least 80mg/ml, at least 90mg/ml, or at least 100mg/ml.

Additional embodiments of pharmaceutical compositions have been described in U.S. patent No. 8,637,642 and international application No. PCT/US2019/056214 (publication No. WO 2020/117373), both of which are incorporated herein by reference in their entirety.

5.5 combination therapy methods

A method for inhibiting tumor cell growth using a pharmaceutical composition provided herein and chemotherapy or radiation or a combination of both, comprising administering a pharmaceutical composition of the invention before, during, or after initiation of chemotherapy or radiation therapy, and any combination thereof (i.e., before and during initiation of chemotherapy and/or radiation therapy, before and after, during and after, or before, during and after). Depending on the treatment regimen and the particular patient needs, the method is performed in a manner that will provide the most effective treatment and ultimately extend the life of the patient. Additional embodiments of such combination therapies have been described in U.S. patent No. 8,637,642 and international application No. PCT/US2019/056214 (publication No. WO 2020/117373), both of which are incorporated herein by reference in their entirety.

5.6 doses of immune checkpoint inhibitor

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

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

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

5.7 dosage of ADC for use in methods

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

In some embodiments, the ADC that describes the method of the various doses in this section (section 5.7) is enrolment Shan Kangwei statin (EV).

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

The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

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

In some embodiments, the antibody drug conjugate formulated in the pharmaceutical compositions provided herein is administered based on the actual body weight of the patient at baseline, and the dose will not change unless the patient's body weight changes by ≡10% or meets the dose-adjustment criteria compared to the baseline of the previous cycle. In some embodiments, the actual weight will be used unless the patient weighs more than 100kg, in which case the dose will be calculated based on a weight of 100 kg. In some embodiments, the maximum dose is 100mg for a patient receiving a dose level of 1.00 mg/kg; and for patients receiving a dose level of 1.25mg/kg, the maximum dose is 125mg.

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

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

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

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

In another embodiment, a single dose of antibody drug conjugate 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 formulated in the pharmaceutical compositions provided herein is administered to a patient at about once monthly (e.g., about 30 day) intervals for a period of time (e.g., one year) to prevent and/or treat cancer, wherein the dose is selected from the group consisting of: about 0.1mg/kg, about 0.5mg/kg, about 0.75mg/kg, about 1mg/kg, about 1.25mg/kg, about 1.5mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 55mg/kg, about 60mg/kg, about 65mg/kg, about 70mg/kg, about 75mg/kg, about 80mg/kg, about 85mg/kg, about 90mg/kg, about 95mg/kg, about 100mg/kg, or combinations thereof (i.e., each monthly dose may be the same or different).

In another embodiment, a single dose of antibody drug conjugate 1, 2, 3, 4, 5 or 6 formulated in the pharmaceutical compositions provided herein is administered to a patient to prevent and/or treat cancer at about two month (e.g., about 60 days) intervals for a period of time (e.g., one year), wherein the dose is selected from the group consisting of: about 0.1mg/kg, about 0.5mg/kg, about 0.75mg/kg, about 1mg/kg, about 1.25mg/kg, about 1.5mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 55mg/kg, about 60mg/kg, about 65mg/kg, about 70mg/kg, about 75mg/kg, about 80mg/kg, about 85mg/kg, about 90mg/kg, about 95mg/kg, about 100mg/kg, or combinations thereof (i.e., each monthly dose may be the same or different).

In another embodiment, a single dose of antibody drug conjugate 1, 2, 3 or 4 formulated in the pharmaceutical compositions provided herein is administered to a patient for a period of time (e.g., one year) at a time interval of about three months (e.g., about 120 days) to prevent and/or treat cancer, wherein the dose is selected from the group consisting of: about 0.1mg/kg, about 0.5mg/kg, about 0.75mg/kg, about 1mg/kg, about 1.25mg/kg, about 1.5mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 35mg/kg, about 40mg/kg, about 45mg/kg, about 50mg/kg, about 55mg/kg, about 60mg/kg, about 65mg/kg, about 70mg/kg, about 75mg/kg, about 80mg/kg, about 85mg/kg, about 90mg/kg, about 95mg/kg, about 100mg/kg, or combinations thereof (i.e., each monthly dose may be the same or different).

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

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

In some more specific embodiments, the antibody drug conjugate formulated in the pharmaceutical compositions provided herein is administered by Intravenous (IV) injection or infusion at a dose of about 0.5mg/kg, about 0.75mg/kg, about 1mg/kg, about 1.25mg/kg, or about 1.5mg/kg of subject body weight, twice every three week period for about 30 minutes. In some embodiments, the antibody drug conjugate formulated in the pharmaceutical composition is administered by Intravenous (IV) injection or infusion for about 30 minutes every three week cycle, day 1 and day 8. In some embodiments, the method further comprises administering the immune checkpoint inhibitor by Intravenous (IV) injection or infusion one or more times over a three week period. In some embodiments, the method further comprises administering the immune checkpoint inhibitor by Intravenous (IV) injection or infusion on day 1 of a three week cycle. In some embodiments, the immune checkpoint inhibitor is palbociclib and wherein the palbociclib is administered in an amount of about 200mg over about 30 minutes. In other embodiments, the immune checkpoint inhibitor is alemtuzumab, and wherein the alemtuzumab is administered in an amount of about 1200mg for about 60 minutes or 30 minutes. In some embodiments, the antibody drug conjugate is administered to a patient with urothelial cancer or bladder cancer who has shown disease progression or recurrence during or after treatment with another cancer treatment. In some embodiments, the antibody drug conjugate is administered to a patient with metastatic urothelial cancer or bladder cancer who has shown disease progression or recurrence during or after treatment with another cancer treatment. In some embodiments, the antibody drug conjugate is administered to a patient with locally advanced urothelial cancer or bladder cancer who has shown disease progression or recurrence during or after treatment with another cancer treatment. In some embodiments, the ADC of the methods described in this paragraph for the various doses is enrolment Shan Kangwei statin (EV).

In other more specific embodiments, the antibody drug conjugate formulated in the pharmaceutical compositions provided herein is administered by Intravenous (IV) injection or infusion at a dose of about 0.5mg/kg, about 0.75mg/kg, 1mg/kg, about 1.25mg/kg, or about 1.5mg/kg of subject body weight, three times every four week period for about 30 minutes. In some embodiments, the antibody drug conjugate formulated in the pharmaceutical composition is administered on days 1, 8, and 15 of every 28-day (four week) cycle. In some embodiments, the antibody drug conjugate formulated in the pharmaceutical composition is administered by Intravenous (IV) injection or infusion for about 30 minutes on days 1, 8, and 15 of every 28-day (four week) cycle. In some embodiments, the method further comprises administering the immune checkpoint inhibitor by Intravenous (IV) injection or infusion one or more times in each four week period. In some embodiments, the immune checkpoint inhibitor is pamphlet Li Zhushan antibody. In other embodiments, the immune checkpoint inhibitor is alemtuzumab. In some embodiments, the antibody drug conjugate is administered to a patient with urothelial cancer or bladder cancer who has shown disease progression or recurrence during or after treatment with another cancer therapy. In some embodiments, the antibody drug conjugate is administered to a patient with metastatic urothelial cancer or bladder cancer who has shown disease progression or recurrence during or after treatment with another cancer therapy. In some embodiments, the antibody drug conjugate is administered to a patient with locally advanced urothelial cancer or bladder cancer who has shown disease progression or recurrence during or after treatment with another cancer therapy. In some embodiments, the ADC of the methods described in this paragraph for the various doses is enrolment Shan Kangwei statin (EV).

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

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

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

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

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

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

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

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

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

In some embodiments of the methods provided herein, the ADC is administered by Intravenous (IV) injection or infusion. In one embodiment, the first ADC dose is administered by IV injection. In another embodiment, the first ADC dose is administered by IV infusion. In another embodiment, the second ADC dose is administered by IV injection. In another embodiment, the second ADC dose is administered by IV infusion. In one embodiment, the first ADC dose is administered by IV injection and the second ADC dose is administered by IV injection. In another embodiment, the first ADC dose is administered by IV infusion and the second ADC dose is administered by IV injection. In another embodiment, the first ADC dose is administered by IV injection and the second ADC dose is administered by IV infusion. In another embodiment, the first ADC dose is administered by IV infusion and the second ADC dose is administered by IV infusion. In some embodiments, the ADC of the methods described in this paragraph for the various doses is enrolment Shan Kangwei statin (EV).

In certain embodiments of the methods provided herein, the ADC is administered by IV injection or infusion three times every four week period. In some embodiments of the methods provided herein, the first ADC dose is administered by IV injection or infusion three times every four week period. In some embodiments of the methods provided herein, the second ADC dose is administered by IV injection or infusion three times every four week period. In some embodiments of the methods provided herein, the first ADC dose is administered by IV injection or infusion three times every four week period and the second ADC dose is administered by IV injection or infusion three times every four week period. In some embodiments, the ADC of the methods described in this paragraph for the various doses is enrolment Shan Kangwei statin (EV).

In some embodiments of the methods provided herein, the ADC is administered by IV injection or infusion on days 1, 8, and 15 of each four week cycle. In some embodiments, the first ADC dose is administered by IV injection or infusion on days 1, 8, and 15 of each four week cycle. In some embodiments, the second ADC dose is administered by IV injection or infusion on days 1, 8, and 15 of each four week cycle. In some embodiments, the first ADC dose is administered by IV injection or infusion on days 1, 8, and 15 of each four week cycle, and the second ADC dose is administered by IV injection or infusion on days 1, 8, and 15 of each four week cycle. In some embodiments, the ADC of the methods described in this paragraph for the various doses is enrolment Shan Kangwei statin (EV).

In certain embodiments of the methods provided herein, the ADC is administered by IV injection or infusion for about 30 minutes three times every four week period. In some embodiments, the first ADC dose is administered by IV injection or infusion for about 30 minutes three times every four week period. In some embodiments, the second ADC dose is administered by IV injection or infusion for about 30 minutes three times every four week period. In some embodiments, the first ADC dose is administered by IV injection or infusion of about 30 minutes three times every four week period and the second ADC dose is administered by IV injection or infusion of about 30 minutes three times every four week period. In some embodiments, the ADC of the methods described in this paragraph for the various doses is enrolment Shan Kangwei statin (EV).

In some embodiments of the methods provided herein, the ADC is administered by IV injection or infusion for about 30 minutes on days 1, 8, and 15 of each four week cycle. In some embodiments of the methods provided herein, the first ADC dose is administered by IV injection or infusion for about 30 minutes on days 1, 8, and 15 of each four week cycle. In some embodiments of the methods provided herein, the second ADC dose is administered by IV injection or infusion for about 30 minutes on days 1, 8, and 15 of each four week cycle. In some embodiments of the methods provided herein, the first ADC dose is administered by IV injection or infusion for about 30 minutes on days 1, 8 and 15 of each four week cycle, and the second ADC dose is administered by IV injection or infusion for about 30 minutes on days 1, 8 and 15 of each four week cycle. In some embodiments, the ADC of the methods described in this paragraph for the various doses is enrolment Shan Kangwei statin (EV).

In other more specific embodiments, the antibody drug conjugate formulated in the pharmaceutical compositions provided herein is administered at a dose of about 1mg/kg, 1.25mg/kg, or about 1.5mg/kg of subject body weight by Intravenous (IV) injection or infusion for about 30 minutes three times every 28 day cycle. In some embodiments, the antibody drug conjugate formulated in the pharmaceutical composition is administered by Intravenous (IV) injection or infusion for about 30 minutes every day 1, day 8, and day 15 of the 28 day cycle. In some embodiments, the method further comprises administering the immune checkpoint inhibitor by Intravenous (IV) injection or infusion one or more times in each four week period. In some embodiments of the methods provided herein, the ADC is administered three times over a 28 day period. In some embodiments of the methods provided herein, the ADC is administered on days 1, 8, and 15 of a 28 day cycle. In some embodiments, the ADC of the methods described in this paragraph for the various doses is enrolment Shan Kangwei statin (EV).

5.8 methods for determining biomarkers

The present disclosure provides that expression of any one of the markers provided herein can be determined by various methods known in the art. In some embodiments, expression of the marker may be determined based on the amount or relative amount of mRNA transcribed from the marker gene. In one embodiment, the expression of the marker gene may be determined based on the amount or relative amount of the protein product encoded by the marker gene. In another embodiment, the expression of the marker gene may be determined based on the level of biological or chemical response induced by the protein product encoded by the marker gene. In addition, in certain embodiments, the expression of a marker gene may be determined based on the expression of one or more genes associated with the expression of the marker gene.

As described above, the level or amount of a gene transcript (e.g., mRNA) of a marker gene may be used as an indication of the level of expression of the marker gene. A variety of different PCR or qPCR protocols are known in the art, including those exemplified herein. In some embodiments, multiple PCR or qPCR methods are applied or adapted to determine mRNA levels of various marker genes. Quantitative PCR (qPCR), also known as real-time PCR, is applied to and adapted for use in some embodiments because it not only provides quantitative measurements, but also reduces time and contamination. As used herein, "quantitative PCR (or" qPCR ")" refers to direct monitoring of the progress of PCR amplification as it occurs without the need to oversample the reaction products. In quantitative PCR, the reaction product can be monitored as it is generated via a signaling mechanism (e.g., fluorescence) and tracked as the signal rises above background levels, but before the reaction reaches a plateau. The number of cycles required to reach a detectable or "threshold" fluorescence level varies directly with the concentration of the amplifiable target at the beginning of the PCR process, allowing the signal intensity to be measured in order to measure the target nucleic acid content in the sample in real time. When qPCR is used to determine mRNA expression levels, an additional step of reverse transcription of mRNA into DNA is performed prior to qPCR analysis. Examples of PCR Methods can be found in the literature (Wong et al, bioTechniques 39:75-85 (2005); D' haene et al, methods 50:262-270 (2010)), which is incorporated herein by reference in its entirety. Examples of PCR assays can also be found in U.S. patent No. 6,927,024, which is incorporated herein by reference in its entirety. An example of an RT-PCR method can be found in U.S. Pat. No. 7,122,799, which is incorporated herein by reference in its entirety. The fluorescent in situ PCR method is described in U.S. patent No. 7,186,507, which is incorporated herein by reference in its entirety.

In one embodiment, qPCR may be performed as follows to determine or measure mRNA levels of marker genes. Briefly, marker bases are assayedThe average Ct (cycle threshold) value (or interchangeably referred to herein as Cq (quantitative cycle)) of the replication qPCR reaction with one or more housekeeping genes. The average Ct value of the marker gene can then be normalized with the Ct value of the housekeeping gene using the following example formula: marker gene Δct= (average Ct of marker genes-average Ct of housekeeping gene a). The relative marker gene delta Ct can then be used to determine the relative level of marker gene mRNA, for example by using the mRNA expression equation = 2 ^-ΔCt . For a summary of Ct and Cq values, see the MIQE guide (Bustin et al The MIQE Guidelines: minimum Information for Publication of Quantitative Real-Time PCR Experiments, clinical Chemistry 55:55:4 (2009)).

For quantification of RNA transcripts of marker genes in a sample as an indication of expression of marker genes, other common methods known in the art may also be used, including northern blotting (northern blotting) and in situ hybridization (Parker and Barnes, methods in Molecular Biology 106:247-283 (1999)); RNase protection assay (Hod, biotechnology 13:852-854 (1992)); microarray methods (Hoheisel et al Nature Reviews Genetics 7:200-210 (2006); jaluria et al Microbial Cell Factories 6:6:4 (2007)); and Polymerase Chain Reaction (PCR) (Weis et al Trends in Genetics 8:263-264 (1992)). RNA In Situ Hybridization (ISH) is a molecular biological technique that is widely used to measure and localize specific RNA sequences, such as messenger RNAs (mrnas), non-coding long RNAs (lncrnas), and micrornas (mirnas), within cells, such as Circulating Tumor Cells (CTCs), or tissue sections, while preserving cell and tissue environment. ISH is a type of hybridization that uses directly or indirectly labeled complementary DNA or RNA strands (e.g., probes) to bind and localize specific nucleic acids, such as DNA or RNA, in a sample, particularly a portion or section of tissue or cells (in situ). The probe type may be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded complementary RNA (sscRNA), messenger RNA (mRNA), microrna (miRNA), ribosomal RNA, mitochondrial RNA, and/or synthetic oligonucleotides. The term "fluorescent in situ hybridization" or "FISH" refers to ISH types using fluorescent labels. The term "chromogenic in situ hybridization" or "CISH" refers to ISH types using chromogenic labels. ISH, FISH and CISH methods are well known to those skilled In the art (see, e.g., stoler, clinics In Laboratory Medicine 10 (1): 215-236 (1990); in situ hybridization. A practical approach, wilkinson, IRL Press, oxford (1992); schwarzacher and hesloop-Harrison, practical In situ hybridization, BIOS Scientific Publishers Ltd, oxford (2000)). RNA ISH thus provides a spatio-temporal visualization and quantification of gene expression within cells and tissues. It has a wide range of applications in research and diagnostics (Hu et al, biomark.Res.2 (1): 1-13, doi:10.1186/2050-7771-2-3 (2014), ratan et al, cureus 9 (6): e1325.doi:10.7759/cureus.1325 (2017), weier et al, expert Rev.mol.Diagn.2 (2): 109-119 (2002)). Fluorescent RNAISH uses fluorescent dyes and fluorescent microscopy for RNA labeling and detection, respectively. Fluorescent RNA ISH can provide for multiplexing of four to five target sequences.

Alternatively, RNA transcripts of the marker gene in the sample may be used as indicators of marker gene expression, as determined by sequencing techniques. Representative methods for sequencing-based gene expression analysis include gene expression series analysis (Serial Analysis of Gene Expression; SAGE) and gene expression analysis by large-scale parallel signature sequencing (MPSS).

In some embodiments, expression of a marker gene can be determined based on the relative abundance of RNA transcripts (including, for example, mRNA) of the marker gene in a total transcribed RNA pool. Such relative abundance of RNA transcripts of marker genes can be determined by next generation sequencing called RNA-seq. In one example of an RNA-seq procedure, RNA from different sources (blood, tissue, cells) is purified, optionally enriched (e.g., with oligonucleotide (dT) primers), converted to cDNA and fragmented. Millions or even billions of short sequence reads were generated using random fragmented cDNA libraries. See Zhao et al BMC genemics 16:97 (2015); zhao et al, scientific Reports 8:4781 (2018); shannng Zhao et al, RNA, published earlier on 13/4/2020, doi:10.1261/rna.074922.120, all incorporated herein by reference in its entirety. The expression level of each mRNA transcript of the marker gene is determined according to the total number of fragments localized after normalization, said total number being proportional to its abundance level. Several normalization schemes are known and used to facilitate the use of RNA transcript abundance as a parameter for determining gene expression, including the number of reads per million per kilobase (RPKM), the number of fragments per million per kilobase (FPKM), and/or the number of transcripts per million per kilobase (TPM). Briefly, RPKM may be calculated as follows: total reads in the sample were counted and the number divided by 1,000,000 (which is a "per million" scale factor); dividing the read count by a "per million" scale factor, which is normalized to the sequencing depth, resulting in the number of Reads Per Million (RPM); and dividing the RPM value by the gene length (in kilobases) to yield RPKM. In addition to replacing reads with fragments, FPKM is closely related to RPKM. RPKM was generated from single-ended RNA-seq, with each read corresponding to a single fragment sequenced. FPKM is generated based on paired-end RNA-seq, where two reads may correspond to a single fragment, or one read may correspond to a single fragment if one read in a pair is not located. The TPM is very similar to the RPKM and FPKM and is calculated as follows: dividing the read count by the length of each gene (in kilobases) to give the number of Reads Per Kilobase (RPK); all RPK values of the samples were counted and this number divided by 1,000,000 to give a "per million" scale factor; dividing the RPK value by the "per million" scale factor gives the TPM. See Zhao et al BMC genemics 16:97 (2015); zhao et al, scientific Reports 8:4781 (2018); shannng Zhao et al, RNA, published earlier on 13/4/2020, doi:10.1261/rna.074922.120, all incorporated herein by reference in its entirety.

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

As described earlier, expression of a marker gene in a sample from a subject can be determined. In some embodiments, the sample is a blood sample, a serum sample, a plasma sample, a bodily fluid (e.g., interstitial fluid, including cancerous tissue fluid), or a tissue (e.g., cancerous tissue or tissue surrounding a cancer). In some embodiments, the sample is a tissue sample. In some embodiments, the tissue sample is a tissue portion isolated or extracted from a mammal, particularly a human. In some embodiments, the tissue sample is a population of cells isolated or extracted from a mammal, particularly a human. In some embodiments, the tissue sample is a sample obtained from a biopsy. In certain embodiments, the sample may be obtained from a variety of organs of a subject (including human subjects). In some embodiments, the sample is obtained from an organ of a subject having cancer. In some embodiments, the sample is obtained from an organ with cancer in a subject with cancer. In other embodiments, the sample (e.g., a reference sample) is obtained from a normal organ of the patient or the second human subject.

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

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

In various IHC assays, the expression of various marker genes can be detected by antibodies directed against the protein product encoded by the marker gene. IHC staining of tissue sections has been shown to be a reliable method of assessing or detecting the presence of proteins in a sample. IHC techniques utilize antibodies to detect and visualize in situ cellular antigens, typically by chromogenic or fluorescent methods. Primary antibodies or antisera, such as polyclonal and monoclonal antibodies that specifically target the protein product encoded by the marker gene, can be used in IHC assays to detect expression of the marker gene. In some embodiments, the tissue sample is contacted with the primary antibody directed against the particular target for a time sufficient for antibody-target binding to occur. As discussed in greater detail earlier, the antibodies can be detected by directly labeling the antibodies themselves with, for example, a radiolabel, a fluorescent label, a hapten label (e.g., biotin), or an enzyme (e.g., horseradish peroxidase or alkaline phosphatase). Alternatively, unlabeled primary antibodies are used in combination with labeled secondary antibodies (including antisera, polyclonal antisera, or monoclonal antibodies) specific for the primary antibodies. IHC protocols and kits are well known in the art and are commercially available. Automated systems for slide preparation and IHC processing are commercially available. A Leica BOND automatic staining instrument and Leica Bond Refine detection system are examples of such automated systems.

In some embodiments, the IHC assay is performed using unlabeled primary antibodies in combination with labeled secondary antibodies in an indirect assay. Indirect assays use two antibodies to detect the protein product encoded by a marker gene in a tissue sample. First, unconjugated primary antibodies are applied to the tissue (first layer) and react with the antigen of interest in the tissue sample. Next, an enzyme-labeled secondary antibody is applied, which specifically recognizes the antibody isotype of the primary antibody (second layer). The secondary antibody reacts with the primary antibody, followed by the application of a substrate-chromogen. The second layer of antibody may be labeled with an enzyme (e.g., peroxidase) that reacts with the chromogen 3,3' -Diaminobenzidine (DAB) to produce a brown precipitate at the reaction site. This approach is sensitive and versatile due to the potential signal being amplified via the signal amplification system.

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

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

Signal amplification systems based on enzyme labeling systems exploit the catalytic activity of enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase to generate high density labels of target proteins or nucleic acid sequences in situ. In one embodiment, the HRP signal may be enhanced using tyramide. In this system, HRP enzymatically converts the labeled tyramide derivative into a highly reactive, short-lived tyramide group. The labeled active tyramide groups were then covalently coupled to residues near the HRP antibody-target interaction site (predominantly the phenol moiety of the protein tyrosine residue), resulting in an amplification of the number of labels at the site with minimal loss of signal localization associated with diffusion. Thus, the signal may be amplified 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 50, 75, or 100 times. As known to those of skill in the art, the label on the tyramide may be any label described herein, including fluorophores, enzymes, haptens, radioisotopes, and/or luminophores. Other enzyme-based reactions may also be utilized to produce signal amplification. For example, enzyme-labeled fluorescence (ELF) signal amplification may be used for alkaline phosphatase, where alkaline phosphatase enzymatically cleaves a weak blue fluorescent substrate (ELF 97 phosphate) and converts it to a bright yellow-green fluorescent precipitate that exhibits exceptionally large Stokes shift (Stokes shift) and excellent photostability. Tyramide-based signal amplification systems and ELF signal amplification are available, for example, from ThermoFisher Scientific (Waltham, mass. USA 02451).

Thus, in some embodiments of the methods provided herein, IHC using a signal amplification system is used to detect the expression level of a marker gene. In some embodiments, the sample is then counterstained to identify cellular and subcellular elements.

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

In other embodiments, the expression level of the protein product encoded by the marker gene may also be detected using antibodies described herein in flow cytometry assays, including Fluorescence Activated Cell Sorting (FACS) assays. Similar to IHC or immunoblot assays, protein expression can be detected in FACS assays using primary antibodies or antisera (e.g., polyclonal and monoclonal antibodies that specifically target the protein product encoded by the marker gene). In some embodiments, the cells are stained with a primary antibody directed against a particular protein of interest for a time sufficient for antibody-antigen binding to occur and the bound antibody is detectable by direct labeling (e.g., fluorescent labeling or hapten labeling, e.g., biotin on the primary antibody) on the primary antibody. In other embodiments, in an indirect assay as described above, unlabeled primary antibodies are used in combination with fluorescently labeled secondary antibodies specific for the primary antibodies. FACS provides a method (one cell at a time) to sort or analyze a mixture of fluorescently labeled biological cells based on specific light scattering and fluorescence characteristics of each cell. Thus, the flow cytometer detects and reports the intensity of the fluorochrome-labeled antibodies, which are indicative of the expression level of the protein of interest. Thus, the expression level of a protein product encoded by a marker gene can be detected using antibodies directed against such protein product. Non-fluorescent cytoplasmic proteins can also be visualized by staining permeabilized cells. Methods for performing FACS staining and analysis are well known to those skilled in the art and are described in Teresa s.hawley and Robert g.hawley in Flow Cytometry Protocols, humana Press,2011 (ISBN 1617379506,9781617379505).

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

In some embodiments of the EIA or ELISA assay, the enzyme is conjugated to a secondary antibody. In other embodiments, a fluorescently labeled secondary antibody may be used in place of an enzymatically labeled secondary antibody in an ELISA assay format to generate a detectable signal. When activated by irradiation with light of a specific wavelength, the fluorochrome-labeled antibody absorbs light energy, induces an excitability state of the molecule, and then emits light of a characteristic color that can be visually detected with an optical microscope. Like EIA and ELISA, the fluorescently labeled antibody is allowed to bind to the primary antibody-target protein complex. After washing away unbound reagent, the remaining ternary complex is then exposed to light of the appropriate wavelength, and the observed fluorescence indicates the presence of the target protein of interest. Immunofluorescence and EIA techniques are well established in the art and are disclosed herein.

For the immunoassays described herein, any of a variety of enzymatic or non-enzymatic labels can be used, provided that enzymatic activity or non-enzymatic labels can be detected, respectively. The enzyme thus produces a detectable signal that can be used to detect the protein of interest. Particularly suitable detectable signals are chromogenic or fluorescent signals. Thus, particularly suitable enzymes for use as labels include enzymes that can utilize chromogenic or fluorogenic substrates. Such chromogenic or fluorogenic substrates can be converted by enzymatic reactions into readily detectable chromogenic or fluorogenic products which can be readily detected and/or quantified using microscopy or spectroscopy. Such enzymes are well known to those skilled in the art and include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, and the like (see Herman, bioconjugate Techniques, academic Press, san Diego (1996)). Other enzymes that utilize well known chromogenic or fluorogenic substrates include various peptidases, wherein chromogenic or fluorogenic peptide substrates can be used to detect proteolytic cleavage reactions. The use of chromogenic and fluorogenic substrates in bacterial diagnostics is also well known, including but not limited to the use of alpha-galactosidase and beta-galactosidase, beta-glucuronidase, 6-phospho-beta-D-galactosidase 6-phosphogalactohydrolase, beta-glucosidase, alpha-glucosidase, amylase, neuraminidase, esterase, lipase, etc. (Manafi et al, microbiol. Rev.55:335-348 (1991)), and such enzymes for which chromogenic or fluorogenic substrates are known can be readily adapted for use in the methods of the present disclosure.

Various chromogenic or fluorogenic substrates that produce a detectable signal are well known to those skilled in the art and are commercially available. Exemplary substrates that can be used to generate a detectable signal include, but are not limited to, 3 '-Diaminobenzidine (DAB) for horseradish peroxidase, 3',5 '-Tetramethylbenzidine (TMB), chloronaphthol (Chloronaphthol) (4-CN) (4-chloro-1-naphthol), 2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS), o-phenylenediamine dihydrochloride (OPD), and 3-amino-9-ethylcarbazole (AEC); 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP), nitroblue tetrazolium (NBT), fast red (fast red TR/AS-MX) and p-nitrophenyl phosphate (PNPP) for alkaline phosphatase; 1-methyl-3-indolyl-beta-D-galactopyranoside and 2-methoxy-4- (2-nitrovinyl) phenyl beta-D-galactopyranoside for beta-galactosidase; beta-glucose2-methoxy-4- (2-nitrovinyl) phenyl beta-D-glucopyranoside for glycosidase; etc. Exemplary fluorogenic substrates include, but are not limited to, 4- (trifluoromethyl) umbelliferyl phosphate for alkaline phosphatase; 4-methylumbelliferyl phosphate bis (2-amino-2-methyl-1, 3-propanediol), 4-methylumbelliferyl phosphate bis (cyclohexylammonium) and 4-methylumbelliferyl phosphate for phosphatases; quantaBlu for horseradish peroxidase ^TM And QuantaRed ^TM The method comprises the steps of carrying out a first treatment on the surface of the 4-methylumbelliferyl β -D-galactopyranoside, fluorescein bis (β -D-galactopyranoside) and naphthofluorescein bis- (β -D-galactopyranoside) for β -galactosidase; 3-acetyl umbelliferyl beta-D-glucopyranoside and 4-methylumbelliferyl beta-D-glucopyranoside for beta-glucosidase; and 4-methylumbelliferyl-alpha-D-galactopyranoside for alpha-galactosidase. Exemplary enzymes and substrates for producing a detectable signal are also described, for example, in U.S. publication 2012/0100540. Various detectable enzyme substrates, including chromogenic or fluorogenic substrates, are well known and commercially available (Pierce, rockford IL; santa Cruz Biotechnology, dallas TX; invitrogen, carlsbad Calif., 42Life Science;Biocare). In general, the substrate is converted to a product, forming a precipitate, which is deposited at the target nucleic acid site. Other exemplary substrates include, but are not limited to, HRP-Green (42 Life Science), betazoid DAB, cardassian DAB, romulin AEC, bajoran Vina Green, deep Space Black ^TM 、Warp Red ^TM Vulcan fast red and ferrogi blue of Biocare (Concord CA; biocare. Net/products/detection/chromagens).

In some embodiments of the immunoassay, the detectable label may be directly coupled to a primary antibody that may be present or a secondary antibody that detects unlabeled primary antibody. Exemplary detectable labels are well known to those skilled in the art and include, but are not limited to, chromogenic or fluorescent labels (see Hermanson, bioconjugate Techniques, academic Press, san Diego (1996)). Exemplary fluorophores suitable for use as labels include, but are not limited to, rhodamine (rhodomine) derivatives, such as tetramethylrhodamine, rhodamine B, rhodamine 6G, sulfonylrhodamine B, texas Red (Texas Red) (sulfonylrhodamine 101), rhodamine 110, and derivatives thereof Things such as tetramethyl rhodamine-5- (or 6), lissamine rhodamine B (lissamine rhodamine B), and the like; 7-nitrobenzo-2-oxa-1, 3-diazole (NBD); fluorescein and its derivatives; naphthalene, such as dansyl (5-dimethylaminonaphthalene-1-sulfonyl); coumarin derivatives, e.g. 7-amino-4-methylcoumarin-3-acetic acid (AMCA), 7-diethylamino-3- [ (4' - (iodoacetyl) amino) phenyl]-4-methylcoumarin (DCIA), alexa fluor dyes (Molecular Probes), etc.; 4, 4-difluoro-4-boron-3 a,4 a-diaza-s-dicyclopentadiene acene (BODIPY) ^TM ) And derivatives thereof (Molecular Probes; eugene oreg.); pyrene and sulfonated pyrene, e.g. Cascade Blue ^TM And derivatives thereof, including 8-methoxypyrene-1, 3, 6-trisulfonic acid, etc.; pyridyl oxazole derivatives and daphnikyl (dapxyl) derivatives (Molecular Probes); fluorescein (3, 6-disulfonate-4-amino-naphthalimide) and derivatives thereof; cyDye ^TM Fluorescent dyes (Amersham/GE Healthcare Life Sciences; piscataway N.J.), and the like. Exemplary chromophores include, but are not limited to, phenolphthalein, malachite green, nitroaromatic hydrocarbons (e.g., nitrophenyl), diazo dyes, dabsyl (4-dimethylaminoazobenzene-4' -sulfonyl), and the like.

The chromogenic or fluorogenic detectable signal bound to the bound primary or secondary antibody may be visualized using methods well known to those skilled in the art (e.g., microscopy or spectroscopy).

The methods provided in this section (section 5.8) can be used in conjunction with various cancer models known in the art. In one embodiment, a mouse xenograft cancer model is used. Briefly, T-24 and UM-UC-3 cells were purchased from ATCC and cultured using recommended medium conditions. T-24hNectin-4 (human connexin-4) and UM-UC-3 connexin-4 cells were generated by transducing the parental cells with a lentivirus containing human connexin-4 using pRCDCMEP-CMV-hNectin-4EF1-Puro construct and selected using puromycin. T-24 connexin-4 (clone 1A 9) cells were implanted into nude mice and passaged through a trocar, allowing approximately 200mm to be reached ³ Tumor volume, and subsequent treatment with a single Intraperitoneal (IP) dose of enrolment Shan Kangwei statin (3 mg/kg) or unbound ADC (3 mg/kg), 7 animals per treatment groupAnd (3) an object. Tracking ICD studies using this model involved collecting tumors on day 5 post-treatment for downstream analysis by RNA-seq, flow, immunohistochemistry (IHC) and Luminex. Tumors were fixed in formalin and prepared into FFPE tissue blocks. The 4 μm blocks were cut and immunohistochemistry was performed using F4/80, CD11 c. Immunohistochemical stained slide sections were scanned with a Leica AT2 digital whole slide scanner and images were analyzed with visioparm software using algorithms tailored for connexin 4, CD11c and F4/80 staining. The algorithm is optimized based on the staining intensity and background staining. Percent positive staining of connexin 4 was calculated and per mm F480 and CD11c were calculated ² Positive cell number.

Tumor sections were lysed in lysis buffer 2 (R&DDirectory number 895347). Cytokines and chemokines in tumor samples were measured using the millepplex MAP mouse cytokine/chemokine bead set (Millipore) and read on the luminerx magix system.

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

The disclosure is generally provided using a positive language to describe the many embodiments. The present disclosure also includes, inter alia, embodiments in which particular subject matter, such as materials or substances, method steps and conditions, protocols, procedures, assays or analyses, is wholly or partially excluded. Thus, although the disclosure is not generally presented herein to the extent that it is not included, aspects of the disclosure are disclosed herein that are not expressly included in the disclosure.

Specific embodiments of the disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of those disclosed embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description, and it is contemplated that such variations may be employed by those skilled in the art as appropriate. Accordingly, it is intended that the present disclosure be practiced otherwise than as specifically described herein, and that the present disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

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

Various embodiments of the present disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure.

6. Examples

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

6.1 example 1-open label randomized phase 3 study (EV-301) for evaluation of enrolment Shan Kangwei statin with chemotherapy in subjects with previously treated locally advanced or metastatic urothelial cancer.

6.1.1Medicament for clinical research

In one embodiment, the ADC provided herein is enrolment Shan Kangwei statin, also known as PADCEV. In one particular embodiment tested in this example (6.1), enrolment Shan Kangwei statin-ejfv comprises an anti-191P 4D12 antibody, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising amino acid residues 20 through 466 of SEQ ID NO. 7 and a light chain comprising amino acid residues 23 through 236 of SEQ ID NO. 8.

Enrolment Shan Kangwei statin-ejfv is an antibody-drug conjugate (ADC) to connexin-4 comprising a fully human anti-connexin-4 IgG1 kappa monoclonal antibody (AGS-22C 3) conjugated to a small molecule microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease cleavable maleimidocaproyl valine-citrulline (vc) linker (SGD-1006). Coupling occurs at cysteine residues comprising interchain disulfide bonds of the antibody, yielding a drug to antibody ratio of about 3.8:1 product. The molecular weight is about 152kDa.

Enrolment Shan Kangwei statin-ejfv has the following structural formula:

approximately 4 molecules of MMAE were attached to each antibody molecule. Enrolment Shan Kangwei statin-ejfv is produced by chemically coupling the antibody and small molecule components. Antibodies are produced by mammalian (chinese hamster ovary) cells and small molecule components are produced by chemical synthesis.

PADCEV (enrolment Shan Kangwei statin-ejfv) for injection is provided in single dose vials for intravenous use as a sterile, preservative-free, white to off-white lyophilized powder. PADCEV in 20 mg/vial and 30 mg/vial form was supplied and required to be reconstituted with sterile water for USP injection (2.3 mL and 3.3mL respectively) to give a clear to slightly milky, colorless to slightly yellow solution with a final concentration of 10mg/mL (see dose and administration (6.1.6.1 (i))). After reconstitution, each vial was allowed to draw 2mL (20 mg) and 3mL (30 mg). Each mL of reconstitution solution contained 10mg of enrolment Shan Kangwei statin-ejfv, histidine (1.4 mg), histidine monohydrated hydrochloride (2.31 mg), polysorbate 20 (0.2 mg) and trehalose dihydrate (55 mg), pH 6.0.

6.1.2Summary of the study

6.1.2.1 outline

(i) Study of the names of drugs

Enrobed Shan Kangwei Duotong (ASG-22 CE)

(ii) Development stage

Stage 3

(iii) Study title

Open label randomized phase 3 study (EV-301) for evaluation of enrolment Shan Kangwei statins with chemotherapy in subjects with previously treated locally advanced or metastatic urothelial cancer

(iv) Planned study period

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

(v) Research objective

(a) Main objective

To compare OS of subjects with locally advanced or metastatic urothelial cancer treated with EV with OS of patients treated with chemotherapy.

(b) Secondary target

To compare the progression free survival (PFS 1) of study therapy for subjects treated with EV versus patients treated with chemotherapy according to the solid tumor response assessment criteria (RECIST) V1.1

To compare the Overall Response Rate (ORR) of EV and chemotherapy according to RECIST V1.1

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

In order to compare EV to chemotherapy Disease Control Rate (DCR) according to RECIST V1.1

To assess safety and tolerability of EV

For assessing quality of life (QOL) and Patient Reporting Outcome (PRO) parameters

(c) Exploration of targets

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

To assess pharmacokinetics of EV (Total antibody (Tab), antibody-drug conjugate (ADC) and monomethyl auristatin E (MMAE))

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

To evaluate PFS (PFS 2) of EV compared to next-line therapy of chemotherapy

Medical resource utilization (HRU)

(vi) Planned total number and location of research centers

There are about 185 research centers in north america, europe, asia-pacific and latin america

(vii) Study population

Subjects with locally advanced or metastatic urothelial cancer previously treated with platinum-based chemotherapy and immune checkpoint inhibitors (CPI)

(viii) Number of subjects to enroll/randomized

About 600 subjects

(ix) Overview of study design

This is a phase 3 study of a randomized group of global open markers in adult subjects with locally advanced or metastatic urothelial cancer who have received platinum-containing chemotherapy and have experienced disease progression or recurrence during or after treatment with immune checkpoint inhibitors. Approximately 600 subjects were randomized to EV (group a) or chemotherapy (group B) at a 1:1 ratio. The subjects will be stratified according to the following: eastern tumor collaboration group behavioral status (Eastern Cooperative Oncology Group Performance Status; ECOG PS), world area, and liver metastasis.

OS is the primary endpoint (primary endpoint). The OS is defined as the time from the start of a random packet to the day of death. Secondary endpoints (Secondary endpoint) include PFS1, ORR, DOR, DCR, security and QOL/PRO.

Subjects in group a will receive EVs on days 1, 8 and 15 of each 28 day cycle. Subjects in group B will receive docetaxel, paclitaxel, or vinflunine on day 1 of each 21-day cycle (as determined by the investigator prior to randomization: vinflunine is the choice of comparator only in countries where it is approved for urothelial cancer). Within the control group, the upper limit of the overall proportion of subjects receiving vinflunine will be about 35%. The subject will continue to receive study treatment until the radiological disease progression or other disruption criteria as determined by the investigator's assessment is met, or the first occurrence is taken at the termination of the study or at the completion of the study. No crossover in the study will be allowed. This study will consist of 3 phases: screening, treatment and follow-up.

Screening will be performed up to 28 days prior to random grouping. The subject will begin at cycle 1 and continue to follow up to 21 or 28 day cycles until one of the disruption criteria is met. The treatment cycle was defined as 28 days for group a and 21 days for group B. Subjects randomized to group a (EV) will receive treatment and evaluation on days 1, 8 and 15 of all treatment cycles. Subjects randomized to group B (docetaxel, paclitaxel, or vinflunine) will receive treatment and evaluation on day 1 of all treatment cycles.

The response of the subjects will be assessed according to RECIST V1.1. Imaging of both groups will be performed every 56 days (+ -7 days) from the first dose of study treatment during the baseline and the entire study period until PFS1 is recorded by radiological disease progression or subjects lose track, die, withdraw study consent, or begin subsequent anti-cancer therapies.

Baseline imaging performed as standard care prior to informed consent can be used as long as it is performed within 28 days prior to the randomized group. All subjects will undergo a bone scan (scintigraphy) at screening/baseline. Subjects who performed positive bone scans at baseline will perform bone scans every 56 days (+ -7 days) or more frequently (if clinically indicated) throughout the study. If the clinical indication is not related to the baseline condition, the subject should conduct a follow-up bone scan. Brain scans (computer tomography with contrast/Magnetic Resonance Imaging (MRI)) will only be performed at screening/baseline as indicated clinically, and repeated as indicated clinically or according to standard care throughout the study period.

QOL assessments and PRO will be collected from all randomized, grouped subjects at the protocol-prescribed time points. The following validation tools will be used: european cancer research and treatment organization (EORTC) quality of life questionnaire (QLQ-C30) and EuroQOL 5 dimension (EQ-5D-5L). Medical resource utilization (HRU) information will be collected at a protocol-specified point in time, with particular focus on the number of subjects not planning to use medical resources related to clinical or AE from subjects assigned to treatment groups a and B.

Pharmacokinetic and ATA blood samples were collected from subjects randomized into group a throughout the study. The concentrations of EV ADC and MMAE in serum or plasma will be measured using a validation assay and ATA assessed. Pharmacokinetic samples will not be collected from subjects randomized to group B. Samples for the exploration of biomarkers will be collected at the time points prescribed by the protocol. Biomarker assessment will not be used for subject selection.

After discontinuing the study medication, subjects will follow up for 30 days (+7 days) following their last dose of medication for safety assessment. If the subject discontinued the study drug prior to progression of the radiological disease (i.e., PFS 1), the subject should enter a post-treatment follow-up period and continue to undergo imaging assessment every 56 days (+ -7 days) until either PFS1 is recorded or the subject begins another anti-cancer treatment, whichever occurs earlier.

Following PFS1, the subject will enter a long-term follow-up period and follow-up according to institutional guidelines, but track the survival and progression status of subsequent therapies (i.e., PFS 2) no less than every 3 months from the day of follow-up visit.

The subject will be followed until PFS2 is recorded, or the subject begins another anti-cancer treatment, subject to earlier occurrence. All subsequent anti-cancer therapies, including date and progression sites of PFS2, will be recorded according to a case report format.

Following PFS2, subjects will enter a survival tracking period and follow the survival status every 3 months until death, loss of tracking, withdrawal of study consent, or termination by the sponsor study. Once the final survival analysis was completed, this study was expected to end.

The license independent data monitoring committee (Independent Data Monitoring Committee; IDMC) was supervising safety and mid-plan efficacy analysis, which will occur after at least 285 OS events (about 65% of total planned events) were observed. The preliminary analysis will be performed at 439 OS events. The IDMC may suggest to the sponsor whether the trial should be terminated, modified, or continued unchanged based on the continued review of the safety data and the interim efficacy analysis. Additional details will be summarized in the IDMC rights.

(x) Inclusion/exclusion criteria

The method comprises the following steps: the subject met the study conditions if all of the following were applicable:

1. prior to any study-related procedures, including withdrawal of forbidden drugs, if applicable, written informed consent and privacy language approved by the Institutional Review Board (IRB)/Independent Ethics Committee (IEC) in accordance with national regulations, such as the health insurance privacy and liability act (HIPAA) authority of the united states study site, must be obtained from the subject.

2. The subject was a legal adult according to local regulations when signing the informed consent.

3. The subject has a histologically or cytologically confirmed urothelial cancer (i.e., bladder cancer, renal pelvis cancer, urinary tract cancer, or urinary tract cancer). Subjects with urothelial cancer (transitional cells) with squamous differentiated or mixed cell types are eligible.

4. The subject must undergo radiological progression or recurrence of locally advanced or metastatic disease during or after CPI (anti-programmed cell death-1 (PD-1) or anti-programmed cell death-ligand 1 (PD-L1)). Since the subjects with toxicity interrupting CPI treatment were eligible, provided they had evidence of disease progression after interruption. CPI need not be the most recent therapy. A subject is eligible if the most recent therapy has progressed/relapsed during or after its most recent therapy in a subject based on a non-CPI-based regimen. Locally advanced disease must not be resected as intended by the treating physician using the cure.

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

6. The subject had a radiological record of metastatic or locally advanced disease at baseline.

7. Archiving tumor tissue samples should be available for presentation to a central laboratory prior to study treatment. If an archived tumor tissue sample is not available, a fresh tissue sample should be provided. If fresh tissue samples cannot be provided due to safety issues, participation in the study must be discussed with the medical monitor.

8. ECOG PS of the subject is 0 or 1

9. The subjects had the following baseline laboratory data:

● Absolute Neutrophil Count (ANC) at 1500/mm or more ³

● Platelet count is greater than or equal to 100X 10 ⁹ personal/L

● Hemoglobin is more than or equal to 9g/dL

● Serum total bilirubin is 1.5×upper normal limit (ULN) or 3×uln for subjects with gilbert disease

● Creatinine clearance (CrCl) > 30mL/min as estimated from institutional standards or as measured by 24 hour urine collection (glomerular filtration rate (GFR) may also be used instead of CrCl)

● Alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) are less than or equal to 2.5 XULN, or

For subjects with liver metastasis +.3XULN

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

10. The female subject must meet any of the following:

● Fertility potential free:

● Postmenopausal prior to screening (defined as at least 1 year without any menses, without other apparent pathological or physiological etiology), or

● Sterility was recorded surgically (e.g., hysterectomy, double sided tubectomy, double sided ovariectomy).

Note that: those patients postmenopausal due to alternative medical reasons were not considered and had to follow the criteria of a fertility potential subject.

● Or, if having fertility potential:

● It was agreed that pregnancy was not attempted during the study and for at least 6 months after the last study medication administration,

● And a negative urine or serum pregnancy test within 7 days prior to day 1 (validated females with false positive results and recorded negative pregnancy status are eligible for participation),

● And if there is a specificity, consent is given to the use of condoms plus 1 form of efficient fertility control, continuously initiated at screening and during the whole study period and at least 6 months after the last study drug administration, according to locally approved criteria.

11. Female subjects must agree that no lactation or donation of ova is performed at the start of screening and during the whole study period and at least 6 months after the last study drug administration.

12. Sexually active male subjects with female partners having fertility potential were eligible for:

● The male condom was agreed to begin at screening and continued throughout the study treatment period and for at least 6 months after the last study drug administration. If a male subject has not undergone vasectomy or is not sterile as defined below, then its female partner begins at the time of screening, utilizing 1 form of efficient fertility control according to locally approved criteria, and continues to do so throughout the study treatment and at least 6 months after the male subject receives its final study drug administration.

* Efficient forms of fertility control include:

■ Always and correctly using established hormonal contraceptives that inhibit ovulation,

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

■ Bilateral tubal occlusion

■ Vasectomy (vasectomy is a high-efficiency contraceptive method, provided that no sperm is confirmed, otherwise an additional high-efficiency contraceptive method should be used)

■ Male infertility due to bilateral orchiectomy or eradication of cystprostatectomy/removal of seminal vesicles

■ Forbidden is considered an efficient method only when it is defined as avoiding non-intercourse throughout the risk period associated with study treatment. There is a need to assess the reliability of abstinence with respect to the duration of the study and the preferred and common lifestyle of the participants.

Note that: in swiss there is insufficient libido to be used as a contraceptive method.

13. Male subjects were not allowed to donate sperm at the beginning of the screening and throughout the study period, and at least 6 months after administration of the final study medication.

14. Male subjects with pregnant or lactating partners must agree to either have a contraception or use the condom during pregnancy or during the time of pregnancy for the partner to lactate during the entire study period and at least 6 months after administration of the final study medication.

15. Subjects agreed not to participate in another intervention study when undergoing treatment in this study.

No exemption from inclusion criteria is allowed.

Exclusion: the subject will exclude participation if any of the following is applicable:

1. the subject suffers from pre-existing sensory or motor neuropathy of grade No. 2.

2. The subject suffers from active Central Nervous System (CNS) metastasis. Subjects with treated CNS metastases were allowed to study if all of the following were true:

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

● If steroid treatment is desired for CNS metastasis, the subject is treated with a stabilizing dose of 20 mg/day or less of prednisone (prednisone) or an equivalent for at least 2 weeks

● Baseline scans showed no signs of new brain metastases or enlarged brain metastases

● The subject did not suffer from leptomeningeal disease

3. Subjects are experiencing clinically significant toxicity (grade 2 or higher, except hair loss) associated with previous treatments, including systemic therapy, radiation therapy or surgery. Subjects with +.2 immunotherapy-related hypothyroidism or hypopituitarism are eligible for selection when they are adequately maintained/controlled at a stable dose of hormone replacement therapy (if indicated). Patients experiencing grade 3 or more immunotherapy-related hypothyroidism or hypopituitarism are excluded. Subjects experiencing immunotherapy-related colitis, uveitis, myocarditis, or pneumonia or subjects with other immunotherapy-related AEs requiring high doses of steroid (> 20 mg/day of prednisone or equivalent) are excluded.

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

5. In the control group, the subjects have received prior chemotherapy for urothelial cancer as well as all available study therapies (i.e., both prior paclitaxel and docetaxel in the area where vinflunine is not approved for therapy; or prior paclitaxel, docetaxel and vinflunine in the area where vinflunine is approved for therapy).

Note that: after reaching the upper limit of vinflunine, subjects who have received both docetaxel and paclitaxel will be excluded.

6. The subject has received more than 1 prior chemotherapy regimen for locally advanced or metastatic urothelial cancer, including chemotherapy for adjuvant or neoadjuvant disease (if recurrence occurs within 12 months of completion of therapy). The substitution of cisplatin with carboplatin does not constitute a new regimen, provided that no new chemotherapeutic agent is added to the regimen.

7. The subject had a history of another malignant disease within 3 years prior to the first dose of study drug, or had any signs of residual disease of the malignant disease previously diagnosed. Subjects with non-melanoma skin cancers, localized prostate cancers that undergo curative treatment and have no signs of progression, low or very low risk (according to standard guidelines) localized prostate cancers that are inadvertently treated with active supervision/observation waiting, or any type of carcinoma in situ (if complete excision is performed) are permissible.

8. At the time of the first dose of EV, the subject is currently receiving systemic antimicrobial therapy against viral, bacterial or fungal infection. Conventional antimicrobial control is tolerated.

9. The subject suffers from known active hepatitis b (e.g., HBsAg reactivity) or active hepatitis c (e.g., HCV RNA detected (qualitative)).

10. Subjects are known to have a history of Human Immunodeficiency Virus (HIV) infection (HIV 1 or 2).

11. The subjects had a history of recorded cerebrovascular events (stroke or transient ischemic attacks), unstable angina, myocardial infarction, or heart symptoms (including congestive heart failure) consistent with the new york heart association (New York Heart Association) group III-IV within 6 months prior to the first dose of study medication.

12. The subjects underwent radiation therapy or major surgery within 4 weeks prior to the first dose of study drug.

13. The subject received chemotherapy, biological agents, study agents, and/or anti-tumor treatment with immunotherapy and was not completed 2 weeks before the first dose of study drug.

14. The subject is known to be allergic to EV or to any excipient contained in EV pharmaceutical formulations (including histidine, trehalose dihydrate and polysorbate 20); or the subject is known to be allergic to a biopharmaceutical prepared in Chinese Hamster Ovary (CHO) cells.

15. The subject is known to be allergic to:

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

● Paclitaxel or any other excipient listed in the product label, including polyethylene glycol glycerol ricinoleate 35 (ph.eur.); and

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

16. Subjects are known to have active keratitis or corneal ulcers. Subjects with superficial punctate keratitis are allowed if their condition is adequately treated from the point of view of the researcher.

17. The subject suffers from other potential medical conditions that, from the point of view of the researcher, would impair the subject's ability to receive or tolerate the planned treatment and follow-up.

18. A history of uncontrolled diabetes within 3 months of the first dose of study medication. Uncontrolled diabetes is defined as hemoglobin A1c (HbA 1 c) > 8% or HbA1c between 7% and <8% with diabetes symptoms (polyuria or polydipsia) not otherwise explained.

No exemption from the exclusion criteria is allowed.

(xi) Medicine: enrolment Shan Kangwei statin: dosage, mode of administration and dosage adjustment

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

EV will be administered on day 1 of each cycle based on the actual body weight of the subject, except that the subject is greater than 100kg in body weight; in such a situation, the dose will be calculated based on a maximum body weight of 100 kg. Group a does not require recalculation of dose based on actual body weight on day 8 and day 15 of each cycle unless mandated by institutional standards.

Depending on the type and severity of the toxicity, the allowable dose was reduced to 1mg/kg (dose level-1) and 0.75mg/kg (dose level-2). Subjects in need of a reduced dose can be further increased by 1 dose level (i.e., subjects reduced to 0.75mg/kg can only be further increased to 1 mg/kg), provided that toxicity does not require discontinuation of study drug and has returned to baseline or grade 1. If toxicity is recurring, no further increment will be allowed. Subjects presenting with grade 2 AE were not allowed to escalate the dose. EV should not be administered to subjects with CrCl <30 mL/min. Dose-modulating recommendations for EV-related toxicity are presented in tables 6 and 7.

Researchers at the study site may tolerate discontinuation of administration due to other EV-related toxicities as appropriate. The discontinuation of administration may last up to 8 weeks (2 cycles). If toxicity in the subject does not otherwise require permanent discontinuation of the administration, discontinuation of the administration from the subject for clinical benefit from treatment may extend beyond 8 weeks. If there is a disruption in dosing, the time course of response assessment will not be adjusted.

Table 6 recommended dose modulation for enrolment Shan Kangwei statin related hematological toxicity

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

Table 7 dose modulation for enrolment Shan Kangwei statin related non-hematological toxicity recommendations

AE: adverse events; EV: enrolment Shan Kangwei statin

* The 3/4 grade electrolyte imbalance/laboratory abnormality, which is not accompanied by clinical sequelae and/or corrected by supplementation/appropriate treatment within 72 hours of its onset, does not require discontinuation of treatment (e.g., grade 4 hyperuricemia). Grade 3 rash, which does not limit self-care activities of daily living or concomitant infections and requires systemic antibiotics, does not require discontinuation of treatment provided the symptoms are not severe and can be treated by supportive treatment.

(xii) Comparative drug

Generally, for drug-related grade 4 hematologic and non-hematologic toxicities ∈3, treatment with the chemotherapy comparator (docetaxel, paclitaxel, or vinflunine) should be discontinued and subsequent doses adjusted as per table 15. The guidelines for the recommended dose adjustment, specific to subjects receiving docetaxel, paclitaxel, or vinflunine, are detailed below. Dose adjustments should also be considered based on local product labeling or product characteristics (SmPC) and an overview of institutional guidelines. For grade 3 or greater hematologic toxicity associated with docetaxel, paclitaxel, or vinflunine, as indicated by institutional guidelines, transfusion or growth factors may be used.

(a) Docetaxel: dosage, mode of administration and dosage adjustment

Docetaxel will be administered intravenously on day 1 of every 21-day cycle. Will be administered over a period of 60 minutes or 75mg/m as required locally ² Starting dose of docetaxel. Other guidelines for docetaxel administration refer to local product labels or institutional guidelines for SmPC and docetaxel.

Docetaxel should not be given total bilirubin>Subjects with ULN, or administration of AST and/or ALT>1.5 XULN and accompanying alkaline phosphatase>2.5 XULN subjects. Subjects with elevated bilirubin or simultaneous abnormalities in transaminase and alkaline phosphatase have an increased risk of developing grade 4 neutropenia, febrile neutropenia, infection, severe thrombocytopenia, severe stomatitis, severe skin toxicity and toxic death. Docetaxel should not yet be administered with neutrophil counts<1500 cells/mm ³ Is a subject of (a). Serious fluid retention has been reported following docetaxel therapy.

The subject should be pre-dosed with a corticosteroid according to institutional guidelines prior to administration of each docetaxel. The effusion of a subject with pre-existing effusion may be worsened by the close monitoring of the subject starting from the first dose. Subjects developing peripheral oedema may be treated with standard measures, such as salt limitation, oral diuretics. The discontinuation of administration may last up to 6 weeks (2 cycles). If toxicity in the subject does not otherwise require permanent discontinuation of the administration, discontinuation of the administration from the subject for clinical benefit from treatment may extend beyond 6 weeks.

Dose adjustments (e.g., severe or cumulative skin reactions) not specified in table 8 should also be considered according to local product labels or SmPC and institutional guidelines.

Table 8 recommended dose adjustment for docetaxel-receiving subjects

ANC: absolute neutrophil count; N/A: inapplicable; t: temperature (temperature)

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

Vinflunine will be administered intravenously on day 1 of every 21 day cycle. Unless otherwise specified below, 320mg/m will be administered over a 20 minute period (or as locally required) ² An initial dose of vinflunine. In the case where WHO/ECOG PS.gtoreq.1 or ECOG PS 0 and previous pelvic irradiation, 280mg/m should be used ² Starts the vinflunine treatment. In the absence of any hematological toxicity causing delay in treatment or dose reduction during the first period, the dose in the subsequent period may be increased to 320mg/m every 21 days ² 。

In subjects with moderate kidney damage (40 mL/min. Ltoreq. CrCl. Ltoreq.60 mL/min), a recommended dose of 280mg/m administered every 21 day period ² Once. When suffering from kidney injury (30 mL/min is less than or equal to CrCl)<40 mL/min) of 250mg/m every 21-day cycle ² Once. In subjects with mild liver injury (Child-Pugh A grade), the recommended dose of vinflunine is 250mg/m administered every 21 day cycle ² Once.

The recommended doses in subjects aged 75 years are as follows:

● In subjects at least 75 years of age but less than 80 years of age, the dose of vinflunine to be administered is 280mg/m every 21-day cycle ² 。

● In subjects aged 80 and older, the dose of vinflunine to be administered is 250mg/m every 21-day cycle ² 。

For other guidelines for administration of vinflunine, reference is made to local product labels or institutional guidelines for SmPC and vinflunine.

The discontinuation of administration may last up to 6 weeks (2 cycles). If toxicity in the subject does not otherwise require permanent discontinuation of the administration, discontinuation of the administration from the subject for clinical benefit from treatment may extend beyond 6 weeks. For specific dose adjustments for subjects receiving vinflunine, reference is made to the acceptable product label.

(c) Paclitaxel: dosage, mode of administration and dosage adjustment

After all procedures/assessments have been completed, paclitaxel should be administered intravenously on day 1 of the 21-day cycle for study treatment. Will take 3 hours to administer as intravenous infusion or 175mg/m as local demand ² An initial dose of paclitaxel. See guidelines for initial dose adjustment. Other guidelines for paclitaxel administration refer to local product labels or institutional guidelines for SmPC and paclitaxel.

All subjects should be given a prior administration of paclitaxel according to institutional guidelines to prevent severe allergic reactions. Such precursor administration may consist of: about 12 and 6 hours prior to paclitaxel, 20mg of dexamethasone was orally administered; 30 to 60 minutes prior to paclitaxel, IV administration of diphenhydramine (or equivalent thereof) 50mg; and IV administration of cimetidine (300 mg) or ranitidine (50 mg) 30 to 60 minutes prior to paclitaxel. Appropriate precursor dosing regimens may be determined by the researcher.

The baseline neutrophil count should not be less than 1500 cells/mm ³ Paclitaxel is administered to the subject. Following the cycle, the subject should not be retreated with paclitaxel until neutrophils return to levels>1500 cells/mm ³ And the platelets return to the level>100000/mm ³ . Has been in the course of paclitaxel therapy<Severe conduction abnormalities were noted in 1% of the subjects and in some cases, the atrial pulse generator was placed. If the subject develops significant conduction abnormalities during paclitaxel infusion, appropriate therapy should be administered and continuous cardiac monitoring should be performed during subsequent paclitaxel therapy.

In the case of mild liver injury (total bilirubin not less than 1.25 ULN), paclitaxel should be added at 135mg/m ² Is started.

The guidelines for the specific recommended dose adjustment for subjects receiving paclitaxel are detailed in table 9 below. Dose adjustments should also be considered according to local product labels or SmPC and institutional guidelines.

The discontinuation of administration may last up to 6 weeks (2 cycles). If toxicity in the subject does not otherwise require permanent discontinuation of the administration, discontinuation of the administration from the subject for clinical benefit from treatment may extend beyond 6 weeks.

Table 9 recommended dose adjustment for subjects receiving paclitaxel

N/A: inapplicable; t: temperature (temperature)

(xiii) Interrupt criteria

Discontinuation of treatment was applicable to subjects enrolled in the study and subjects permanently discontinued in the study treatment for any reason.

The subject may freely withdraw from study treatment and/or study for any reason and at any time, without giving the reason to do so and without penalty or bias. The researcher may also freely discontinue the subject from study treatment at any time or terminate the subject from participating in the study if the subject's clinical condition permits. If subjects discontinue the study with persistent Adverse Events (AEs) or unresolved laboratory results significantly outside of the reference range, the researcher will attempt to track until the condition is stable or no longer clinically significant.

The following are treatment disruption criteria for individual subjects:

● The subject developed a radiological disease progression.

● The subject is required to receive another systemic anti-cancer treatment against the underlying or new cancer.

● The subject developed unacceptable toxicity.

● The female subject is pregnant.

● The investigator decided to discontinue treatment in accordance with the best benefit of the subject.

● The subject refuses further treatment.

● Based on the assessment of the researcher or medical monitor, the subject does not follow the regimen.

● Despite reasonable efforts by researchers to locate subjects, subjects were not visited.

● Death.

Subjects who discontinued treatment prior to radiological disease progression will enter a post-treatment follow-up period and continue imaging assessment every 56 days (+ -7 days) until PFS1 is recorded by radiological disease progression or the subject begins another anti-cancer treatment, whichever occurs earlier.

Following PFS1, the subject will enter a long-term follow-up period and follow-up according to institutional guidelines, but track the survival and progression of the next line of therapy no less than every 3 months from the date of follow-up visit until PFS2 is recorded or the subject begins another anti-cancer therapy, whichever occurs earlier.

The subject will then enter a survival tracking period. The survival status of the subjects will be tracked every 3 months until any disruption criteria for OS are met. The subject will interrupt the post-OS treatment follow-up period if any of the following occurs:

● Subjects refused to participate in further studies (i.e., withdraw consent).

● Death.

● The study was terminated.

(xiv) Concomitant drug limitations or requirements:

if the researcher decides that any of the following drugs is needed to provide proper medical support to the subject, the subject must exit from further research treatment administration:

● Other research drugs

● Chemotherapy or other drugs intended to provide anti-tumor activity. This is not applicable to subjects with a history of breast cancer at the time of assisted endocrine therapy, or to subjects using agents intended for the treatment of bone metastasis (e.g., bisphosphonates or RANK ligand inhibitors).

● Radiation therapy

● Note that: after negotiating with the sponsor, radiation therapy to symptomatically isolated lesions or to bones may be considered on a case-by-case basis in special cases. According to RECIST V1.1, the irradiated lesion must be a non-target lesion and the subject must have a significantly measurable disease outside the irradiated region.

Group A (enroute Shan Kangwei Duojin)

● Adverse reactions should be closely monitored in subjects receiving either a potent cytochrome P450 (CYP) 3A4 inhibitor or a P-pg inhibitor concurrently with EV.

Group B (docetaxel)

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

Group B (vinflunine)

● Subjects receiving vinflunine should be avoided from using strong inhibitors or inducers of the CYP3A4 enzyme.

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

Group B (Pacific paclitaxel)

● Care should be taken when paclitaxel is administered with strong inhibitors or inducers of CYP3A4 and CYP2C 8.

For concomitant drug limitations or requirements of docetaxel, paclitaxel, and vinflunine, reference is made to the local drug instructions.

(xv) Duration of treatment

Subjects will be allowed to receive EVs or comparators until meeting the study disruption criteria or upon termination or completion of the study, subject to the first occurrence.

(xvi) Assessment of endpoint

Primary

●OS

Secondary minor

● According to RECIST V1.1, PFS1

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

● DCR (CR+PR+ Stable Disease (SD)) according to RECIST V1.1

● DOR according to RECIST V1.1

● Safety variables (e.g., AE, laboratory tests, vital sign measurements, 12-lead electrocardiography, and ECOG PS)

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

Exploratory property

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

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

● Incidence of ATA for EV

● According to RECIST V1.1, PFS2

●HRU

(xvii) The statistical method comprises the following steps:

approximately 600 subjects were randomized into 2 treatment groups at a 1:1 ratio: group a (EV) and group B (docetaxel, paclitaxel, or vinflunine). The random packet will be layered as follows:

● Liver transfer (Yes/No)

● ECOG PS (0 relative to 1)

● World area (United states, western Europe and elsewhere in the world)

(a) Sample sizing

Approximately 600 subjects (with 10% elimination rate and 1 metaphase analysis) were randomized into groups at a 1:1 ratio to receive EV or chemotherapy.

● The main end point is: OS (operating System)

● Single-sided 2.5% type I error; 85% assay force (Power)

● The OS assumes that: risk ratio (HR) =0.75 (EV vs. median OS of chemotherapy group 10.7m vs 8 m)

● When about 65% of deaths occur, a formal metaphase efficacy analysis will be performed.

● Preliminary analysis will be performed at about 439 OS events

For the planned metaphase efficacy analysis, a group sequence design (group sequential design) using the O' Brien-Fleming boundary performed by the Lan-DeMets method will be employed to control overall unilateral type 0.025I error. If the interim analysis confirms statistically significant results of EV on efficacy, the study can be stopped and concluded for the efficacy.

The full analysis set (Full Analysis Set; FAS) will be used for efficacy analysis of OS and PFS 1. All subjects randomized in FAS will be included. For the time to event endpoint, including OS and PFS1 log rank test stratified by random packet stratification factor (including liver transfer), the baseline ECOG PS and world region at baseline will be used to compare 2 treatment groups. The risk ratios from the hierarchical Cox proportional risk regression model and corresponding 95% confidence intervals will also be presented. Median OS, PFS1 and DOR will be estimated using Kaplan-Meier method and will be reported with corresponding 95% confidence intervals per treatment group.

The orc and DCR between treatment groups will be compared using the Cochran-Mantel-Haenszel test and stratified by the same stratification factors as used in the event analysis time. The difference in response rate between treatment groups will be estimated and corresponds to a 95% confidence interval.

(b) Safety of

The security analysis set (Safety Analysis Set; SAF) will be used for security analysis. All subjects randomized and receiving study drug will be included in the SAF. The frequency of AEs and severe AEs will be outlined in terms of med dra system organ class and preference. Furthermore, summary statistics of the following security parameters will be provided:

● Laboratory values

● Vital sign measurement

●ECOG PS

(c) Pharmacokinetics of drugs

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

6.1.3Study goals, designs and endpoints

6.1.3.1 study objectives

(i) Main objective

(ii) Secondary target

To assess safety and tolerability of EV

(iii) Exploration of targets

To assess pharmacokinetics of EV (TAb, ADC and MMAE)

To assess the incidence of ATA

To assess PFS (PFS 2) as assessed by RECIST V1.1 by a researcher examining next line therapy in subjects treated with EV versus docetaxel, paclitaxel, or vinflunine

Medical resource utilization (HRU)

6.1.3.2 study design and dose philosophy

(i) Study design

This is a phase 3 study of a randomized group of global open markers in adult subjects with locally advanced or metastatic urothelial cancer who have received platinum-containing chemotherapy and have experienced disease progression or recurrence during or after treatment with immune checkpoint inhibitors. CPI treated subjects are eligible for discontinuation of toxicity provided they have evidence of disease progression following discontinuation.

Approximately 600 subjects were randomized to EV (group a) or chemotherapy (group B) at a 1:1 ratio. The subjects will be stratified according to the following: eastern tumor cooperative group behavioral status (ECOG PS), world area, and liver metastasis.

OS is the primary endpoint. The OS is defined as the time from the start of a random packet to the day of death. Secondary endpoints include PFS1, ORR, DOR, DCR, safety and QOL/PRO.

Subjects in group a will receive EVs on days 1, 8 and 15 of each 28 day cycle. Subjects in group B will receive docetaxel, paclitaxel, or vinflunine on day 1 of each 21-day cycle as determined by the investigator prior to randomization (vinflunine is the choice as a comparator only in countries where it is approved for urothelial cancer). Within the control group, the upper limit of the overall proportion of subjects receiving vinflunine will be about 35%. The subject will continue to receive study treatment until the radiological disease progression or other disruption criteria as determined by the investigator's assessment is met, or the first occurrence is taken at the termination of the study or at the completion of the study. No crossover in the study will be allowed. During the study treatment, subjects assigned to the chemotherapy group will not be allowed to switch to a different chemotherapy treatment. This study will consist of three phases: screening, treatment and follow-up.

Screening will be performed up to 28 days prior to random grouping. The screening assessment may be repeated over a 28 day screening period.

Subjects do not necessarily IRT "screening failed" and re-enter screening with a new subject ID, provided that the subject enrolled within a 28 day window from which informed consent was signed. If more than 28 days from the date of signing the informed consent, the subject must fail IRT screening. A new agreement must be signed and the subject enters the screening with a new subject ID. The subjects can be screened only once again.

The subject will begin at cycle 1 and continue to follow up to 21 or 28 days until one of the disruption criteria is met or the first occurrence is reached at the termination of the study or completion of the study. The treatment cycle was defined as 28 days for group a and 21 days for group B.

The response of the subjects will be assessed according to RECIST V1.1. Imaging of both groups will be performed every 56 days (+ -7 days) from the first dose of study treatment during the baseline and the entire study period until PFS1 is recorded by radiological disease progression or subjects lose track, die, withdraw study consent, or begin subsequent anti-cancer therapies. Baseline imaging performed as standard care prior to informed consent can be used as long as it is performed within 28 days prior to the randomized group. All subjects will undergo a bone scan (scintigraphy) at screening/baseline. Subjects who performed positive bone scans at baseline will perform bone scans every 56 days (+ -7 days) or more frequently (if clinically indicated) throughout the study. If the clinical indication is not related to the baseline condition, the subject should conduct a follow-up bone scan. Brain scanning, computer Tomography (CT) with contrast/Magnetic Resonance Imaging (MRI), will only be performed at screening/baseline as clinically indicated and repeated as clinically indicated or according to standard of care throughout the study.

QOL assessments and PRO will be collected from all randomized, grouped subjects at the protocol-prescribed time points. The following validation tools will be used: european cancer research and treatment organization (EORTC) quality of life questionnaires (QLQ-C30) and EuroQOL 5-dimensional 5-level questionnaires (EQ-5D-5L). HRU information will be collected at protocol-specified time points, with particular focus on the number of subjects not planning to use medical resources related to clinical or AE from subjects assigned to treatment groups a and B.

Pharmacokinetic and ATA blood samples were collected from subjects randomized into group a throughout the study. The concentrations of EV ADC and monomethyl auristatin E (MMAE) in serum or plasma will be measured using a validated assay and ATA assessed. Pharmacokinetic samples will not be collected from subjects randomized to group B. Samples for the exploration of biomarkers will be collected at the time points prescribed by the protocol.

Following PFS2, subjects will enter a survival tracking period and follow the survival status every 3 months until death, loss of tracking, withdrawal of study consent, or termination by the sponsor study. Once the final survival analysis was completed, this study was expected to end. Subjects will be eligible to continue to receive treatment in this study until they meet the disruption criteria as outlined in section 6.1.7 disruption, or upon termination of the study or completion of the study, whichever occurs first.

The privileged IDMC will govern the safety and mid-plan efficacy analysis, which will occur after at least 285 OS events (about 65% of total planned events) are observed. The preliminary analysis will be performed at 439 OS events. The IDMC may suggest to the sponsor whether the trial should be terminated, modified, or continued unchanged based on the continued review of the safety data and the interim efficacy analysis. Additional details will be summarized in the IDMC rights.

(ii) Dose rationale

(a) Enrolment Shan Kangwei statin

EV will be administered at a dose of 1.25mg/kg in the form of an approximately 30 minute intravenous infusion on days 1, 8 and 15 of each 28 day cycle. This dose and regimen has demonstrated an acceptable safety profile and encouraging clinical activity in phase 1 studies evaluating ascending dose levels of 0.5, 0.75, 1 and 1.25mg/kg, with the ascending (expansion) group at dose levels of 0.75, 1 and 1.25 mg/kg. The maximum tolerated dose (maximum tolerated dose; MTD) was not reached in this study. At a dose level of 1mg/kg, 2 dose limiting toxicities (dose-limiting toxicity; DLT) were observed: grade 3 anal pain (grade 2 later changed by researchers) and grade 4 hyperuricemia without clinical sequelae associated with irradiation review (recovery) are considered. No DLT was observed at 1.25mg/kg, and doses above 1.25mg/kg were not tested.

The incidence of some of the most frequent drug-related AEs (e.g., diarrhea and rash), while primarily grade 1 to 2 and clinically treatable, increases with increasing dose levels.

Furthermore, for 1.25mg/kg and lower dose levels, the dose reduction due to AE was more frequent. Safety assessments of patients with metastatic urothelial cancer and non-metastatic urothelial cancer showed that all TEAEs, AEs leading to withdrawal, and TEAEs of grade 3 to 4 were comparable in frequency between all dose levels.

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

Based on pharmacokinetic data from phase 1 studies, the half-life of EV was-1 to 2 days. At any dose level, no significant (< 30%) ADC accumulation was observed during the current dosing regimen (on days 1, 8 and 15 of every 28 day cycle). MMAE accumulation was observed over the minimum (< 50%) period. It is expected that the dosing schedule in the study will maintain ADC exposure over each 28 day period, contributing to a good balance of activity and safety as observed in phase 1 trials.

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

(b) Comparison agent

There is no recognized standard of care following CPI treatment for locally advanced or mUC; however, prior to CPI approval, taxanes are often used after platinum-based therapies, as suggested in the treatment guidelines. In addition, vinflunine was approved in europe for treatment mUC after failure of previous platinum-based protocols.

Multi-centered, randomly grouped activity-controlled trial KEYNOTE-045 in patients with locally advanced or mUC with disease progression at or after platinum-containing chemotherapy compares palboc Li Zhushan anti every 3 weeks with the investigator's choice paclitaxel, docetaxel or vinflunine. Bellmutt J et al, N Engl J med.2017;376:1015-26.DOI:10.1056/NEJMoa1613683.

Although no labeled taxane guidelines for administration are available in this context, this is the only large random grouping of reported combined survival data for these agents, so current phase 3 enrolment monoclonal antibodies and chemistryThe therapy study will use the same comparator selections and their corresponding dosages. Patients randomized to chemotherapy groups in KEYNOTE-045 study were treated with paclitaxel (175 mg/m ² ) Docetaxel (75 mg/m) ² ) Or vinflunine (320 mg/m) ² ) Treatment was administered intravenously every 3 weeks. The upper limit of the overall proportion of subjects receiving vinflunine in the control group is approximately 35% (keyote protocol).

6.1.3.3 end point

(i) Primary endpoint

OS

(ii) Secondary endpoint

PFS1 is detected by RECIST V1.1

ORR (CR+PR) by RECIST V1.1

DCR (CR+PR+SD) by RECIST V1.1

DOR by RECIST V1.1

Safety variables (e.g., AE, laboratory tests, vital sign measurements, 12-lead ECG and ECOG PS)

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

(iii) Exploration of endpoint

Plasma or serum concentrations of TAb, ADC and MMAE

Incidence of ATA for EV

PFS2

HRU

6.1.4Study population

Selection of 6.1.4.1 study population

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

6.1.4.2 inclusion criteria

The subject met the study conditions if all of the following were applicable:

prior to any study-related procedures (including withdrawal of forbidden drugs, if applicable), written informed consent and privacy language approved by the Institutional Review Board (IRB)/Independent Ethics Committee (IEC) in accordance with national regulations (e.g., health insurance privacy and liability act authority of the united states study site) must be obtained from the subject.

The subject was a legal adult according to local regulations when signing the informed consent.

The subject has a histologically or cytologically confirmed urothelial cancer (i.e., bladder cancer, renal pelvis cancer, urinary tract cancer, or urinary tract cancer). Subjects with urothelial cancer (transitional cells) with squamous differentiated or mixed cell types are eligible.

The subject must undergo radiological progression or recurrence of locally advanced or metastatic disease during or after CPI (programmed cell death protein-1 (PD-1) or anti-programmed cell death-ligand 1 (PD-L1)). CPI treated subjects are eligible for discontinuation of toxicity provided they have evidence of disease progression following discontinuation. CPI need not be the most recent therapy. A subject is eligible if the most recent therapy has progressed/relapsed during or after its most recent therapy in a subject based on a non-CPI-based regimen. Locally advanced disease must not be resected as intended by the treating physician using the cure.

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

The subject had a radiological record of metastatic or locally advanced disease at baseline.

Archiving tumor tissue samples should be available for presentation to a central laboratory prior to study treatment. If an archived tumor tissue sample is not available, a fresh tissue sample should be provided. If fresh tissue samples cannot be provided due to safety issues, participation in the study must be discussed with the medical monitor.

The subject has an ECOG PS of 0 or 1.

The subjects had the following baseline laboratory data:

● Absolute Neutrophil Count (ANC) at 1500/mm or more ³

● Platelet count is greater than or equal to 100X 10 ⁹ personal/L

● Hemoglobin is more than or equal to 9g/dL

● Alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) are less than or equal to 2.5 XULN or less than or equal to 3 XULN for subjects with liver metastasis

The female subject must meet any of the following:

● Fertility potential free:

● Or, if having fertility potential:

Female subjects must agree that no lactation or donation of ova is performed at the start of screening and during the whole study period and at least 6 months after the last study drug administration.

Sexually active male subjects with female partners having fertility potential were eligible for:

* Efficient forms of fertility control include:

● Always and correctly using established hormonal contraceptives that inhibit ovulation,

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

● Bilateral tubal occlusion

● Vasectomy (vasectomy is a high-efficiency contraceptive method, provided that no sperm is confirmed, otherwise an additional high-efficiency contraceptive method should be used)

● Male infertility due to bilateral orchiectomy or eradication of cystprostatectomy/removal of seminal vesicles

● Forbidden is considered an efficient method only when it is defined as avoiding non-intercourse throughout the risk period associated with study treatment. There is a need to assess the reliability of abstinence with respect to duration of the study and preference and common lifestyle of participants

Note that: inadequate abstinence in swiss for use as a contraceptive method

Male subjects were not allowed to donate sperm at the beginning of the screening and throughout the study period, and at least 6 months after administration of the final study medication.

Male subjects with pregnant or lactating partners must agree to either have a contraception or use the condom during pregnancy or during the time of pregnancy for the partner to lactate during the entire study period and at least 6 months after administration of the final study medication.

Subjects agreed not to participate in another intervention study when undergoing treatment in this study.

No exemption from inclusion criteria is allowed.

6.1.4.3 exclusion criteria

The subject will exclude participation if any of the following is applicable:

the subject suffers from pre-existing sensory or motor neuropathy of grade No. 2.

The subject suffers from active Central Nervous System (CNS) metastasis. Subjects with treated CNS metastases were allowed to study if all of the following were true:

● The subject did not suffer from leptomeningeal disease

Subjects are experiencing clinically significant toxicity (grade 2 or higher, except hair loss) associated with previous treatments, including systemic therapy, radiation therapy or surgery. Subjects with +.2 immunotherapy-related hypothyroidism or hypopituitarism are eligible for selection when they are adequately maintained/controlled at a stable dose of hormone replacement therapy (if indicated). Patients experiencing grade 3 or more immunotherapy-related hypothyroidism or hypopituitarism are excluded. Subjects experiencing immunotherapy-related colitis, uveitis, myocarditis, or pneumonia or subjects with other immunotherapy-related AEs requiring high doses of steroid (> 20 mg/day of prednisone or equivalent) are excluded.

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

In the control group, the subjects have received prior chemotherapy for urothelial cancer as well as all available study therapies (i.e., both prior paclitaxel and docetaxel in the area where vinflunine is not approved for therapy; or prior paclitaxel, docetaxel and vinflunine in the area where vinflunine is approved for therapy). Note that: after reaching the upper limit of vinflunine, subjects who have received both docetaxel and paclitaxel will be excluded.

The subject has received more than 1 prior chemotherapy regimen for locally advanced or metastatic urothelial cancer, including chemotherapy for adjuvant or neoadjuvant disease (if recurrence occurs within 12 months of completion of therapy). The substitution of cisplatin with carboplatin does not constitute a new regimen, provided that no new chemotherapeutic agent is added to the regimen.

The subject had a history of another malignant disease within 3 years prior to the first dose of study drug, or had any signs of residual disease of the malignant disease previously diagnosed. Subjects with non-melanoma skin cancers, localized prostate cancers that undergo curative treatment and have no signs of progression, low or very low risk (according to standard guidelines) localized prostate cancers that are inadvertently treated with active supervision/observation waiting, or any type of carcinoma in situ (if complete excision is performed) are permissible.

At the time of the first dose of EV, the subject is currently receiving systemic antimicrobial therapy against viral, bacterial or fungal infection. Conventional antimicrobial control is tolerated.

The subject suffers from known active hepatitis b (e.g., HBsAg reactivity) or active hepatitis c (e.g., HCV RNA detected (qualitative)).

Subjects are known to have a history of Human Immunodeficiency Virus (HIV) infection (HIV 1 or 2).

The subjects had a history of recorded cerebrovascular events (stroke or transient ischemic attacks), unstable angina, myocardial infarction, or heart symptoms (including congestive heart failure) consistent with the new york heart association (New York Heart Association) group III-IV within 6 months prior to the first dose of study medication.

The subjects underwent radiation therapy or major surgery within 4 weeks prior to the first dose of study drug.

The subject received chemotherapy, biological agents, study agents, and/or anti-tumor treatment with immunotherapy and was not completed 2 weeks before the first dose of study drug.

The subject is known to be allergic to EV or to any excipient contained in EV pharmaceutical formulations (including histidine, trehalose dihydrate and polysorbate 20); or the subject is known to be allergic to a biopharmaceutical prepared in Chinese Hamster Ovary (CHO) cells.

The subject is known to be allergic to:

Subjects are known to have active keratitis or corneal ulcers. Subjects with superficial punctate keratitis are allowed if their condition is adequately treated from the point of view of the researcher.

The subject suffers from other potential medical conditions that, from the point of view of the researcher, would impair the subject's ability to receive or tolerate the planned treatment and follow-up.

A history of uncontrolled diabetes within 3 months of the first dose of study medication. Uncontrolled diabetes is defined as hemoglobin A1c (HbA 1 c) > 8% or HbA1c between 7% and <8% with diabetes symptoms (polyuria or polydipsia) not otherwise explained.

No exemption from the exclusion criteria is allowed.

6.1.5Treatment of

Identification of 6.1.5.1 pharmaceutical products

(i) Research medicament

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

(ii) Comparative drug

The comparison drugs will be supplied by the pharmacy at the site of responsibility of each clinical trial site. If the site is unable to purchase or utilize a local comparison drug supply, the sponsor may centrally provide the supply when appropriate. The sites were allowed to use universal docetaxel and paclitaxel approved by the respective regulatory authorities. For storage and handling conditions and warning statement, reference is made to the product labels of docetaxel, paclitaxel and vinflunine.

For specific dosing instructions, the study reference section 6.1.6.1 studies the dosing and administration of drugs and other drugs.

6.1.5.2 package and label

The EV (ASG-22 CE) used in this study will be responsible for preparation, packaging and labeling by qualified staff according to standard operating procedures (Standard Operating Procedure; SOP), good manufacturing practice (Good Manufacturing Practice; GMP) guidelines, ICH GCP guidelines and applicable local laws/regulations.

Each carton and vial will carry a label that identifies the contents as a pharmaceutical product in compliance with regulatory guidelines, GMP, and local legal regulations.

In the case of supply by sponsors, the comparison drugs used in this study will be labeled according to SOP GMP guidelines, ICH GCP guidelines, and applicable local laws/regulations.

The qualified personnel will perform the final release of the drug according to the requirements of EUDirective 2003/94/EC appendix 13.

6.1.5.3 study drug treatment

Current ICH GCP guidelines require researchers to ensure that study drugs delivered from sponsors are received by researchers and/or designated personnel, and:

● Such a delivery is recorded and is recorded as,

● The study drug was handled and stored according to the labeled storage conditions,

● The study drug had appropriate expiration/retest and was administered to the study subject only according to the protocol, and

● Any unused study drug is returned to the sponsor or standard procedures for alternative treatment of unused study drug are followed.

The study site responsible person or study drug storage manager should take care of the following study drug responsibilities:

● The study drug storage manager should store the study drug and assume its responsibility following the disposition study drug program written by the sponsor.

● Study drug storage manager should prepare and maintain a study drug receipt, study site inventory, a record of each subject's use, and return unused study drug to sponsor or replacement process. These records should include date, number, batch/serial number, expiration date (if applicable) and unique codes assigned to study drugs and subjects.

● The study drug storage manager should prepare and maintain records that properly record the doses prescribed by the subject's delivery regimen, and adjust all study drugs delivered by the sponsor.

6.1.5.4 Blind method (blank)

This is an open label study.

Although the study is an open-label study, in order to maintain experimental integrity, analysis or overview by randomized group treatment assignment or actual treatment assignment will be limited and recorded while the study is in progress and recorded prior to the primary rigid lock (primary hard lock). The interim analysis will be performed outside the independent data analysis center (independent data analysis center; IDAC). Details will be included in the SAP.

6.1.5.5 assignment and allocation

Subjects will be randomized to treatment groups via interactive response techniques (Interactive Response Technology; IRT) at a 1:1 ratio according to a randomized group schedule. All subjects meeting the eligibility criteria will be randomized. The field personnel will administer the treatment according to the IRT system's assignment. Specific procedures for random grouping via IRT are contained in the study procedure manual. The researcher must select one of the group B choices to be used in the case of randomized subjects into group B before randomized subjects are randomized. Within the control group, the upper limit of the overall proportion of subjects receiving vinflunine will be about 35%.

6.1.6Treatment and assessment

6.1.6.1 study of administration and administration of drugs and other drugs

(i) Dosage/dosing regimen and period of administration

(a) Enrolment Shan Kangwei statin

EV will be administered at a dose of 1.25mg/kg in the form of an approximately 30 minute intravenous infusion on days 1, 8 and 15 of each 28 day cycle. In the absence of IRR, the infusion rates of all subjects should be calculated to achieve an infusion period of about 30 minutes. EV is not administered in intravenous push or bolus form. EV should not be mixed with other drugs. At least 1 week must elapse between each EV dose.

Weight-based dosing was calculated using the actual weight of the subject on day 1 of each cycle. Group a does not require recalculation of dose based on actual body weight on day 8 and day 15 of each cycle unless mandated by institutional standards. For subjects weighing more than 100kg, weight-based administration was not applicable; the dose for these subjects was based on 100kg. The maximum allowable dose for this study was 125mg.

The subject weight must be measured during all relevant assessment time points as described in the event time course.

The subject should be observed during the enrolment Shan Kangwei statin administration and at least 60 minutes after the infusion period of the first 3 cycles. All supportive measures meeting optimal patient care should be given throughout the study, according to institutional standards.

Redness, swelling, pain and infection at the injection site were closely monitored during and at any time after administration. The subject should be advised to report redness or discomfort immediately upon administration or after infusion.

(b) Comparative drug

The chemotherapeutic agent will be administered following local product labeling or product feature profile (SmPC) and institutional guidelines, and taking care to prevent extravasation according to institutional standards and as described in the following: "Chemotherapy and Biotherapy Guidelines and Recommendations for Practice" (Polovich M et al Chemotherapy and biotherapy guidelines and recommendations for practic 4 th edition Pittsburgh: oncology Nursing Society;2014.473 p.) and "Management of Chemotherapy Extravasation: ESMO-EONS Clinical Practice guides," Fidalgo JA et al Ann Oncol.2012;23 (7):167-73.

A male subject randomized to receive docetaxel, vinflunine, or paclitaxel should seek medical advice regarding cryopreservation of sperm due to possible sterility prior to receiving treatment.

Docetaxel (docetaxel)

Docetaxel study treatment should be administered as intravenous infusion on day 1 of every 21-day cycle after all procedures/assessments are completed, including the required precursor dosing according to local standard of care prior to day 1. Unless otherwise specified in sections 6.1.6.1 (i) (b) and 6.1.6.1 (ii) (c), this will be at 75mg/m ² Docetaxel is administered. Other guidelines for docetaxel administration refer to centrally supplied local product labels or SmPC and institutional guidelines.

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

All subjects should begin 1 day prior to docetaxel administration, and a corticosteroid, such as 16mg per day dexamethasone (e.g., 8mg twice daily) is administered orally for 3 days according to local standard-of-care precursors, to reduce the incidence and severity of fluid retention and the severity of allergic reactions. Appropriate precursor dosing regimens may be determined by the researcher.

Vinflunine

Vinflunine study treatment should be administered as intravenous infusion on day 1 of every 21 day cycle after all procedures/assessments are completed. Unless otherwise specified in section 6.1.6.1 (ii) (d), will be at 320mg/m ² Vinflunine is administered. For guidelines for initial dose adjustment, see 6.1.6.1 (ii) (d) vinflunine dose adjustment. Subjects should be observed during vinflunine administration and at least 30 minutes after the infusion period of the first 3 cycles. All supportive measures meeting optimal patient care should be given throughout the study, according to institutional standards. For other guidelines for administration of vinflunine, reference is made to local product labels or institutional guidelines for SmPC and vinflunine.

Pacific paclitaxel

Paclitaxel study treatment should be administered as intravenous infusion on day 1 of every 21-day cycle after all procedures/assessments are completed. Unless otherwise specified in section 6.1.6.1 (ii) (e), it will be at 175mg/m ² Paclitaxel is administered. For initial dose adjustment, see section 6.1.6.1 (ii) (e) paclitaxel dose adjustment. Paclitaxel should be administered within 3 hours or according to local guidelines. Subjects should be observed during paclitaxel administration and at least 30 minutes after the infusion period of the first 3 cycles. All supportive measures meeting optimal patient care should be given throughout the study, according to institutional standards. Other guidelines for paclitaxel administration refer to local product labels or institutional guidelines for SmPC and paclitaxel.

All subjects should be pre-dosed prior to paclitaxel administration to prevent severe allergic reactions. Such precursor administration may consist of: about 12 and 6 hours prior to paclitaxel, 20mg of dexamethasone was orally administered; 50mg of diphenhydramine (or equivalent thereof) is administered intravenously 30 to 60 minutes prior to paclitaxel; and 30 to 60 minutes before paclitaxel, administering cimetidine (300 mg) or ranitidine (50 mg) intravenously. Appropriate precursor dosing regimens may be determined by the researcher.

(ii) Study of increase or decrease in dosage of drug

(a) Enrolment Shan Kangwei statin

Depending on the type and severity of the toxicity, the allowable dose was reduced to 1mg/kg (dose level-1) and 0.75mg/kg (dose level-2). Subjects in need of a reduced dose may be further escalated to 1 dose level (e.g., subjects reduced to 0.75mg/kg can only be further escalated to 1 mg/kg) provided that toxicity does not require discontinuation of study drug and has returned to baseline or grade 1. If toxicity is recurring, no further increment will be allowed. Subjects presenting with grade 2 AE were not allowed to escalate the dose.

EV should not be administered to subjects with CrCl <30 mL/min. Dose-modulating recommendations for EV-related toxicity are presented in tables 13 and 14. Researchers at the study site may tolerate discontinuation of administration due to other EV-related toxicities as appropriate. The discontinuation of administration may last up to 8 weeks (2 cycles). If toxicity in the subject does not otherwise require permanent discontinuation of the administration, discontinuation of the administration from the subject for clinical benefit from treatment may extend beyond 8 weeks. If there is a disruption in dosing, the time course of response assessment will not be adjusted.

Table 13 recommended dose modulation for enrolment Shan Kangwei statin related hematological toxicity

Table 14 dose modulation for enrolment Shan Kangwei statin related non-hematological toxicity recommendations

AE: adverse events; EV: enrolment Shan Kangwei statin

* Grade 3/4 electrolyte imbalance/laboratory abnormalities (except hyperglycemia) that are not accompanied by clinical sequelae and/or corrected by supplementation/appropriate treatment within 72 hours of their onset do not require discontinuation of treatment (e.g., grade 4 hyperuricemia). Grade 3 rash, which does not limit self-care activities of daily living or concomitant infections and requires systemic antibiotics, does not require discontinuation of treatment provided the symptoms are not severe and can be treated by supportive treatment.

* Ophthalmic examinations should be performed by an ophthalmologist. In countries where optometrists can perform the examination and prescribe, the examination can actually be performed using optometrists.

Enrobed Shan Kangwei statin related rashes

In phase 1 studies, rashes and similar dermatological AEs were common in patients treated with EV and were more frequently seen at the highest dose. Although the exact etiology of the skin viruses associated with EV is not clear at this time, rashes may be the target toxicity due to the expression of connexin-4 in the skin. The most common skin AE types reported in ASG-22CE-13-2 are maculopapules, rashes, skin exfoliation and skin pigmentation disorders. Most occur early (during cycle 1) and some are accompanied by itching. Mild rashes due to EV should be treated with local supportive care as needed. For itch, topical corticosteroids have been used as well as antihistamines as needed. Grade 3 rash, which does not limit self-care activities of daily living or concomitant infections and requires systemic antibiotics, does not require discontinuation of treatment provided the symptoms are not severe and can be treated by supportive treatment.

Treatment of hyperglycemia

The researcher should monitor blood glucose levels and if any symptoms of hyperglycemia are observed, it is recommended to perform additional assessments, including a thorough assessment of the infection. In addition, if the steroid is used to treat any other condition, the blood glucose level may require additional monitoring. If an increase in blood glucose level is observed, the subject should be treated according to local care standards and may consider a referral to the endocrinology department.

If the glucose level of a subject, especially a subject with a history of or suffering from diabetes or hyperglycemia, becomes difficult to control or if it experiences symptoms of dark hyperglycemia (e.g. frequent urination, increased thirst, blurred vision, fatigue and headache), it is recommended that the subject should immediately inform his physician.

Subjects entering the study with elevated HbA1c (> 6.5%) at baseline should be referral to the appropriate provider for glucose treatment during cycle 1. Blood glucose should be checked prior to each administration and for blood glucose >250mg/dL (13.9 mmol/L) (grade 3 or higher) administration should remain stopped. Once the subject's blood glucose has improved to +.2 and the subject is clinically and metabolically stable, administration can continue. Blood glucose associated with EV >500mg/dL (27.8 mmol/L) (grade 4) was considered to require discontinuation of treatment. If the subject experiences new onset diabetes, the subject is evaluated with metabolome, urone, glycosylated hemoglobin, and C-peptide to assess new onset type 1 diabetes in the previous CPI background.

Treatment of enrolment Shan Kangwei statin infusion related reactions (Infusion Related Reaction; IRR)

IRR may occur during infusion of study treatment. Infusion should be performed at a site equipped with appropriate equipment and personnel for treatment when a systemic allergic reaction occurs. All supportive measures meeting optimal patient care should be given throughout the study, according to institutional standards. Supportive measures may include administering a drug against IRR.

Subjects who have undergone IRR may be administered a precursor for subsequent infusions. Precursor administration may include analgesic (e.g., acetaminophen or an equivalent), antihistamine (e.g., diphenhydramine hydrochloride), and corticosteroid administered about 30 to 60 minutes prior to each infusion or according to institutional standards. If the subject experiences IRR in the context of a pre-administration, continued treatment with enrolment Shan Kangwei statin must be discussed with the medical monitor before the next planned dose.

If systemic allergic reactions occur, study therapeutic administration should be discontinued immediately and permanently.

(b) Dose modulation for treatment with docetaxel, paclitaxel, or vinflunine

In general, for grade 4 drug-related hematologic toxicities and ≡3 non-hematologic toxicities, treatment with the chemotherapy comparator (docetaxel, paclitaxel, or vinflunine) should be stopped and subsequent doses adjusted as per table 15 below. Dose adjustment will apply to all subsequent doses. Specific dose-setting guidelines for docetaxel, paclitaxel, and vinflunine are found in the following articles sections 6.1.6.1 (ii) (b), 6.1.6.1 (ii) (c), and 6.1.6.1 (ii) (d). Dose adjustments should also be considered according to local product labels or SmPC and institutional guidelines. For grade 3 or greater hematologic toxicity associated with docetaxel, paclitaxel, or vinflunine, as indicated by institutional guidelines, transfusion or growth factors may be used.

(c) Docetaxel dose modulation

Docetaxel should not be given total bilirubin>Subjects with ULN, or administration of AST and/or ALT>1.5 XULN and accompanying alkaline phosphatase>2.5 XULN subjects. Subjects with elevated bilirubin or simultaneous abnormalities in transaminase and alkaline phosphatase have an increased risk of developing grade 4 neutropenia, febrile neutropenia, infection, severe thrombocytopenia, severe stomatitis, severe skin toxicity and toxic death. Docetaxel should not be administered with neutrophil counts either<1500 cells/mm ³ Is a subject of (a).

Serious fluid retention has been reported following docetaxel therapy. The subject should be pre-dosed with a corticosteroid prior to each docetaxel injection administration to reduce the incidence and severity of fluid retention. The effusion of a subject with pre-existing effusion may be worsened by the close monitoring of the subject starting from the first dose. Subjects with peripheral oedema can be treated with standard measures, such as salt limitation, oral diuretics. The discontinuation of administration may last up to 6 weeks (2 cycles). If toxicity in the subject does not otherwise require permanent discontinuation of the administration, discontinuation of the administration to the subject in response to the treatment may extend beyond 6 weeks. The guidelines for recommended dose adjustment for subjects receiving docetaxel are detailed in table 16 below. Dose adjustments (e.g. severe or cumulative skin reactions) and initial doses not specified below should also take into account local product labels or SmPC and institutional guidelines.

Docetaxel dosage adjustment guidelines suggested in table 16

ANC: absolute neutrophil count; N/A: inapplicable; t: temperature (temperature)

(d) Vinflunine dosage adjustment

In the case of WHO/ECOG PS of 1 or ECOG PS of 0 and prior pelvic irradiation, 280mg/m should be used ² Starts the vinflunine treatment. In the absence of any hematological toxicity causing delay in treatment or dose reduction during the first period, the dose in the subsequent period may be increased to 320mg/m every 21 days ² . In subjects with moderate kidney damage (40 mL/min. Ltoreq. CrCl. Ltoreq.60 mL/min), a recommended dose of 280mg/m administered every 21 day period ² Once. When suffering from kidney injury (30 mL/min is less than or equal to CrCl)<40 mL/min) of 250mg/m every 21-day cycle ² Once.

In subjects with mild liver injury (Child-Pugh A grade), the recommended dose of vinflunine is 250mg/m administered every 21 day cycle ² Once. For other dose adjustments, reference is made to local product labels or SmPC and institutional guidelines.

The recommended doses in subjects aged 75 years are as follows:

● Subjects aged 80 years and older In (2), the dose of vinflunine to be administered is 250mg/m per 21-day cycle ² 。

At 280mg/m ² In subjects with initial vinflunine and AE undergoing dose-adjustment, the dose should be reduced to 250mg/m after the 1 st occurrence and regression ² And is interrupted after the 2 nd occurrence. At 250mg/m ² Vinflunine should be discontinued in the subject who initiated vinflunine and experienced AE in need of dose adjustment.

After administration of vinflunine, cases of reversible posterior encephalopathy syndrome (Posterior Reversible Encephalopathy Syndrome; PRES) have been observed. Typical clinical symptoms (with varying degrees) are: neurological (headache, confusion, epilepsy, vision disorders), systemic (hypertension) and gastrointestinal (nausea, vomiting). Radiological signs are white matter abnormalities in the posterior region of the brain. In subjects who develop PRES neurological signs, vinflunine must be discontinued. The dosage adjustments of subjects receiving vinflunine are detailed in table 17 below. The discontinuation of administration may last up to 6 weeks (2 cycles). If toxicity in the subject does not otherwise require permanent discontinuation of the administration, discontinuation of the administration from the subject for clinical benefit from treatment may extend beyond 6 weeks. For other information, reference is made to vinflunine SmPC。

Table 17 vinflunine dosage modulation

ANC: absolute neutrophil count; N/A: inapplicable; t: temperature (temperature)

¹ Adverse events the common terminology standard of the national cancer institute for grade 2 constipation is defined as requiring laxatives, grade 3 as obstinate constipation (obstinate) requiring manual drainage or bowel movement, and grade 4 as obstructive or toxic megacolon. Mucositis grade 2 is defined as "moderate", grade 3 as "severe", and grade 4 as "life threatening".

(e) Pacific paclitaxel dose modulation

The baseline neutrophil count should not be less than 1500 cells/mm ³ Paclitaxel is administered to the subject. Following the cycle, the subject should not be retreated with paclitaxel until neutrophils return to levels>1500 cells/mm ³ And the platelets return to the level>100000/mm ³ . Has been in the course of paclitaxel therapy<Severe conduction abnormalities were noted in 1% of the subjects and in some cases, the atrial pulse generator was placed. If a subject develops significant conduction abnormalities during paclitaxel infusion, appropriate therapy should be administered and continuous cardiac monitoring should be performed during subsequent paclitaxel therapy, provided that the subject does not need to be discontinued.

In the case of mild liver injury (total bilirubin not less than 1.25 ULN), paclitaxel should be added at 135mg/m ² Is started. Dosage adjustment of subjects receiving paclitaxel is detailed below. The discontinuation of administration may last up to 6 weeks (2 cycles). If toxicity in the subject does not otherwise require permanent discontinuation of the administration, discontinuation of the administration to the subject in response to the treatment may extend beyond 6 weeks. The guidelines for recommended dose adjustment for subjects receiving paclitaxel are detailed in table 18 below. For other dose adjustments, reference is made to local product labels or SmPC and institutional guidelines.

Pacific paclitaxel dose adjustment guidelines suggested in Table 18

ANC: absolute neutrophil count; N/A: inapplicable; t: temperature (temperature)

(iii) Previous and concomitant therapies (both medicinal and non-medicinal therapies)

● Other research drugs

● Radiation therapy

Note that: after negotiating with the sponsor, radiation therapy to symptomatically isolated lesions or to bones may be considered on a case-by-case basis in special cases. According to RECIST V1.1, the irradiated lesion must be a non-target lesion and the subject must have a significantly measurable disease outside the irradiated region.

(a) Group A (enroute Shan Kangwei Duojin)

Adverse reactions should be closely monitored in subjects receiving either a potent cytochrome P450 (CYP) 3A4 inhibitor or a P-pg inhibitor concurrently with EV.

(b) Group B (docetaxel)

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

(c) Group B (vinflunine)

The use of strong inhibitors of the CYP3A4 enzyme by subjects receiving vinflunine should be avoided.

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

(d) Group B (Pacific paclitaxel)

Care should be taken when paclitaxel is administered with strong inhibitors or inducers of CYP3A4 and CYP2C 8.

For concomitant drug limitations or requirements of docetaxel, paclitaxel, and vinflunine, reference is made to the local drug instructions. Unless specified otherwise, all concomitant treatments will be recorded in the eCRF or electronic data source.

(iv) Compliance with treatment

The dosages and schedules of EV, paclitaxel, docetaxel, and vinflunine administered to each subject will be recorded on the appropriate electronic case report table (eCRF) at each cycle. The reason for dose delay, reduction or omission will also be recorded.

If a toxic or adverse event occurs on day 1 of any cycle and EV cannot be administered, the start of the cycle may be delayed. If toxicity occurs on either day 8 or day 15 of any cycle and a dose of >3 days needs to be maintained, the dose must be eliminated, not delayed. If the subject only receives day 1 and needs to skip days 8 and 15, the subject can restart the next cycle as early as day 22 (new day 1) if toxicity subsides later.

6.1.6.2 demographic data and baseline characteristics

(i) Demographic data

Demographic information will be collected for all subjects according to local regulations and will include date of birth, gender, race, and history of smoking (package year).

(ii) Medical history

The medical history will include all major medical conditions other than urothelial cancer that have resolved prior to informed consent or are in progress at the time of consent. Details to collect include date of onset and date of recovery, if applicable, adverse event common terminology standard (CTCAE) level.

(iii) Diagnosis, severity and duration of disease of interest

For urothelial cancer, information including, but not limited to, the following will be collected during the screening period and entered into eCRF:

● Date of initial diagnosis, histological type, histopathological or cytopathological diagnosis of primary carcinoma

● Date of diagnosis of locally advanced or metastatic or recurrent disease

● TNM classification and disease stage at screening

● Sampling method and tumor tissue type for connexin-4 mutation analysis

● Previous treatments for potential diseases (including drugs, radiation therapy and surgery)

(iv) Behavior state

The ECOG PS scale will be used to assess behavioral status. Refer to section 6.1.9.9.OKen et al, am J Clin Oncol.1982;5:649-55.

6.1.6.3 efficacy assessment

The reaction and progress will be assessed using RECIST V1.1 6.1.9.8.

Imaging of both groups will be performed every 56 days (+ -7 days) from the first dose of study treatment at screening/baseline and throughout the study period. Baseline imaging performed as standard care prior to informed consent can be used as long as it is performed within 28 days prior to the randomized group. All subjects will undergo a bone scan (scintigraphy) at screening/baseline. Subjects who performed positive bone scans at baseline will perform bone scans every 56 days (+ -7 days) or more frequently (if clinically indicated) throughout the study. If clinically indicated throughout the study period, even if not positive at baseline, subjects with negative bone scans at baseline should undergo bone scans. Brain scans (CT with contrast/MRI) were only performed if clinically indicated at screening/baseline, and repeated clinically indicated throughout the study or according to the standard of care. If the subject discontinued the study drug prior to radiological disease progression, the subject should continue imaging assessment every 56 days (+ -7 days) until disease progression is noted, or the subject begins another anti-cancer treatment, based on an earlier occurrence.

CT scans with contrast (chest and abdomen) are the preferred mode of tumor assessment. Magnetic resonance imaging is acceptable if local standard practice or if the subject is contraindicated for CT scanning (e.g., the subject is allergic to contrast agents). All other approved RECIST scanning methods, such as x-rays, are optional. Additional description of imaging assessment can be found in the study procedure manual.

The assessment will include tumor measurements of target lesions, non-target lesions, and any new lesions. The overall assessment will be characterized for a given point in time assessment.

At the end of the subject study, the best overall response to the study protocol will be characterized. To ensure comparability, the same techniques should be used for screening and subsequent assessment of the reaction. If possible, images of any 1 subject should be assessed by the same investigator for the duration of the study. For subjects with known brain metastases at the time of entry into the study, it is recommended that repeated imaging also include the brain, and that lesions be tracked throughout the study using the same method used to detect brain lesions at baseline.

The site of disease progression including targeted, non-targeted and/or new lesions should be recorded in eCRF. Additional imaging may be performed at any time to confirm suspected progression of the disease.

This study will be analyzed based on the results of local (investigator site) radioactivity assessment, including date of progression and death. Since imaging scans may be required for future regulatory purposes or may take into account independent review of all or representative samples of the scans after completion of the PFS1 analysis, copies of all scans will be collected throughout the study and centrally stored by the coordination provider. The images of all randomly grouped subjects will be sent to the imaging provider according to the frequencies of table 10.

(i) Evaluation of target lesions

(a) Complete reaction

CR is defined as the disappearance of all targeted and non-targeted lesions. Any pathological lymph node (whether targeted or non-targeted) must be reduced to <10mm on the short axis compared to the baseline measurement.

(b) Partial reaction

PR is defined as the sum of the target lesion diameters (longest axis for non-nodular lesions and short axis for nodular lesions) reduced by at least 30% using the sum of the baseline diameters as a reference.

(c) Stabilizing disease

SD is defined as the sum of the smallest diameters at the time of administration of study drug taken as a reference, neither sufficiently reduced to meet PR nor sufficiently increased to meet progressive disease.

(d) Progressive disease

PD is defined as the sum of target lesion diameters (longest axis for non-nodular lesions and lesions for nodular lesions) increased by at least 20% using the minimum sum at study (including baseline sum if the baseline sum is the minimum sum at study) as a reference. In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5 mm. The appearance of 1 or more new lesions is also considered progress.

(ii) Evaluation of non-target lesions

In order to achieve a clear progression based on non-target lesions, there must be a significant worsening of the overall level of non-target disease, thereby increasing the overall tumor burden sufficiently to be worth interrupting therapy even in the presence of SD or PR of the target lesion. A modest increase in the size of 1 or more non-target lesions is often insufficient to define a clear progression.

(a) Complete reaction

For CR of non-target lesions, all non-target lesions of the subject must disappear and all lymph nodes must be non-pathological in size (short axis <10 mm).

(b) non-CR/non-PD

non-CR/non-PD of a non-target lesion is defined as surviving 1 or more non-target lesions.

(c) Progressive disease

PD of a non-target lesion is defined as the clear progression of an existing non-target lesion or the appearance of 1 or more new lesions.

(iii) Assessment of time point response

The overall response status of subjects with measurable disease at baseline at each time point will be reported according to the tables in sections 6.1.9.6 and 6.1.9.7.

6.1.6.4 pharmacokinetic assessment

PK samples from subjects receiving EV will be collected during the treatment period and at follow-up visit after treatment to determine ADC and MMAE concentrations. If the subject is presented to the clinic but not administered, pre-dose PK sample collection should be performed.

Blood samples (7 mL/sample) should be collected at the time points indicated in the evaluation schedule (table 10) for pharmacokinetic analysis. The blood sample should be collected via a peripherally placed intravenous cannula or by direct venipuncture.

Blood should not be drawn from the arm or well used to study drug infusion.

Bioassays of TAb, ADC and MMAE in serum or plasma were performed at a bioanalytical laboratory specified by the sponsor using a validated method.

6.1.6.5 Security assessment

(i) Vital sign

Vital signs including systolic and diastolic blood pressure (mmHg), radial pulse rate (heart beats/min) and temperature will be obtained and recorded according to the assessment schedule (table 10). All vital sign measurements will be obtained with the subject in a sitting or supine position.

If clinically significant vital sign changes compared to baseline (pre-treatment) were noted, the changes would be recorded as AEs on the AE page of eCRF. Clinical significance will be defined as a change in vital signs with a medical relevance as deemed by the researcher to be likely to lead to a change in medical care. The researcher will continue to monitor the subject until the parameters return to < 1 grade or baseline (pre-treatment) values, or until the researcher decides that tracking is no longer medically necessary.

(ii) Adverse events

For information on AE collection and data handling, see section 6.1.6.6 (adverse events and other security aspects).

(a) Adverse events of possible hepatic origin

If the AE of the subject enrolled in the study and receiving the study drug is accompanied by an elevated liver function test value ((LFT), e.g., AST, ALT, bilirubin, etc.) or suspected to be due to liver dysfunction, then for detailed information on liver abnormalities, monitoring and assessment, see section 6.1.9.2 liver safety monitoring and assessment.

Subjects with liver-derived AEs with LFT abnormalities should be carefully monitored.

(iii) Laboratory assessment

Table 19 below is a table of laboratory tests to be performed during the study conducted. See assessment time course for study visit collection dates.

TABLE 19

Laboratory tests will be performed prior to dosing according to the assessment schedule and sent to a central laboratory for analysis. All screening experiments must be sent to the central laboratory, but if screening results from the central laboratory are not available at the time of planning random groupings, local laboratory results can be used to determine eligibility. In the event that the received central laboratory result after the random grouping is not within the qualifying parameters, the subject will still be considered eligible and will not be considered a protocol deviation if the local laboratory result meets the qualifying criteria. Local laboratory results supporting eligibility and dosing decisions must be entered into the clinical database. If a local laboratory is used to support dosing decisions, local laboratory tests will include whole blood count and classification, glucose, serum creatinine, ALT, and AST. In the case of multiple laboratory data over this period, the most recent data should be used. Laboratory assessments collected beyond-7 days of random grouping should be repeated. Additional assessment may be done centrally or locally to monitor AE or as required by dose adjustment requirements.

Creatinine clearance assessment or GFR estimation should be performed according to institutional guidelines prior to administration of vinflunine and EV as suggested in section 6.1.6.1 (ii) (increase or decrease in dose of study drug).

Additional laboratory tests should be performed according to institutional care standards. Clinical significance beyond the scope of laboratory study results was determined and recorded by researchers/assistant researchers as qualified physicians.

(iv) Physical examination

Standard, complete physical examination will be performed at the time of screening to assess general appearance, skin, eyes, ears, nose, throat, neck, heart vessels, chest and lung, abdomen, musculoskeletal, neurological status, mental status and lymphatic system. For subsequent and end of treatment (EOT) visits, the physical examination may be more directional, but should include examination of the lung, abdomen, skin and cardiovascular system. Physical examination will be performed at visit as outlined in the assessment schedule (table 10). Each physical examination will include body weight; height is only required at screening. If at any study visit a clinically significant deterioration of the study results from baseline was noted, the change would be recorded as AE on AE eCRF. Clinical significance is defined as any change in the outcome of a physical study with a medical relevance that can lead to a change in medical care. The researcher will continue to monitor the subject until the parameters return to < 1 grade or baseline conditions, or until the researcher decides that tracking is no longer medically necessary.

(v) Ophthalmic examination

There is a need for an ophthalmic assessment of subjects with recent ocular discomfort (within 3 months of screening). Assessment should include the following: vision, slit lamp, pressure measurement examination and distention fundus examination. Previous ophthalmic examinations performed within 3 months of screening were acceptable provided that the symptoms were not new since the start of the examination. The ophthalmic assessment during treatment should be made according to standard of care or upon clinical indication (e.g., subject developing new or worsening ocular symptoms). During the study, subjects experiencing corneal adverse events were required to undergo an EOT slit lamp examination. EOT slit lamp checks must be performed for > 4 weeks from the last dose.

Additional eye examinations were performed as indicated clinically.

(vi) Electrocardiogram

The standard 12 lead ECG will be run and rated according to the rating schedule in table 10 using the local standard procedure. Clinically significant abnormal study results should be recorded as medical history at the time of screening.

6.1.6.6 adverse events and other security aspects

(i) Definition of adverse events

AE is any adverse medical event in a subject that is temporally related to the use of a drug, whether or not it is considered to be related to a medical product. Thus, an AE may be any adverse and unexpected sign (including abnormal laboratory findings), symptom, or disease (new or exacerbated) that is temporally associated with the use of a pharmaceutical product.

To identify any events that may be related to the study procedure and that may lead to a change in the performance of the study, the sponsor collects AEs even though the subject has not received study medication. AE collection was started after signing of informed consent and will be collected until 30 days after the last dose of study medication.

(a) Abnormal laboratory study results

Any abnormal laboratory test results (e.g., hematology, clinical chemistry, or urine sample analysis) or other safety assessment (e.g., ECG, radiological scan, vital sign measurement, physical examination), including those that are worsening compared to baseline, are considered clinically significant at the medical and scientific discretion of the researcher and are not related to underlying disease, will be reported as (S) AEs.

Any clinically significant abnormal laboratory study results or other abnormal safety assessment associated with a potential disease need not be reported as (S) AE unless judged by the researcher to be more severe than expected for the subject' S condition.

Repeated abnormal laboratory tests or other safety assessments do not constitute an AE in the absence of any of the above criteria. Any abnormal test result determined to be an error need not be reported as AE.

(b) Potential cases of drug-induced liver injury

For a detailed description of drug-induced liver injury (Drug Induced Liver Injury; DILI), refer to section 6.1.9.2 liver safety monitoring and assessment. Abnormal values in aspartate Aminotransferase (AST) and/or alanine Aminotransferase (ALT) that meet the criteria outlined in section 6.1.9.2 liver safety monitoring and assessment, in the presence of other causes of liver injury, are treated as potential cases of drug-induced liver injury (potential case of hod's Law) either simultaneously or concomitantly with abnormal elevation of total bilirubin, and are always treated as important medical events and reported according to section 6.1.6.6 (v) (report of serious adverse events).

(c) Disease progression and study endpoint

According to this scheme, the following events will not be considered as (S) AEs:

● Disease progression: events that included defined study endpoints that clearly met the expected progression pattern of the underlying disease were not recorded as AEs unless death was caused. These data will be retrieved as efficacy assessment data as outlined in section 6.1.6.3 efficacy assessment. If there is any uncertainty as to whether the event is due to the expected disease progression and/or if there is evidence that there is a causal relationship between the study drug and the event, it should be reported as (S) AE. All deaths within 30 days of the last dose of study drug must be reported as SAE.

● Pre-planning and selection of hospitalization or procedures for diagnostic, therapeutic or surgical procedures of pre-existing lesions that do not deteriorate during the course of clinical trials. These procedures were collected according to the eCRF completion guideline.

(ii) Definition of Serious Adverse Events (SAE)

An AE is considered "severe" if it appears to the researcher or sponsor that it results in any of the following results:

● Resulting in death of

● Life threatening (an AE occurrence places a subject at immediate risk of death if it appears to a researcher or sponsor, it is considered "life threatening". It does not include AEs that would cause it to appear in a more severe form, possibly leading to death)

● Resulting in a persistent or significant disability/disability or substantial disruption of the ability to perform normal vital functions

● Causes congenital anomalies or birth defects

● Hospitalization is required (except for the planned procedure allowed according to the study) or prolonged (except for the prolonged planned hospitalization not caused by AE). Hospitalization for treatment/observation/examination caused by AE is considered serious. )

● Other medically significant events (defined in the following paragraphs)

In deciding whether to expedite notification of suitability for other situations, medical and scientific judgment may be employed, such as important medical events that may not immediately endanger life or result in death or hospitalization but may endanger the subject or may require intervention to prevent one of the other outcomes listed in the definition above. These events, including those that may lead to disablement/disablement, are generally considered severe. Examples of such events are emergency room or home emergency treatment of allergic bronchospasm; does not cause hospitalized blood cachexia or convulsion; or drug dependence or development of drug abuse.

The sponsor has an event list that classifies it as a "always serious" event. If an AE of an event deemed "always serious" according to this classification is reported, other information about the event may be requested.

(iii) Criteria for causal relationship with study drugs

Researchers are obligated to assess the relationship between study drug and each occurrence of each (S) AE. The researcher will use clinical judgment to determine the relationship. Researchers should also use the information provided herein and/or product information for commercially available products. The investigator was asked to provide an explanation of the causal relationship assessment for each SAE and must record this on the SAE worksheet.

Causal relationship assessment is one of the criteria used in determining regulatory reporting requirements. The researcher may correct his/her causality assessment based on the new information about SAE and should send SAE tracking reports and update eCRF with the new information and updated causality assessment.

After review of the relevant data, the causal relationship between the study drug and each (S) AE will be determined by the question "is there a reasonable likelihood that you think that the event might be caused by the study drug? The answer is assessed either by "yes" or "no".

When performing causal relationship assessment, the following factors should be considered when deciding whether evidence exists and/or whether a (S) AE might be caused by the study drug to have a 'reasonable likelihood' (rather than an unexclusive relationship) or whether evidence exists to reasonably reject causal relationships:

● Exposure to study drug and onset and/or regression of (S) AE. Is the subject actually received the study drug? Is there a reasonable time relationship between (S) AE occurrence and study drug administration?

● Rationality; that is, the event may be caused by the study drug? Biological and/or pharmacological mechanisms, half-life, literature evidence, drug classes, preclinical and clinical study data, and the like are considered.

● Discontinuation (dechange)/dose reduction/recovery (rechange):

is o (S) AE resolved or improved after stopping administration or reducing the dose of the suspected drug? The effect of treatment on the event at the time of assessing withdrawal from the dosing experience is also considered.

Is (S) AE relapsed when a suspected drug is reintroduced after having stopped?

● Laboratory or other test results; assessment of the relationship between (S) AE and study drug (e.g. based on values before, during and after treatment) is supported by the particular laboratory study

● Independent of the available alternative explanation for drug exposure; such as other concomitant medications, past medical history, concurrent or potential diseases, risk factors including medical and family medical history, seasons, location, etc., and strength of alternate interpretation

There may be situations where SAE occurs and the researcher has minimal information to include in the initial report of the sponsor. However, it is very important that researchers consistently assess the causality of each event before the SAE data is initially transmitted to the sponsor. The causal relationship assessment of 'no' is considered with limited or insufficient information about the event for which an informed decision is made and without any indication or evidence of the establishment of causality. In such cases, it is contemplated that researchers may obtain other information about the event as soon as possible and re-evaluate the causal relationship after receiving the other information.

(iv) Criteria defining severity of adverse events

AE including abnormal clinical laboratory values will be ranked using NCI-CTCAE guidelines (version 4.03). Items not specified in NCI-CTCAE version 4.03 will be rated according to the criteria in table 20 below and entered into eCRF.

Table 20

Level of	Evaluation criterion
		1-Mild degree	Note asymptomatic or mild symptoms, clinical or diagnostic observations; no intervention is indicated.
2-moderate degree	Indicating a local or non-invasive intervention.
		3-severe degree	Medically significant but not immediately life threatening, hospitalizing or prolonged hospitalization.
4-life threatening	Life threatening outcome, indicative of emergency intervention
		5-death	Death associated with AE

Researchers will rate the severity of each adverse event using the following definition

● Light: undisturbed normal daily activities

● And (3) moderately: influence on normal daily activities

● Severe: cannot perform daily activities

(v) Reporting of serious adverse events

The collection of AEs and accelerated notification of SAE will begin after receiving informed consent and will continue until 30 days after the last administration of study drug.

In the case of SAE, the researcher must immediately (within 24 hours of awareness) contact the sponsor by fax or email.

The researcher must complete the SAE worksheet containing all the information required by local and/or regional regulations and submit it to the sponsor immediately (within 24 hours of awareness) by email or facsimile. If the SAE worksheet is not possible or fax is not possible within 24 hours, the local drug safety contact should be told by telephone.

The following minimum information is required:

● International study number (International Study Number; ISN)/study number,

● The number, sex and age of the subject,

● The date of the report is given,

● SAE (event, severity criteria) description,

● Causal relationships (including causes) with study drugs, and

● Provided medicament (if present)

(vi) Tracking of adverse events

All AEs occurring during or after the subject has discontinued the study will be tracked until regression or decision is no longer clinically significant, or until it becomes chronic to the point that it can be adequately characterized by the researcher.

If after a protocol-defined AE collection period (see section 6.1.6.6 (i) definition of adverse events), the AE progresses to SAE or the researcher learns of any (S) AE (including death) he/she thinks there is a reasonable likelihood that it is relevant to study medication or study participation, the researcher must notify the sponsor in real time.

(vii) Monitoring of common severe adverse events

Common SAE are those that are normally expected to occur in the study population but are not related to drug exposure. SAE classified as "common" are provided in section 6.1.9.3, reference to common severe adverse events. The list does not change the researcher's reporting obligation, nor does it change its obligation to perform causal relationship assessment, or prevent reporting from meeting the AE requirements of the SAE definition as detailed above. The purpose of this list is to alert researchers that some events reported as SAE may not require expedited notification to regulatory authorities based on the "common SAE" classification as specified in chapter 6.1.9.3 for serious adverse events. The sponsor will monitor any frequency changes of these events throughout the study. Any change to this list will be communicated to the sites participating in the clinical trial. Researchers must report the individual occurrence of serious adverse events as set forth in section 6.1.6.6 (v) report of these events.

(viii) Special cases

Certain special cases observed in connection with study drug (e.g., incorrect administration (e.g., incorrect dose of study drug, comparator, or background therapy) are collected in eCRF as a protocol deviation from the chapter 6.1.8.8 significant protocol deviation, or may require special reporting, as described below.

If a special case is associated with or results in an AE, the AE will be assessed separately from the special case and captured as AE in the eCRF. If AE meets the SAE definition, SAE reports as described in section 6.1.6.6 (v) report of serious adverse events, and details of the relevant special case will be included in the clinical description on the SAE worksheet.

The special cases are:

● Pregnancy with a new pattern of medicine

● Misuse and overdose

● Misuse/abuse

● Occupational exposure

● Suspected drug-drug interactions

(a) Pregnancy with a new pattern of medicine

If a female subject is pregnant during the study dosing period or within 6 months of discontinuing dosing, the researcher will report information to the sponsor using the pregnancy report form according to the timeline in the report of severe adverse events of chapter 6.1.6.6 (v) and in eCRF.

Researchers will attempt to collect pregnancy information for any female partner of a male subject pregnant during the study dosing period or within 6 months of discontinuation of dosing and report the information to the sponsor using a pregnancy report form according to the timeline in the report of severe adverse events in section 6.1.6.6 (v).

The date of the expected or end of pregnancy, the last menstruation, estimated pregnancy date, pregnancy outcome and neonatal data etc. should be included in this information.

Although pregnancy itself is not considered AE or SAE, any pregnancy complications or termination (including selective termination) of pregnancy in female study subjects will be reported as AE in eCRF or as SAE according to section 6.1.6.6 (v) as a report of serious adverse events. For (S) AEs experienced by the female partner of the male subject, the (S) AEs were reported via a pregnancy report form.

Other information about pregnancy outcomes when also classified as SAE are as follows:

● "spontaneous abortion (Spontaneous abortion)" includes miscarriage (miscarriage), abortion (abortions) and miscarriage (abortions).

● Neonatal or infant death within 1 month after birth will be reported as SAE, regardless of its relationship to study drug.

● If the infant dies more than 1 month after birth, a relationship between reported death and intrauterine exposure to study medication is judged "possible" by the researcher.

● Congenital anomalies (including anomalies in aborted fetuses)

Unless congenital anomalies are identified prior to spontaneous abortion or miscarriage, congenital defects in embryos or fetuses should be assessed by visual inspection. The (S) AE experienced by the neonate/infant should be reported via a pregnancy report form. Generally, the tracking will be no longer than 6 to 8 weeks after the expected date of delivery.

(b) Misuse, overdose and "Off-Label Use"

If medication errors, overdosing, or "off-indication use" (i.e., use beyond what is stated in the regimen) are suspected, reference is made to section 6.1.8.8 for significant regimen deviations. Any relevant (S) AE should be reported in eCRF. If AE meets the SAE definition, SAE should also be reported as described in section 6.1.6.6 (v) report of serious adverse events, along with details of medication errors, overdosing, or "off-indication use".

In the event of suspected EV overdosing or accidental infusion in the form of bolus injection, the subject should receive supportive care and monitoring. The medical monitor/expert should be contacted, as appropriate.

No specific procedure is available for the treatment of EV overdose or accidental infusion in the form of bolus injections, and only supportive treatment can be given. If the EV is accidentally overdosed or accidentally infused as a bolus, the researcher/assistant researcher will provide emergency procedures and/or general maintenance therapies to ensure the safety of the subject based on the symptoms.

In the case of suspected overdosing of paclitaxel, docetaxel or vinflunine, reference is made to approved drug instructions, smPC or local product information provided by the manufacturer for each agent.

Overdosing events should be recorded in eCRF together with the actual dose administered.

(c) Misuse/abuse

If misuse or abuse of the study medication is suspected, the researcher must immediately (within 24 hours of awareness) deliver the special case worksheet to the sponsor by fax or email. Any relevant (S) AE should be reported in eCRF. If AE meets the SAE definition, SAE should also be reported as described in section 6.1.6.6 (v) report of serious adverse events and details of misuse or abuse of study drugs.

(d) Occupational exposure

If occupational exposure to the study medication occurs (e.g., inadvertent exposure to the study medication while the on-site staff is ready for administration to the patient), the researcher must immediately (within 24 hours of awareness) deliver the special case worksheet to the sponsor by fax or email. Any relevant (S) AEs that are relevant to or that cause the occurrence of a particular situation in the subject should be reported on the special situation worksheet.

(e) Suspected drug-drug interactions

If drug-drug interactions associated with the study drug are suspected, the researcher must immediately (within 24 hours of awareness) deliver the special case worksheet to the sponsor by fax or email. Any relevant (S) AE should be reported in eCRF. If AE meets the SAE definition, the SAE should also be reported as well as details of suspected drug-drug interactions as described in section 6.1.6.6 (v) report of serious adverse events.

(ix) New information for research of supply influence

When new information needed to conduct a clinical study becomes available appropriately, the sponsor will inform all researchers and authorities participating in the clinical study. Researchers should inform IRBs/IEC of such information when needed.

The researcher will also inform the subject that the subject will need to sign updated informed consent in order to continue the clinical study.

(x) Deviations in protocols and other actions taken to avoid risk of life of the subject

Researchers cannot deviate from the protocol except for emergency situations to avoid subject risk. When a researcher does not follow a regimen in order to avoid a subject's risk of urgency, the researcher should take the following actions.

1. The content of the deviation and its cause are described in the written notice and immediately sent to the sponsor and the research site responsible for the delivery of the file that sets forth the deviation or correction and cause. A copy of the notification is maintained.

2. The earliest possible business with the sponsor is about the need for a revision scheme. Approval for the draft of the revised regimen was obtained from IRB and study site responsible and written approval from sponsor.

6.1.6.7 test drug concentration

Blood samples for PK and ATA will be collected throughout the study according to the sample collection schedule provided in table 11. The concentrations of ADC, total antibodies (TAb) and MMAE in serum or plasma will be measured using a validation assay. PK samples will be collected and archived for possible analysis of other EV-related species. Verification assays will also be used to determine ATA levels in serum.

For information on the collection, handling, storage and transport of samples, reference is made to laboratory manuals.

6.1.6.8 other measurements, assessments or methods

(i) Exploratory biomarkers

Procedures for collecting, handling and transporting samples will be specified in the laboratory manual.

The biomarkers in the blood of section 6.1.6.8 (ii) and 6.1.6.8 (iii) other biomarkers of the sample described in the biomarkers in pre-treatment tumor tissue, including DNA, RNA, and protein, can be analyzed to investigate possible relationships with the resistance or sensitivity mechanisms to the study treatment, dynamic changes associated with the study treatment, and methods of diagnostic assays associated with the study treatment.

After the database is closed, the samples may be stored at the facility of the research sponsor or at the contract laboratory facility for up to 15 years, at which point the samples will be destroyed.

(ii) Biomarkers in blood

Plasma and Peripheral Blood Mononuclear Cell (PBMC) samples collected at baseline and post-baseline time points can be analyzed for markers of immune function, immune cell subsets, and cytokines. Plasma and PBMC samples can be used for additional exploratory analysis as described in section 6.1.6.8 (i) exploratory biomarkers.

(iii) Biomarkers in pre-treatment tumor tissue

Submission of unstained charged slides (at least 10 and up to 15 slides) to FFPE tumor mass or fresh sections at screening should be provided (unless prior approval is obtained from the sponsor). Archiving tissues or pre-treatment fresh tumor tissues (obtained from fresh biopsies) is acceptable. For details, see the laboratory manual. Samples of tumor tissue can be analyzed for connexin-4 expression, markers for disease subtypes, and markers associated with tumor immune microenvironment. Tumor tissue samples can be used for additional exploratory analysis as described in section 6.1.6.8 (i) exploratory biomarkers.

(iv) Blood samples for future pharmacogenomic analysis (retrospective)

Pharmacogenomics (PGx) studies can be performed in the future to analyze or determine genes associated with clinical response, pharmacokinetics, and toxicity/safety issues. After random grouping (see evaluation time course), 4mL whole blood samples were collected for possible retrospective PGx analysis. The sample is transported to the sponsor-specified reserve CRO.

Details about the sample collection, labeling, storage and shipping procedures will be provided in the separate laboratory manuals.

For additional details on the reserve procedure, see section 6.1.9.4 retrospective PGx.

(v) EORTC core quality of life questionnaire QLQ-C30

QLQ-C30 was developed to measure QOL aspects associated with patients with a wide range of cancers who participated in clinical trials. Sneeuw KC et al, J Clin epidemic 1998;51:617-31; aaronson NK et al, jnattl Cancer inst.1993;85:365-76. The core scale of the current version (QLQ-C30, version 3) is a 30-item questionnaire consisting of:

● Functional domains (body, role, cognition, emotion, social);

● 3 symptoms scales (fatigue, pain, nausea and vomiting);

● Individual projects for symptoms (shortness of breath, loss of appetite, sleep disorders, constipation, diarrhea) and financial impact of the disease; and

● 2 global items (health, overall QOL).

(vi) EuroQOL-5 dimensions

EQ-5D-5L is a standardized tool developed by the EuroQOL group for use as a general, preference-based measure of health. It is applicable to numerous health conditions and treatments and provides a simple descriptive overview and a single index value for the health condition. EQ-5D-5L is a 5-self-reported metric for function and health, which assesses 5-dimensional health including mobility, self-care, daily activity, pain/discomfort, and anxiety/depression. Each dimension contains 5 levels (no problem, minor problem, moderate temperature, severe problem, extreme problem). The unique EQ-5D-5L health status is defined by combining 1 hierarchy for each of the 5 dimensions. The questionnaire also records the respondents' self-scoring health status on a visual analog scale with vertical scale (0 to 100). The reactions to the 5 items were also converted to weighted health status indices (utility scores) based on values derived from the general population samples. Herdman M et al, qual Life res.2011;20 (10):1727-36.

(vii) Medical resource utilization (HRU)

HRU information will be collected, particularly focusing on the number of subjects scheduled to use medical resources related to clinical events or AEs for all subjects (section 6.1.9.7).

6.1.6.9 total blood volume

The total amount of blood collected for study assessment for each subject will vary depending on how long the subject remains treated.

At any time during the study, if any laboratory abnormalities in the subject or disease assessment are found, additional blood may be drawn for monitoring.

Additional blood beyond standard monitoring to be drawn for this study will include drawing for: qualification evaluation; hematology and chemistry assessments at defined time points for a particular study; pharmacokinetics; and bioanalytical sampling.

The maximum blood volume collected was about 130.0mL in cycle 1 for subjects randomized to group a and 37.0mL in cycle 1 for subjects randomized to group B. The maximum blood volume collected for subjects participating in cycles 2 to 6 and completing EOT visit was 219.0mL for subjects randomized to group a and 164.0mL for subjects randomized to group B.

6.1.7Interrupt

Interruption of 6.1.7.1 individual subjects

Discontinuing treatment is a subject enrolled in the study and permanently discontinuing study treatment for any reason.

The subject may freely discontinue study treatment and/or withdraw from the study for any reason and at any time, without giving the reason to do so and without penalty or bias. The researcher may also freely discontinue the subject from study treatment at any time or terminate the subject from participating in the study if the subject's clinical condition permits.

All subjects who discontinued study treatment will remain in the study and must continue to track the regimen-specific tracking procedure as outlined in the assessment schedule (table 10) until the subject explicitly withdraws consent to any further communication with the person he/she or the participant previously authorized to provide this information.

If the subject discontinued the study with a sustained AE or outstanding laboratory results that were significantly outside of the reference range, the researcher would attempt to provide tracking until the condition was stable or no longer clinically significant.

The following are treatment disruption criteria for individual subjects:

● The subject developed a radiological disease progression.

● The subject developed unacceptable toxicity.

● The female subject is pregnant.

● The investigator decides that discontinuing treatment is of optimal benefit to the subject

● The subject refuses further treatment.

● Death.

Subjects who discontinued study medication before reaching PFS1 and entered a post-treatment follow-up period will discontinue the post-treatment period if any of the following occurs:

● Based on the assessment of the investigator, the subject developed a radiological progressive disease (i.e., PFS 1)

● The subject initiates a new systemic anticancer therapy (first-line anticancer therapy after discontinuing study drug)

● Death of

● Subjects refused to participate in further studies (i.e., withdraw consent)

● Despite reasonable efforts by researchers to locate subjects, subjects remain out of visit

The subject will discontinue the long-term tracking period (for PFS 2) if any of the following occurs:

● The subject initiates a new systemic anticancer therapy (two-line anticancer therapy after discontinuing study drug)

● Based on the investigator assessment, the subject presented evidence of PD

● Subjects refused to participate in further studies (i.e., withdraw consent)

● Death of

● The sponsor ends the long-term trace collection period

The subject will discontinue the survival tracking period if any of the following occurs:

● Subjects refused to participate in further studies (i.e., withdraw consent)

● Death of

● The sponsor ends the survival tracking collection period

6.1.8Statistical method

Statistical analysis plans (Statistical Analysis Plan; SAP) will be written to provide analytical details, as well as specifications for tables, lists, and drawings to be prepared. SAP will be completed prior to enrollment in the first subject. Any changes in the planned analysis in SAP will be accommodated in the clinical study report.

Generally, continuous data will be summarized using descriptive statistics (subject number, mean, SD, min, median, and max) and frequency and percentage of category data.

6.1.8.1 sample size

Approximately 600 subjects were randomized into 2 treatment groups at a 1:1 ratio: EV (group a) and chemotherapy (group B). Random groupings will be stratified by baseline ECOG PS (0 and 1), world area (western europe, the united states, or elsewhere in the world), and liver metastasis (yes or no). Assuming a 10% decrease rate for hr=0.75 (median OS in groups a and B of 10.7 months and months, respectively), this sample size would provide 85% assay force to detect statistically significant differences at an overall type I error rate of 0.025 on one side at the final analysis at the planned 439 death events and 1 interim analysis at 65% of the total planned events (285 death events).

Determining the sample size by the primary endpoint OS; 600 subjects will provide assay force exceeding 90% to detect statistically significant differences with respect to the selected secondary endpoint: PFS1 (assuming median PFS1 in group a and group B for 6 months and 4 months, respectively), ORR and DCR (assuming 15% treatment difference in ORR and DCR between group a and group B).

6.1.8.2 analysis set

The detailed criteria of the analysis set will be arranged in SAP or classification specifications and the assignment of subjects to the analysis set will be determined before the database is hard locked.

(i) Full analysis set

The full analysis set (full analysis set; FAS) will consist of all subjects randomized. This analysis set is compliant with the Intent To Treat (ITT) principle, which includes all randomly grouped subjects, FAS being equivalent to ITT population. This will be a preliminary analysis set of efficacy analyses, except for response-related efficacy endpoints.

(ii) Security analysis set

The safety analysis Set (SAF), which consists of all subjects receiving any amount of study drug, will be used for safety analysis.

(iii) Reaction sets can be evaluated

The response set (response evaluable set; RES) can be assessed as all subjects in FAS and with measurable disease at baseline and tracked for at least 6 months after random grouping. RES will be used for primary efficacy analysis of reaction related endpoints (e.g., ORR and DCR).

(iv) Pharmacokinetic analysis set

Pharmacokinetic analysis set (PKAS) includes a subject receiving an active drug, to whom at least one blood sample is collected and to which measurement of the TAb, ADC and MMAE serum/plasma concentrations is determined, and the sampling time and dosing time of day of sampling are known.

6.1.8.3 demographic data and baseline characteristics

Demographic data and baseline characteristics will be summarized in terms of treatment groups and population. The ethnic sources will only be summarized in the demographic table. Descriptive statistics will include the number of subjects, the average value of consecutive endpoints, standard deviation, minimum, median and maximum values, and the frequency and percentage of category endpoints.

(i) Subject configuration

The number and percentage of subjects completing and interrupting treatment and the reason for interrupting treatment will be presented for all randomized groups of subjects and subjects in the SAF, by treatment group and population. A similar table for screening configuration, time period configuration for study, and tracking configuration will also be presented for all randomized, grouped subjects by treatment group and population. All configuration details and the date of the first and last evaluation of each subject will be enumerated.

(ii) Previous and concomitant medications

All previous and concomitant medications will be presented in the list. The frequency of concomitant medications (prescriptions, non-prescriptions and nutritional supplements) will be summarized in terms of Preferred Terms (PT) for the treatment group and SAF. The drugs will be encoded using the WHO drug dictionary. The medications will be counted by the number of subjects taking each medication. Subjects taking the same drug multiple times will only count one time for that drug. Drugs will be presented in decreasing frequency order based on the total number of subjects taking each drug.

(iii) Medical history

The medical history of each subject will be presented in a list. The detailed medical history for each subject will be obtained during the screening period and will be summarized in terms of treatment groups and overall.

6.1.8.4 efficacy analysis

Efficacy analysis of OS and PFS was performed for FAS. Results of the statistical test are interpreted based on FAS. Efficacy analysis of response-related endpoints, such as ORR and DCR, were performed for RES.

The family I error rate of this study was strongly controlled at 2.5% (single sided), which allowed the study to state that it was positive for the primary endpoint OS of the FAS population. The OS was formally tested at both mid-term and final analysis. At mid-term or final analysis, the grading (according to the order PFS1- > ORR- > DCR) performs a formal hypothesis test for the selected secondary endpoints (including PFS1, ORR, and DCR) only when the OS analysis is rejected. Details about the significance level at the mid-term and final analysis of each efficacy endpoint (OS, PFS1, ORR, and DCR) are illustrated in SAP.

(i) Analysis of primary endpoints

(a) Preliminary analysis

The primary efficacy endpoint of OS was analyzed using a log rank test layered by ECOG PS (0 and 1), world area (western europe, the united states, or elsewhere in the world), and liver metastasis (yes or no). The analysis was performed using randomized treatment and layers for randomized grouping. In addition, a hierarchical Cox proportional hazards model was used to estimate the risk ratio and corresponding 95% confidence interval. The median OS was estimated using Kaplan-Meier method and will be reported as treatment group with the corresponding 95% confidence interval.

Preliminary analysis was performed using FAS.

(b) Sensitivity analysis

Additional analysis may be performed as appropriate, such as an non-layered log rank test to adjust for cross-effects and OS analysis. Details are illustrated in SAP.

(ii) Analysis of secondary endpoints

(a) Progression free survival

For each subject, PFS1 was defined as the time from the day of random grouping until the day of radiological disease progression (according to RECIST V1.1) or until death due to any cause. If the subject is neither progressing nor dying, the subject is checked on the last radiological assessment date (cenored). Before the initiation of an anti-cancer therapy, subjects receiving any other anti-cancer therapy for the disease prior to radiological progression are examined at the final radiological assessment day.

Efficacy endpoints of PFS1 were analyzed using a log rank test layered by ECOG PS (0 and 1), world area (western europe, the united states, or elsewhere in the world), and liver metastasis (yes or no). The analysis was performed using randomized treatment and layers for randomized grouping. In addition, a hierarchical Cox proportional hazards model was used to estimate the risk ratio and corresponding 95% confidence interval. Median PFS1 was estimated using Kaplan-Meier method and reported as treatment group with corresponding 95% confidence interval.

Preliminary analysis was performed using FAS. Additional sensitivity assays for PFS1 were also performed. Details are illustrated in SAP.

(b) Total reaction rate

Total response rate (ORR) is defined as the proportion of subjects with complete or partial objective response based on RECIST V1.1. ORR between groups a and B were compared using a layered CMH test. The same layering factors used in event analysis time will be used for layered CMH verification. The difference in response rate between treatment groups and corresponding 95% confidence intervals were estimated. In addition, the ORR for each group is estimated and the corresponding 95% confidence interval will be constructed. Preliminary analysis was performed using RES.

(c) Duration of reaction

Duration of response (DOR) is defined as the time from the day (followed by confirmation, as assessed by a researcher) of first response CR/PR (according to RECIST V1.1) to the day of radiological progression or death in subjects who reached CR or PR. If the subject is not progressing or dying, the subject is examined on the day of the last radiological assessment or on the day of the first CR/PR (if no other post-baseline radiological assessment is available after the first CR/PR). Median DOR was estimated using the Kaplan-Meier method and reported as treatment group with the corresponding 95% confidence interval.

(d) Disease control rate (Disease Control Rate; DCR)

DCR is defined as the proportion of subjects with complete or partial objective response or stable disease based on RECIST V1.1. DCR between groups a and B were compared using a layered CMH test. The same layering factors used in event analysis time are used for layered CMH verification. The difference in response rate between treatment groups and corresponding 95% confidence intervals were estimated. In addition, the DCR for each group is estimated and the corresponding 95% confidence interval will be constructed. Preliminary analysis was performed using RES.

(e) qOL and PRO parameters

The FAS was subjected to descriptive QOL and PRO analyses. The completion rate of each questionnaire is summarized. Additional analysis is discussed in detail in the statistical analysis plan.

(iii) Subgroup analysis

Analysis for OS, PFS1 and ORR was repeated with ECOG PS (0 and 1), world area and liver metastases, respectively, using FAS.

In addition, subgroup analysis is performed for the selected endpoint to determine whether the therapeutic effect is constant. Subgroups may include, but are not limited to, the following:

● Age group (< 65 years and > 65 years; <75 years and > 75 years)

● Sex (female and male)

● Previous platinum (cisplatin and carboplatin and both)

● The number of previous systemic therapy lines in locally advanced or metastatic background (1 line to 2 lines and ≡3 lines)

● Optimal response to recent CPI (responders and non-responders) CPI recent treatment (yes or no)

● Baseline hemoglobin (greater than or equal to 10 and less than 10 g/dL)

● Histological (urothelial carcinoma/transitional cell and mixed urothelial carcinoma and others)

● Primary tumor site (upper urinary tract and bladder/other)

● Smoking status (never and once and current)

● Brain transfer state (previous brain transfer and previous no brain transfer)

● Choice of researchers for paclitaxel/docetaxel or vinflunine

● Baseline connexin-4 IHC score (< 150 and 150-225 and > 225)

● Previous taxane (yes or no)

● PD-L1 CPS (< 10 and > 10)

(iv) Analysis of exploratory endpoints

The exploratory analysis of efficacy endpoints is discussed in the statistical analysis program.

Serum or plasma TAb, ADC and MMAE concentrations were summarized using PK analysis sets with descriptive statistics for each PK sampling time point. These data can be combined with data from prior studies of population PK and PK/PD analysis. The relationship between TAb, ADC, MMAE and PD endpoint, safety or efficacy was explored.

6.1.8.5 the incidence of ATA is summarized per visit and overall using a security analysis set.

6.1.8.6 Security analysis

SAF is used to conduct all security analyses. All treated subjects were analyzed according to the treatment they received.

(i) Adverse events

AE is encoded using the medical dictionary for medicine management (MedDRA).

TEAE is defined as AE observed or worsened after the start of administration of study drug.

The number and percentage of subjects with treatment-induced AEs, SAE, treatment-stopped AEs and study drug-related AEs are summarized by system organ category, preferred terminology and treatment group. The number and percentage of AEs were also summarized in terms of severity. All AEs are listed.

Study drug-related TEAE is defined as any TEAE that a researcher considers causal. Also outlined are AEs of interest as classified by custom MedDRA query and/or standard MedDRA query.

(ii) Laboratory assessment

For quantitative laboratory measurements, the results of subjects in SAF and their changes from baseline were summarized by treatment group and time point using descriptive statistics.

The change in the normal range from baseline to each time point during treatment in laboratory tests was also tabulated. Laboratory data are shown in the list.

(iii) Vital sign

With descriptive statistics of subjects in SAF, vital sign results and changes from baseline at scheduled visits were summarized by treatment group. Vital sign data is displayed in a list.

(iv) Conventional 12-lead electrocardiogram

12-lead ECG results were summarized by treatment group and time point.

(v) ECOG behavioral state

Summary statistics (number and percentage of subjects) for each class of ECOG PS at each assessment are provided. The change from baseline to final visit or early termination is also summarized. A negative change score indicates improvement. A positive score indicates performance degradation.

(vi) Exposure-response relationship analysis

The relationship between TAb, ADC and MMAE concentrations and certain efficacy or safety endpoints can be analyzed in a exploratory manner. Additional details of these assays are described in the exposure-response assay program.

6.1.8.7 pharmacokinetic analysis

Individual and summary tables of serum TAb and ADC and plasma MMAE concentrations are provided, as well as a list of blood collection times and concentrations.

Summary statistics are provided, including n, mean, SD, geometric mean, minimum, median, maximum, and CV%. If all values are below the lower quantification limit (below the lower limit of quantification; BLOQ), the BLOQ value is set to not calculated. In the case that more than half of the individual data in the group are BLOQ, the geometric mean is not calculated. Additional model-based analysis may be performed and described in the individual population PK analysis program.

6.1.8.8 significant protocol bias and other analysis

All randomized group subjects will be summarized by treatment group and population as well as by location for significant protocol bias as defined by chapter 6.1.8.8 significant protocol bias. The data list will be provided by location and subject.

Significant solution bias criteria will be specifically identified in summary tables and lists. The unique identifier will be as follows:

● PD 1-entry study, even if it does not meet the entry criteria,

● PD 2-reached (Developed) exit criteria during the study and did not exit,

● PD 3-received either incorrect treatment or incorrect dosage,

● PD 4-receives concomitant therapy with exclusion.

6.1.8.9 metaphase analysis (and early interruption of clinical studies)

Mid-plan efficacy analysis occurs after about 285 OS events (about 65% of total planned events) are observed. The OS will test at a single-sided 0.00541 significant level according to the O' Brien-Fleming boundary as implemented by Lan-detens alpha consumption function. Lan KKG, deMets DL. Biometrika.1983;70:659-63.IDMC may suggest to terminate the trial in mid-term analysis based on statistically significant OS results favoring EV. When total mortality reached 439, the final OS analysis will be performed at a single-sided 0.02332 significance level. If the exact number of events at the mid and final analysis is different from the plan, the significance level is adjusted accordingly based on the O' Brien-Fleming method and the Lan-DeMets alpha consumption function.

The interim analysis will be performed by IDAC and reviewed by IDMC. In addition, security data review during the trial will be performed periodically by the IDMC. For example, IDMC will review safety data after the first 50 subjects have been randomized and taken study medication for about 3 months. The complete procedure for IDMC security review and interim analysis will be described in the individual IDMC privilege and interim analysis plans.

6.1.8.10 treatment of missing data, outliers, access windows and other information

The calculation method of missing data and the definition of windows to be used for analysis will be summarized in the SAP, if applicable.

6.1.9Appendix of clinical study

6.1.9.1 alert list of concomitant medications

The following list describes drugs and foods in the form of common strong inhibitors/inducers of CYP3A, CYP2C8 and P-glycoprotein (P-gp) inhibitors that should be avoided, carefully used or closely monitored. This list should not be considered to include all; individual medication labels are queried for specific information. If there is concern or problems with concomitant use of any of the drugs listed below, discussion with the sponsor is encouraged.

Table 22 overview of possible drug responses

EV: enrolment Shan Kangwei spines; p-gp: p-glycoprotein

Other information on inhibitors/inducers can be found in FDA guidelines (drug interactions and labels). fda. Gov/Drugs/development appurvalprocess/development resources/drug interactionsLabeling/ucm093664.Ht m# table5-2

6.1.9.2 liver safety monitoring and assessment

Clinical studies enrolled with active drug therapy and revealed that any subject whose serum transaminase (AT) increased to >3×uln (in subjects with liver metastasis, >5×uln) or bilirubin >2×uln should undergo detailed testing for liver enzymes, including AT least ALT, AST, ALP and TBL. The test should be repeated within 72 hours of informing the test results. For studies using a central laboratory, alerts regarding moderate and severe liver abnormalities were generated by the central laboratory to inform researchers, study monitors, and study groups. If the subject has any symptoms suggesting liver and gall dysfunction, the subject should be queried.

(i) Definition of liver abnormalities

The confirmed abnormalities will be characterized as moderate and severe if ULN is:

in addition, for any of the following, the subject should be considered to have severe liver abnormalities:

● ALT or AST >8 XULN.

● ALT or AST >5×uln for more than 2 weeks (in the absence of liver metastasis).

● ALT or AST >3 XULN and International normalized ratio (International Normalized Ratio; INR) >1.5 (if INR test is applicable/evaluated).

● ALT or AST >3 XULN, with concomitant fatigue, nausea, vomiting, pain or tenderness in the upper right abdomen, fever, rash and/or eosinophilia (> 5%).

Researchers may decide that abnormal liver function results other than those described above may be considered moderate or severe abnormalities and require additional monitoring and tracking.

(ii) Tracking program

The confirmed moderate and severe liver function abnormalities should be well characterized by obtaining appropriate expert consultations, detailed related medical history, physical examinations and laboratory tests. The venue should complete a liver abnormality case report form (LA-CRF) that has been conducted worldwide and can be activated for any study or appropriate documentation. Subjects with confirmed abnormal liver function tests should be tracked as described below.

If abnormal stabilization occurs or study medication has been discontinued and the subject is asymptomatic, the LFT for the confirmed moderate abnormality should be repeated 2 to 3 times weekly, followed by once weekly or less.

In the absence of another etiology, severe liver function abnormalities as defined above may be considered an important medical event and may be reported as SAE. The initiator should be contacted and all subjects notified that severe liver dysfunction, possibly attributable to the study drug, was observed.

To further assess abnormal liver laboratory findings, researchers are expected to:

● A more detailed history of symptoms and previous or concurrent disease is obtained. Symptoms and new onset disease are recorded as "AE" in (e) CRF. Care should be taken to avoid pain and illness that may lead to secondary liver abnormalities, such as hypotensive events and decompensated heart disease. Nonalcoholic steatohepatitis is found in obese hyperlipoproteinemia and/or diabetic patients and may be associated with fluctuating AT levels. The researcher should ensure that the medical history form captures any afflictions prior to study enrollment that may be relevant in assessing liver function.

● A history of concomitant medication use (including over-the-counter, supplemental and replacement medications), drinking, recreational use, and special diets is obtained. Drug (including dose) is infused (e) into the CRF. Information about alcohol, other substance use and diet should be entered on LA-CRF or an appropriate file.

● A medical history of exposure to the environmental chemistry is obtained.

● Based on the subject's medical history, other tests may be appropriate, including:

acute viral hepatitis (type A, type B, type C, type D, type E or other infectious agents),

ultrasound or other imaging to assess biliary disease,

Other laboratory tests, including INR, direct bilirubin.

● Taking into account gastrointestinal or liver study consultation.

● The results of any additional tests and possible etiologies are submitted on the LA-CRF or an appropriate file.

(iii) Study disruption

In the absence of an increase in LFT, such as viral hepatitis, pre-existing or acute liver disease, the presence of liver metastasis, or exposure to other agents associated with liver damage, the subject may discontinue the study. The researcher may decide that continued research participation does not meet the best interests of the subject. The discontinuation of treatment should be considered if the following occurs:

● ALT or AST >8 XULN.

● ALT or AST >5×uln for more than 2 weeks (in subjects without liver metastasis).

● ALT or AST >3 XULN and TBL >2 XULN or INR >1.5 (if INR test is applicable/evaluated).

● ALT or AST >5 XULN and (TBL >2 XULN in patients with liver metastases).

In addition, if it is not possible to closely monitor subjects with moderate or severe liver laboratory tests, the medication should be discontinued.

* Hold's law defines:

1. Evidence that drugs can cause damage to hepatocyte types is generally shown by a higher rate of transaminase rise in humans than controls, 3 x and greater above the upper normal limit (2 x rise is too common in treated and untreated patients to distinguish).

2. Cases of bilirubin increase (to at least 2×uln) in a population accompanied by an increase in transaminase to at least 3×uln (but which is almost always higher) and no evidence of intrahepatic or extrahepatic bilirubin blockage (elevated alkaline phosphatase) or gilbert syndrome. Tambour alogie law (sample r.hy's law): severe hepatotoxicity was predicted. Pharmacoepidemiol Drug saf.2006;15 (S4) 241-3

3. Drug-induced hepatocyte damage is typically manifested by a 3-fold or higher elevation of ALT or AST compared to (non-hepatotoxic) control drug or placebo.

4. Among test subjects showing such AT elevation (typically AT well above 3×uln), one or more subjects also showed an elevation of serum TBL to >2×uln without initial findings of cholestasis (serum ALP elevation).

5. No other causes were found that could explain the combination of AT and TBL elevation, such as hepatitis a, b or c virus; pre-existing or acute liver disease; or another drug capable of causing the observed damage. Guide for industry, "Drug-Induced Liver Injury: premarketing Clinical Evaluation", issued by the FDA in 2009, 7.

6.1.9.3 common serious adverse events

The following is a list of SAE that the sponsor believes to be related to the disease condition under study. The list does not change our reporting obligations or prevent reporting from meeting the AE requirements of SAE definition as detailed in the definition of serious adverse events in section 6.1.6.6 (ii). The purpose of this list is to alert researchers that some events reported as SAE may not require expedited notification to authorities based on the classification of "common SAE". Researchers need to follow the requirements detailed in section 6.1.6.6 (v) report of serious adverse events.

● Pain in urinary tract

● Bladder disorders

● Pain in urination

● Urinary tract hemorrhage (hematuria)

● Obstruction of urinary tract

6.1.9.4 retrospective PGx sub-study

(i) Introduction to the invention

PGx studies aim to provide information about: based on genetic variation, naturally occurring changes in the genes and/or expression of a subject can affect what treatment options are best suited for the subject. The relationship between gene profile and kinetics, efficacy or toxicity of drugs can be better understood through technical studies PGx, such as genotyping, genetic sequencing, statistical genetics and whole genome association studies. Since many diseases may be affected by 1 or more genetic variations, PGx studies can identify which genes are involved in determining the manner in which a subject may or may not respond to a drug.

(ii) Target object

Future PGx studies that can be performed using the obtained blood samples are exploratory. The goal of this study would be to analyze or determine genes associated with clinical response, pharmacokinetics, and toxicity/safety issues.

By analyzing genetic variations, it is possible to predict the response of individual subjects to treatment in terms of efficacy and/or toxicity.

(iii) Subject participation

Subjects who have agreed to participate in this study may participate in this PGx sub-study. As part of this sub-study, the subject must provide written consent before providing any blood sample that may be later used for genetic analysis.

(iv) Sample collection and storage

Subjects who agreed to participate in this sub-study will provide a tube of approximately 4-6mL of whole blood, according to the sponsor instructions. Each sample will be identified by a unique subject number (first code).

(v) PGx analysis

The sponsor will initiate PGx analysis where evidence suggests that genetic variants may affect the kinetics, efficacy and/or safety of the drug.

(vi) PGx sample/data placement

After hard lock of the study database, all PGx samples collected will be stored for up to 15 years. If no analysis is required, the whole blood sample will be destroyed after the planned storage period. Subjects had rights to withdraw consent at any time. When a withdrawal notification is received from the subject, the PGx sample will be destroyed.

(vii) Disclosure of information to a subject

Exploratory PGx analysis may be performed after the clinical study has ended, if applicable. The results of the genetic analysis will not be provided to any researchers or subjects nor will they be requested at a later date.

6.1.9.5EORTC-QLQ-C30 (version 3)

We are interested in something about your and your health. Please answer all questions by circling the numbers most suitable for you. There is no "correct" or "incorrect" answer. The information you provide will be kept strictly secret.

Please fill in your name abbreviations:

your birthday (year, month, day):

date of today (year, month, day):

for the following problems, please loop around between 1 and 7 to select the number most suitable for you

29. How you will evaluate your population during the past weekHealth condition？

30. How you will evaluate your population during the past weekQuality of life？

6.1.9.6EQ-5D-5L

EQ-5D-5L is shown in FIG. 2C.

6.1.9.7 health resource utilization

Is you going to the Emergency Room (ER) from your last study visit?

And ≡no (if no, go to 4)

And ≡yes (if yes, go to 2)

How many times you have gone to the emergency room from your last study visit?

For each emergency room visit, the following is done:

From your last study visit, do you have to be admitted (stay more than 24 hours) without first entering the Emergency Room (ER)?

And no (if no, go to 7)

And ≡yes (if yes, go to 5)

How many times admitted (stay more than 24 hours; no previous ER referral?

For each admission (more than 24 hours stay; no prior ER referral) visit, the following is done:

	residence time in hospitals (days)
		Hospital visit 1
Hospital visit 2
		Hospital visit 3

Do you have had to visit the general practitioner (attending physician) from your last study visit?

And no (if no, go to 9)

And ≡yes (if yes, go to 8)

How many times you have visited the general practitioner (attending physician)?

From your last study visit, do you go to a doctor (e.g., oncologist, rheumatologist, endocrinologist, plastic surgeon, etc.) to visit?

Whether or not (

And ≡yes (if yes, go to 10)

How many times you have visited the practitioner (e.g., oncologist, rheumatologist, endocrinologist, orthopedic surgeon, etc.)?

6.1.9.8RECIST V1.1

Table a-time point reaction: patients with target (+/-non-target) disease.

Table B-time point reaction: patients with only non-target diseases.

Table C-best overall response when CR and PR need to be confirmed.

Reproduced by: eisenhauer EA, therasse P, bogaerts J, schwartz LH, sargent D, ford R et al New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J cancer.2009;45:228-47.

6.1.9.9ECOG behavior State

ECOG: eastern tumor cooperative group

Reproduced by: OKEN MM, creech RH, tormey DC, horton J, davis TE, mcFadden ET ET al Toxicity and response criteria of the Eastern Cooperative Oncology group.am J Clin Oncol.1982;5:649-55.

6.1.10Description of terms or abbreviations used in clinical studies

6.1.11Results of the study

The treatment configurations for the clinical studies described herein are shown in table 23 below:

table 23 treatment configuration

Data cut-off to 2020-7-15	Enrolment Shan Kangwei statin	Chemotherapy treatment	Totals to
				Full analysis set	301	307	608
Treatment of	296	291	587
				Untreated treatment	5	16	21
Number of deaths	134	167	301

Patient demographics for the clinical study described herein are shown in table 24 below:

table 24 demographics and baseline characteristics, stratification factors and medical history

* Due to rounding, the percentage may not total 100.

The eastern tumor co-operating group (ECOG) behavioral state scores were in the range of 0 to 4, with higher scores indicating greater disability.

The bellmutt risk score is in the range of 0 to 3, depending on the presence of the following risk factors: hemoglobin levels of less than 10g per deciliter, ecog behavioral state scores above 0, and liver metastasis.

Other histological types include adenocarcinoma, squamous cell carcinoma, and pseudosarcoma differentiation.

The optimal response in a patient with a response is defined as a confirmed complete or partial response; in patients without a response, the optimal response is defined as stable disease or progressive disease.

The study exposures of the clinical study described herein are shown in table 25 below:

table 25 study exposure

In a metaphase analysis comprising a total of 301 deaths, total survival OS for the clinical study described herein was analyzed for a Full Analysis Set (FAS) population, as shown in table 26 below:

table 26 summary of total survival in FAS

* Predetermined efficacy boundary = 0.00679 (adjusted according to observed 301 deaths)

After a median follow-up of 11.1 months, the risk of death of enrolment Shan Kangwei statins was 30% lower than that of chemotherapy (risk ratio, 0.70;95% confidence interval [ CI ],0.56 to 0.89; p=0.001), indicating that enrolment Shan Kangwei statins have a significantly longer overall survival.

The total survival in Kaplan Meier curve-FAS is shown in figure 4.

The total survival of the subgroups is shown in figure 5. The median total survival in the enrolment Shan Kangwei statin group was 12.88 months (95% CI,10.58 to 15.21) and 8.97 months (95% CI,8.05 to 10.74) in the chemotherapy group. The estimated percentage of patients surviving 12 months in the enrolment Shan Kangwei statin group was 51.5% (95% CI,44.6 to 58.0) and 39.2% (95% CI,32.6 to 45.6) in the chemotherapy group.

As an additional measure of efficacy, FAS populations were analyzed for Progression Free Survival (PFS) in a metaphase analysis comprising a total of 432 PFS events, as shown in table 27. Further, the PFS is shown in fig. 6.

TABLE 27 progression free survival, overview of investigator assessment-FAS

The results of subgroup analysis showed that there was a progression free survival benefit of using enrolment Shan Kangwei statins between the multiple subgroups as shown in figure 7. This forest map shows the progression free survival assessed by researchers in a predetermined key subgroup of the intended treatment population consisting of all randomized patients. For "all subjects," the reported results were based on a stratification analysis with the following stratification factors: ECOG PS, regional and liver metastases.

The results of the sensitivity analysis are consistent with those of the preliminary analysis, as shown in table 28.

TABLE 28 sensitivity analysis of total survival

* The hierarchical Cox proportional hazards model was analyzed for 1000 startup simulation datasets. The confidence interval for 1000 simulations was 2.5% to 97.5%. The P-value was calculated from 1000 simulations.

(95% CI) is based on a stratified Cox scale risk model with treatment as an explanatory variable. Layering factors are ECOG PS, regional and liver metastasis. Weights are calculated from two logarithmic models. One model included only baseline covariates (age group, primary tumor site, and number of prior normals in locally advanced or metastatic background). Another model included two baseline covariates and time dependent variables (diameter sum and ECOG assessment). The single-sided P value is based on a weight-tiered log rank test.

CI represents a confidence interval; ECOG PS: scoring the behavioral state of the eastern tumor cooperative group; HR: risk ratio.

ORR and DOR were analyzed and are shown in table 29.

Table 29ORR and DOR, researchers assessment-reactive population (RES)

* Disease control rate is defined as the proportion of patients with a confirmed complete response, a confirmed partial response, or stable disease (at least 7 weeks) as the best overall response.

In all patients with confirmed complete or partial response.

The definition of the optimal overall reaction is according to RECIST v1.1. The complete or partial reaction was confirmed by two scans separated by at least 4 weeks. The shortest duration of stable disease was 7 weeks. CI represents a confidence interval; and RECIST: response assessment criteria in solid tumors.

The results of the subgroup analysis are consistent with those of the preliminary analysis, as shown in fig. 8. This forest map shows the total response rate assessed by researchers in a predetermined key subgroup of an evaluable response population consisting of all randomly grouped patients with measurable disease at baseline. For "all subjects," the reported results were based on non-stratified analysis. Complete responses were observed in 4.9% (14/288) of patients in the enrolment Shan Kangwei statin group and 2.7% (8/296) of patients in the chemotherapy group. Disease control (P < 0.001) was observed in 71.9% (95% CI,66.3 to 77.0) and 53.4% (95% CI,47.5 to 59.2), respectively.

In patients with complete or partial response, the median duration of response in the enrolment Shan Kangwei statin group was 7.39 months and 8.11 months in the chemotherapy group, as shown in fig. 9.

Grade 3 or higher treatment-related adverse events occurred in 51.4% of patients in the enrolment Shan Kangwei statin group and 49.8% in the chemotherapy group. After adjustment for therapeutic exposure, the ratio in the enrolment Shan Kangwei statin and chemotherapy groups was 2.4 and 4.3 events per patient per year, respectively. An overview of adverse events adjusted by patient-year is shown in table 30.

Table 30 overview of adverse events adjusted by patient-year

* TRAE indicates a reasonable likelihood that the event may be caused by study treatment as assessed by a researcher. Adverse events are considered treatment-related if the relationship is absent.

E represents the number of events, PY represents patient-year, TEAE represents treatment-induced adverse events, and TRAE represents treatment-related adverse events.

Treatment-related adverse events that resulted in treatment modulation are shown in table 31. In particular, grade 3 or higher treatment-related adverse events occurring in at least 5% of patients include: in the enrolment Shan Kangwei statin group, maculopapules (7.4%), fatigue (6.4%) and decreased neutrophil count (6.1%); and in the chemotherapy group, neutrophil count decreased (13.4%), anemia (7.6%), white blood cell count decreased (6.9%), neutropenia (6.2%), and febrile neutropenia (5.5%). Treatment-related adverse events leading to dose reduction, discontinuation of treatment, or cessation of treatment occurred in 32.4%, 51.0%, and 13.5% of patients in the enrolment Shan Kangwei statin group, respectively, and in 27.5%, 18.9%, and 11.3% of the chemotherapy group, respectively.

Table 31 treatment-related adverse events leading to treatment modulation

The percentage of events in either treatment group is reported by the preferred term to be ≡2% (listed in order of highest frequency in the enrolment Shan Kangwei statin group).

Adverse events and deaths induced by treatment were analyzed and are shown in table 32. All adverse events occurring during the treatment are listed in table 30.

Table 32 summary of treatment-induced adverse events and death

Adverse events of particular concern were analyzed and are shown in table 33.

Table 33 treatment-induced adverse events (MedDRAV23.0) with special safety concerns

Clinical studies provided herein compare PADCEV with chemotherapy in adult patients with locally advanced or metastatic urothelial cancer previously treated with platinum-based chemotherapy and PD-1/L1 inhibitors. Knot of clinical studyThe results show that, compared with chemotherapy,(enrolment Shan Kangwei statin-ejfv) meets its primary end of total survival.

In clinical studies, PADCEV significantly improved total survival (OS), with a 30% reduction in mortality risk (risk ratio (HR) =0.70, (95% Confidence Interval (CI): 0.56,0.89); p=0.001). PADCEV also significantly improved Progression Free Survival (PFS), secondary endpoint, with 39% reduced risk of disease progression or death (hr=0.61 (95% CI:0.50, 0.75); p < 0.00001).

For patients in the PADCEV group tested, adverse events were consistent with those listed in the U.S. prescription information, with rash, hyperglycemia, decreased neutrophil count, fatigue, anemia, and anorexia as the most frequent grade 3 higher adverse events occurring in more than 5% of patients.

Treatment-related adverse events in the safety population are shown in table 34. The incidence of treatment-related adverse events was generally higher, but similar in both groups (93.9% in the enrolment Shan Kangwei statin group and 91.8% in the chemotherapy group).

Table 34 treatment related adverse events (safety population)

* The safety population includes all patients who received any amount of the test drug. Including treatment-related adverse events that occur in at least 20% of patients in either treatment group, or grade 3 or higher in at least 5% of patients in either treatment group. Treatment-related adverse events are those events for which there is a reasonable likelihood of being caused by the trial treatment, as assessed by the researcher. An event is considered to be treatment-related if data regarding the relationship to the treatment is missing.

A total of 113 patients (55 out of the enrolment Shan Kangwei statin group and 58 out of the chemotherapy group) had pre-existing peripheral neuropathy.

Skin reactions and peripheral neuropathy are the most frequent, particularly interesting treatment-related adverse events in the case of enrolment Shan Kangwei statins, as shown in table 35.

The time to onset of a treatment-related adverse event of particular interest is shown in table 36.

Table 36 time of onset of treatment-related adverse events of particular interest

* The median (range) of times of first onset of grade 2 or more peripheral neuropathy in the enrolment Shan Kangwei statin and chemotherapy groups are respectively: 4.435 (0.36, 12.02) months; and 1.725 (0.07,9.89) months.

NA indicates inapplicability.

Treatment at baseline conditions induced peripheral neuropathy and hyperglycemia is shown in table 37.

Table 37

* The baseline state indicates the health condition (medical history or ongoing) of the patient prior to receiving any study treatment at the time it entered trial. Patients with pre-existing ≡2 grade sensory or motor neuropathy or uncontrolled history of diabetes within 3 months of the first study medication were excluded from the trial. Uncontrolled diabetes is defined as greater than or equal to 8% or between 7% and <8% hemoglobin A1c, with associated diabetic symptoms (polyuria or polydipsia) not otherwise explained. In addition to these exclusion criteria, patients with pre-existing neuropathy and hyperglycemia were allowed to enter the trial.

BMI represents body mass index and PN represents peripheral neuropathy.

Adverse events that caused death (excluding disease progression) during treatment occurred in 11 patients in each group, regardless of the relationship to treatment; after adjustment for therapeutic exposure, the incidence remained the same. Treatment-related adverse events, which resulted in the assessment of dead researchers, occurred in 7 patients (2.4%) in the enrolment Shan Kangwei statin group (multiple organ dysfunction syndrome [ in 2 patients ], and liver dysfunction, hyperglycemia, pelvic abscesses, pneumonia, and septic shock [ in 1 patient each ]); and in 3 patients (1.0%) in the chemotherapy group (neutropenic sepsis, sepsis and whole blood cytopenia [ in 1 patient each ]). Demographics of dead patients in the enrolment Shan Kangwei statin group are provided in table 38.

Table 38 details of patients for adverse events in the enrolment Shan Kangwei statin group leading to death

* A "treatment-related" adverse event indicates a reasonable likelihood that the event may be caused by the study treatment, as assessed by a researcher. Adverse events are considered treatment-related if the relationship is absent.

Adverse events (n=11) of "treatment-induced" also include all events considered treatment-related adverse events.

BMI represents body mass index.

6.2 example 2-analysis of refractory subgroups according to the clinical study described in section 6.1.

In the clinical study described in section 6.1, enrolment Shan Kangwei Statins (EVs) showed superior total survival (OS) in patients with previously treated locally advanced or metastatic urothelial cancer (la/mUC) compared to Standard Chemotherapy (SC). This subgroup analysis evaluates EV efficacy/safety in those patients with poor prognostic factors, e.g., age ∈65 years, presence of liver metastasis, primary upper urinary tract disease, and non-response to checkpoint inhibitors (CPI).

The method comprises the following steps: in this open label trial, la/mUC patients who had previously received platinum-based chemotherapy and had progressed during/after PD-1/L1 inhibitor were randomized at 1:1 to EV or the investigator's SC selection (docetaxel, paclitaxel, vinflunine). The primary endpoint of the OS for subgroup analysis and the secondary endpoint of the Progression Free Survival (PFS) and total reaction rate (ORR) assessed by the investigator according to RECIST v1.1 were predefined. Kaplan-Meier analysis compares OS and PFS between treatments for selected refractory subgroups as follows: age not less than 65 years, liver metastasis, primary upper urinary tract disease and checkpoint inhibitor (CPI) non-response. ORR and security within subgroups were also evaluated. The Cox proportional hazards model is used to estimate the risk ratio. The cocchran-Mantel-Haenszel test was used to compare the response and disease control rates between groups.

Results: a total of 608 patients were randomly grouped into EV (n=301) or SC (n=307); the median trace was 11.1 months. The OS benefit of EVs is maintained between a large subset (e.g., patients with liver metastases and patients that were unresponsive to previous immune checkpoint inhibitors (i.e., PD-1/L1; "CPI anergy" or "CPI unresponsors")) with longer median OS observed in patients taking EVs relative to patients taking SCs, as shown in table 39 and fig. 10A-10D. For the primary upper urinary tract disease subgroup, the median OS of EV was longer relative to SC and consistent with the median OS in the total population, as shown in table 39 and fig. 10A-10D.

PFS is also longer and ORR is higher in all subgroups of EVs compared to SC. In particular, similar benefits of PFS were also observed in subgroups of EVs relative to SC, as shown in table 39 and fig. 11A-11D. For the primary upper urinary tract disease subgroup, the median PFS of EV was longer relative to SC and consistent with the median PFS in the total population, as shown in table 39 and fig. 11A-11D. In all randomized, grouped patients with measurable disease at baseline, the total response rate (ORR) was higher in each subgroup of EVs relative to SC, and a agreement was presented between treatments in all subgroups, as shown in table 39.

Safety and tolerability: the overall ratio of treatment-induced and treatment-related Adverse Events (AEs) is similar between treatments in the subgroups. For example, the total rate of AEs in patients ≡65 years old treated with EV is 97.4%, and the total rate of AEs in patients ≡65 years old treated with SC is 98.9%. The total ratio of AEs in patients with liver metastasis treated with EV was 97.8% and the total ratio of AEs in patients with liver metastasis treated with SC was 96.7%. The total ratio of AEs in patients with primary upper urinary tract disease treated with EV was 99.0%, and the total ratio of AEs in patients with primary upper urinary tract disease treated with SC was 99.0%. The total ratio of AEs in patients who were previously unresponsive to treatment with PD-1/L1 inhibitor and treated with EV was 97.5%, and the total ratio of AEs in patients who were previously unresponsive to treatment with PD-1/L1 inhibitor and treated with SC was 99.5%.

Among the treatments in the subgroup, the treatment-related AEs were comparable, as shown in fig. 12. AE in all patients receiving any amount of test drug was evaluated. When adjusted for therapeutic exposure, the ≡3 treatment-related AEs in EV groups occurred less frequently in all four subgroups relative to SC groups (i.e., in ≡65 years old patients, in patients with liver metastases, in patients with primary upper urinary tract disease, and in patients who were nonreactive to previous treatment with PD-1/L1 inhibitors), as shown in fig. 13. The occurrence of ≡3 grade treatment-related AEs in the refractory subgroups were generally aligned and consistent with those observed in the overall EV-301 safety population, as shown in table 40.

Conclusion: patients with locally advanced or metastatic urothelial cancer (La/mUC) who received EV had poor prognostic factors consistently had longer OS, longer PFS and higher ORR than patients who received SC. The safety profiles of study treatments were consistent with those of the previous study and those of the overall study population; no new security signal was observed. These data support the use of EV in previously treated patients with la/mUC, including those with poor prognostic factors.

Sequence listing

<110> Ai Gengsi company (AGENSYS, INC.)

Sida company (SEAGEN INC.)

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Asp Val Leu Asp Pro Gln Glu Asp Ser Gly Lys Gln Val Asp Leu Val

335 340 345

tca gcc tcg gtg gtg gtg gtg ggt gtg atc gcc gca ctc ttg ttc tgc 1349

Ser Ala Ser Val Val Val Val Gly Val Ile Ala Ala Leu Leu Phe Cys

350 355 360

ctt ctg gtg gtg gtg gtg gtg ctc atg tcc cga tac cat cgg cgc aag 1397

Leu Leu Val Val Val Val Val Leu Met Ser Arg Tyr His Arg Arg Lys

365 370 375

gcc cag cag atg acc cag aaa tat gag gag gag ctg acc ctg acc agg 1445

Ala Gln Gln Met Thr Gln Lys Tyr Glu Glu Glu Leu Thr Leu Thr Arg

380 385 390

gag aac tcc atc cgg agg ctg cat tcc cat cac acg gac ccc agg agc 1493

Glu Asn Ser Ile Arg Arg Leu His Ser His His Thr Asp Pro Arg Ser

395 400 405 410

cag ccg gag gag agt gta ggg ctg aga gcc gag ggc cac cct gat agt 1541

Gln Pro Glu Glu Ser Val Gly Leu Arg Ala Glu Gly His Pro Asp Ser

415 420 425

ctc aag gac aac agt agc tgc tct gtg atg agt gaa gag ccc gag ggc 1589

Leu Lys Asp Asn Ser Ser Cys Ser Val Met Ser Glu Glu Pro Glu Gly

430 435 440

cgc agt tac tcc acg ctg acc acg gtg agg gag ata gaa aca cag act 1637

Arg Ser Tyr Ser Thr Leu Thr Thr Val Arg Glu Ile Glu Thr Gln Thr

445 450 455

gaa ctg ctg tct cca ggc tct ggg cgg gcc gag gag gag gaa gat cag 1685

Glu Leu Leu Ser Pro Gly Ser Gly Arg Ala Glu Glu Glu Glu Asp Gln

460 465 470

gat gaa ggc atc aaa cag gcc atg aac cat ttt gtt cag gag aat ggg 1733

Asp Glu Gly Ile Lys Gln Ala Met Asn His Phe Val Gln Glu Asn Gly

475 480 485 490

acc cta cgg gcc aag ccc acg ggc aat ggc atc tac atc aat ggg cgg 1781

Thr Leu Arg Ala Lys Pro Thr Gly Asn Gly Ile Tyr Ile Asn Gly Arg

495 500 505

gga cac ctg gtc tga cccaggcctg cctcccttcc ctaggcctgg ctccttctgt 1836

Gly His Leu Val

510

tgacatggga gattttagct catcttgggg gcctccttaa acacccccat ttcttgcgga 1896

agatgctccc catcccactg actgcttgac ctttacctcc aacccttctg ttcatcggga 1956

gggctccacc aattgagtct ctcccaccat gcatgcaggt cactgtgtgt gtgcatgtgt 2016

gcctgtgtga gtgttgactg actgtgtgtg tgtggagggg tgactgtccg tggaggggtg 2076

actgtgtccg tggtgtgtat tatgctgtca tatcagagtc aagtgaactg tggtgtatgt 2136

gccacgggat ttgagtggtt gcgtgggcaa cactgtcagg gtttggcgtg tgtgtcatgt 2196

ggctgtgtgt gacctctgcc tgaaaaagca ggtattttct cagaccccag agcagtatta 2256

atgatgcaga ggttggagga gagaggtgga gactgtggct cagacccagg tgtgcgggca 2316

tagctggagc tggaatctgc ctccggtgtg agggaacctg tctcctacca cttcggagcc 2376

atgggggcaa gtgtgaagca gccagtccct gggtcagcca gaggcttgaa ctgttacaga 2436

agccctctgc cctctggtgg cctctgggcc tgctgcatgt acatattttc tgtaaatata 2496

catgcgccgg gagcttcttg caggaatact gctccgaatc acttttaatt tttttctttt 2556

ttttttcttg ccctttccat tagttgtatt ttttatttat ttttattttt attttttttt 2616

agagatggag tctcactatg ttgctcaggc tggccttgaa ctcctgggct caagcaatcc 2676

tcctgcctca gcctccctag tagctgggac tttaagtgta caccactgtg cctgctttga 2736

atcctttacg aagagaaaaa aaaaattaaa gaaagccttt agatttatcc aatgtttact 2796

actgggattg cttaaagtga ggcccctcca acaccagggg gttaattcct gtgattgtga 2856

aaggggctac ttccaaggca tcttcatgca ggcagcccct tgggagggca cctgagagct 2916

ggtagagtct gaaattaggg atgtgagcct cgtggttact gagtaaggta aaattgcatc 2976

caccattgtt tgtgatacct tagggaattg cttggacctg gtgacaaggg ctcctgttca 3036

atagtggtgt tggggagaga gagagcagtg attatagacc gagagagtag gagttgaggt 3096

gaggtgaagg aggtgctggg ggtgagaatg tcgcctttcc ccctgggttt tggatcacta 3156

attcaaggct cttctggatg tttctctggg ttggggctgg agttcaatga ggtttatttt 3216

tagctggccc acccagatac actcagccag aatacctaga tttagtaccc aaactcttct 3276

tagtctgaaa tctgctggat ttctggccta agggagaggc tcccatcctt cgttccccag 3336

ccagcctagg acttcgaatg tggagcctga agatctaaga tcctaacatg tacattttat 3396

gtaaatatgt gcatatttgt acataaaatg atattctgtt tttaaataaa cagacaaaac 3456

ttgaaaaa 3464

<210> 2

<211> 510

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<221> misc_feature

<222> (1)...(510)

<223> 191P4D12

<400> 2

Met Pro Leu Ser Leu Gly Ala Glu Met Trp Gly Pro Glu Ala Trp Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Ala Ser Phe Thr Gly Arg Cys Pro Ala Gly

20 25 30

Glu Leu Glu Thr Ser Asp Val Val Thr Val Val Leu Gly Gln Asp Ala

35 40 45

Lys Leu Pro Cys Phe Tyr Arg Gly Asp Ser Gly Glu Gln Val Gly Gln

50 55 60

Val Ala Trp Ala Arg Val Asp Ala Gly Glu Gly Ala Gln Glu Leu Ala

65 70 75 80

Leu Leu His Ser Lys Tyr Gly Leu His Val Ser Pro Ala Tyr Glu Gly

85 90 95

Arg Val Glu Gln Pro Pro Pro Pro Arg Asn Pro Leu Asp Gly Ser Val

100 105 110

Leu Leu Arg Asn Ala Val Gln Ala Asp Glu Gly Glu Tyr Glu Cys Arg

115 120 125

Val Ser Thr Phe Pro Ala Gly Ser Phe Gln Ala Arg Leu Arg Leu Arg

130 135 140

Val Leu Val Pro Pro Leu Pro Ser Leu Asn Pro Gly Pro Ala Leu Glu

145 150 155 160

Glu Gly Gln Gly Leu Thr Leu Ala Ala Ser Cys Thr Ala Glu Gly Ser

165 170 175

Pro Ala Pro Ser Val Thr Trp Asp Thr Glu Val Lys Gly Thr Thr Ser

180 185 190

Ser Arg Ser Phe Lys His Ser Arg Ser Ala Ala Val Thr Ser Glu Phe

195 200 205

His Leu Val Pro Ser Arg Ser Met Asn Gly Gln Pro Leu Thr Cys Val

210 215 220

Val Ser His Pro Gly Leu Leu Gln Asp Gln Arg Ile Thr His Ile Leu

225 230 235 240

His Val Ser Phe Leu Ala Glu Ala Ser Val Arg Gly Leu Glu Asp Gln

245 250 255

Asn Leu Trp His Ile Gly Arg Glu Gly Ala Met Leu Lys Cys Leu Ser

260 265 270

Glu Gly Gln Pro Pro Pro Ser Tyr Asn Trp Thr Arg Leu Asp Gly Pro

275 280 285

Leu Pro Ser Gly Val Arg Val Asp Gly Asp Thr Leu Gly Phe Pro Pro

290 295 300

Leu Thr Thr Glu His Ser Gly Ile Tyr Val Cys His Val Ser Asn Glu

305 310 315 320

Phe Ser Ser Arg Asp Ser Gln Val Thr Val Asp Val Leu Asp Pro Gln

325 330 335

Glu Asp Ser Gly Lys Gln Val Asp Leu Val Ser Ala Ser Val Val Val

340 345 350

Val Gly Val Ile Ala Ala Leu Leu Phe Cys Leu Leu Val Val Val Val

355 360 365

Val Leu Met Ser Arg Tyr His Arg Arg Lys Ala Gln Gln Met Thr Gln

370 375 380

Lys Tyr Glu Glu Glu Leu Thr Leu Thr Arg Glu Asn Ser Ile Arg Arg

385 390 395 400

Leu His Ser His His Thr Asp Pro Arg Ser Gln Pro Glu Glu Ser Val

405 410 415

Gly Leu Arg Ala Glu Gly His Pro Asp Ser Leu Lys Asp Asn Ser Ser

420 425 430

Cys Ser Val Met Ser Glu Glu Pro Glu Gly Arg Ser Tyr Ser Thr Leu

435 440 445

Thr Thr Val Arg Glu Ile Glu Thr Gln Thr Glu Leu Leu Ser Pro Gly

450 455 460

Ser Gly Arg Ala Glu Glu Glu Glu Asp Gln Asp Glu Gly Ile Lys Gln

465 470 475 480

Ala Met Asn His Phe Val Gln Glu Asn Gly Thr Leu Arg Ala Lys Pro

485 490 495

Thr Gly Asn Gly Ile Tyr Ile Asn Gly Arg Gly His Leu Val

500 505 510

<210> 3

<211> 1432

<212> DNA

<213> Homo sapiens (Homo sapiens)

<220>

<221> CDS

<222> (32)...(1432)

<220>

<221> misc_feature

<222> (1)...(1432)

<223> Ha22-2 (2, 4) 6.1 heavy chain

<400> 3

ggtgatcagc actgaacaca gaggactcac c atg gag ttg ggg ctg tgc tgg 52

Met Glu Leu Gly Leu Cys Trp

1 5

gtt ttc ctt gtt gct att tta gaa ggt gtc cag tgt gag gtg cag ctg 100

Val Phe Leu Val Ala Ile Leu Glu Gly Val Gln Cys Glu Val Gln Leu

10 15 20

gtg gag tct ggg gga ggc ttg gta cag cct ggg ggg tcc ctg aga ctc 148

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

25 30 35

tcc tgt gca gcc tct gga ttc acc ttc agt agc tat aac atg aac tgg 196

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Asn Met Asn Trp

40 45 50 55

gtc cgc cag gct cca ggg aag ggg ctg gag tgg gtt tca tac att agt 244

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser

60 65 70

agt agt agt agt acc ata tac tac gca gac tct gtg aag ggc cga ttc 292

Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

75 80 85

acc atc tcc aga gac aat gcc aag aac tca ctg tct ctg caa atg aac 340

Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser Leu Gln Met Asn

90 95 100

agc ctg aga gac gag gac acg gct gtg tat tac tgt gcg aga gca tac 388

Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ala Tyr

105 110 115

tac tac ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc 436

Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

120 125 130 135

tca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc 484

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

140 145 150

aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac 532

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

155 160 165

tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc 580

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

170 175 180

agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac 628

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

185 190 195

tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag 676

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

200 205 210 215

acc tac atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg gac 724

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

220 225 230

aag aga gtt gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg 772

Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro

235 240 245

tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc 820

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

250 255 260

cca aaa ccc aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca 868

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

265 270 275

tgc gtg gtg gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac 916

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

280 285 290 295

tgg tac gtg gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg 964

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

300 305 310

gag gag cag tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc 1012

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

315 320 325

ctg cac cag gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc 1060

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

330 335 340

aac aaa gcc ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa 1108

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

345 350 355

ggg cag ccc cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gag 1156

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

360 365 370 375

gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc 1204

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

380 385 390

tat ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag 1252

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

395 400 405

aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc 1300

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

410 415 420

ttc ctc tat agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg 1348

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

425 430 435

aac gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac 1396

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

440 445 450 455

acg cag aag agc ctc tcc ctg tcc ccg ggt aaa tga 1432

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

460 465

<210> 4

<211> 466

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<221> misc_feature

<222> (1)...(466)

<223> Ha22-2 (2, 4) 6.1 heavy chain

<400> 4

Met Glu Leu Gly Leu Cys Trp Val Phe Leu Val Ala Ile Leu Glu Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Ser Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln

115 120 125

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

130 135 140

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

145 150 155 160

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

165 170 175

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

195 200 205

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

210 215 220

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

225 230 235 240

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Lys

465

<210> 5

<211> 735

<212> DNA

<213> Homo sapiens (Homo sapiens)

<220>

<221> CDS

<222> (25)...(735)

<220>

<221> misc_feature

<222> (1)...(735)

<223> Ha22-2 (2, 4) 6.1 light chain

<400> 5

agtcagaccc agtcaggaca cagc atg gac atg agg gtc ccc gct cag ctc 51

Met Asp Met Arg Val Pro Ala Gln Leu

1 5

ctg ggg ctc ctg ctg ctc tgg ttc cca ggt tcc aga tgc gac atc cag 99

Leu Gly Leu Leu Leu Leu Trp Phe Pro Gly Ser Arg Cys Asp Ile Gln

10 15 20 25

atg acc cag tct cca tct tcc gtg tct gca tct gtt gga gac aga gtc 147

Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val

30 35 40

acc atc act tgt cgg gcg agt cag ggt att agc ggc tgg tta gcc tgg 195

Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Gly Trp Leu Ala Trp

45 50 55

tat cag cag aaa cca ggg aaa gcc cct aag ttc ctg atc tat gct gca 243

Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile Tyr Ala Ala

60 65 70

tcc act ttg caa agt ggg gtc cca tca agg ttc agc ggc agt gga tct 291

Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser

75 80 85

ggg aca gat ttc act ctc acc atc agc agc ctg cag cct gaa gat ttt 339

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe

90 95 100 105

gca act tac tat tgt caa cag gct aac agt ttc cct ccc act ttc ggc 387

Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro Thr Phe Gly

110 115 120

gga ggg acc aag gtg gag atc aaa cga act gtg gct gca cca tct gtc 435

Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val

125 130 135

ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga act gcc tct 483

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

140 145 150

gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag 531

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

155 160 165

tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc 579

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

170 175 180 185

aca gag cag gac agc aag gac agc acc tac agc ctc agc agc acc ctg 627

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

190 195 200

acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa 675

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

205 210 215

gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg 723

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

220 225 230

gga gag tgt tag 735

Gly Glu Cys

235

<210> 6

<211> 236

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<221> misc_feature

<222> (1)...(236)

<223> Ha22-2 (2, 4) 6.1 light chain

<400> 6

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Gly Ile Ser Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Phe Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ala Asn Ser Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 7

<211> 466

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<221> misc_feature

<222> (1)...(466)

<223> Ha22-2 (2, 4) 6.1 heavy chain

<400> 7

Met Glu Leu Gly Leu Cys Trp Val Phe Leu Val Ala Ile Leu Glu Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Ser Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln

115 120 125

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

130 135 140

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

145 150 155 160

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

165 170 175

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

180 185 190

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

195 200 205

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

210 215 220

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp

225 230 235 240

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

Gly Lys

465

<210> 8

<211> 236

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<221> misc_feature

<222> (1)...(236)

<223> Ha22-2 (2, 4) 6.1 light chain

<400> 8

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Ser Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Gly Ile Ser Gly Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Phe Leu Ile Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ala Asn Ser Phe Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 9

<211> 5

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<223> CDR1 of heavy chain

<400> 9

Ser Tyr Asn Met Asn

1 5

<210> 10

<211> 17

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<223> CDR2 of heavy chain

<400> 10

Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 11

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<223> CDR3 of heavy chain

<400> 11

Ala Tyr Tyr Tyr Gly Met Asp Val

1 5

<210> 12

<211> 11

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<223> CDR1 of light chain

<400> 12

Arg Ala Ser Gln Gly Ile Ser Gly Trp Leu Ala

1 5 10

<210> 13

<211> 7

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<223> CDR2 of light chain

<400> 13

Ala Ala Ser Thr Leu Gln Ser

1 5

<210> 14

<211> 9

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<223> CDR3 of light chain

<400> 14

Gln Gln Ala Asn Ser Phe Pro Pro Thr

1 5

<210> 15

<211> 4

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> joint

<400> 15

Gly Phe Leu Gly

1

<210> 16

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> VH CDR1 according to IMGT

<400> 16

Gly Phe Thr Phe Ser Ser Tyr Asn

1 5

<210> 17

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> VH CDR2 according to IMGT

<400> 17

Ile Ser Ser Ser Ser Ser Thr Ile

1 5

<210> 18

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> VH CDR3 according to IMGT

<400> 18

Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val

1 5 10

<210> 19

<211> 6

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> VL CDR1 according to IMGT

<400> 19

Gln Gly Ile Ser Gly Trp

1 5

<210> 20

<211> 3

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> VL CDR2 according to IMGT

<400> 20

Ala Ala Ser

1

<210> 21

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> VL CDR3 according to IMGT

<400> 21

Gln Gln Ala Asn Ser Phe Pro Pro Thr

1 5

<210> 22

<211> 117

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> heavy chain variable region (VH), amino acid 20 (glutamic acid) to amino acid 136 (serine) of SEQ ID NO:7

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asn Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 23

<211> 108

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> light chain variable region (VL), amino acid 23 (aspartic acid) to 130 (arginine) of SEQ ID NO:8

<400> 23

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Gly Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105

Claims

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

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

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

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

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

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

4. The method of any one of claims 1-3, wherein the subject has a response duration of at least or about 7 months after the treatment.

5. The method of any one of claims 1-3, wherein the subject has a duration of response after the treatment in the range of 5-9 months.

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

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

8. The method of claim 1, wherein the subject has a progression free survival in the range of 4 to 9 months after the treatment.

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

10. The method of claim 1, wherein the subject has a total survival of at least or about 9 months after the treatment.

11. The method of claim 2, wherein the subject has a total survival of at least or about 12 months after the treatment.

12. The method of claim 3, wherein the subject has a total survival of at least or about 11 months after the treatment.

13. The method of any one of claims 1-3, wherein the subject has a total survival in the range of 9-19 months after the treatment.

14. The method of any one of claims 1-3, wherein the population of subjects is treated by the method, and wherein the percentage of subjects in the treated population that have a complete response is at least or about 4%.

15. The method of any one of claims 1-3, wherein the population of subjects is treated by the method, and wherein the percentage of subjects in the treated population that have a partial response is at least or about 35%.

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

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

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

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

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

21. The method of any one of claims 1-3, wherein the population of subjects is treated by the method, and wherein the duration of response in the treated population is in the range of 5 to 9 months.

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

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

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

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

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

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

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

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

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

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

32. The method of claim 1, wherein the total response rate of the population of subjects treated with the method is at least or about 35%.

33. The method of claim 2, wherein the total response rate of the population of subjects treated with the method is at least or about 43%.

34. The method of claim 3, wherein the total response rate of the population of subjects treated with the method is at least or about 39%.

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

36. The method of any one of claims 1 to 3, wherein the duration of the response of the population of subjects treated with the method is from 5 to 9 months.

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

38. The method of any one of claims 1 to 3, wherein the progression free survival of the population of subjects treated with the method is 4 to 9 months.

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

40. The method of claim 2 or 3, wherein the progression free survival of the population of subjects treated with the method is from 5 to 9 months.

41. The method of claim 1, wherein the median total survival of the population of subjects treated with the method is at least or about 9 months.

42. The method of claim 2, wherein the median total survival of the population of subjects treated with the method is at least or about 12 months.

43. The method of claim 3, wherein the median total survival of the population of subjects treated with the method is at least or about 11 months.

44. The method of any one of claims 1 to 3, wherein the total survival of the population of subjects treated with the method is 9 to 19 months.

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

46. The method of any one of claims 1-45, wherein the cancer is urothelial cancer, and wherein the human subject has locally advanced or metastatic urothelial cancer.

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

(i) Absolute Neutrophil Count (ANC) of not less than 1500/mm ³ ；

(ii) Platelet count is not less than 100X 10 ⁹ a/L;

(iii) Hemoglobin is not less than 9g/dL;

(iv) Serum bilirubin does not exceed an Upper Limit of Normal (ULN) of 1.5 times or 3 times ULN for patients with gilbert disease;

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

48. The method of claim 47, wherein the subject has all of conditions (i) to (vi) of claim 47.

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

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

51. The method of any one of claims 1-50, wherein the subject does not have active central nervous system metastasis.

52. The method of any one of claims 1-51, wherein the subject does not have uncontrolled diabetes.

53. The method of claim 52, wherein the uncontrolled diabetes is determined by hemoglobin A1c (HbA 1 c) being no less than 8% or HbA1c being between 7% and 8% with associated diabetes symptoms that are not otherwise explained.

54. The method of claim 53, wherein the associated diabetic symptoms comprise or consist of: polyuria, polydipsia, or both polyuria and polydipsia.

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

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

57. The method of claim 55, wherein the PD-1 inhibitor is nivolumab or pamphlet Li Zhushan antibody.

58. The method of claim 56, wherein the PD-L1 inhibitor is selected from the group consisting of: alemtuzumab, avistuzumab and Dewaruzumab.

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

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

61. The method of any one of claims 1 to 60, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 23.

62. The method of any one of claims 1 to 61, wherein the antibody comprises: a heavy chain comprising an amino acid sequence in the range of amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID No. 7; and a light chain comprising an amino acid sequence in the range of amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO. 8.

63. The method of any one of claims 1 to 61, wherein the antigen binding fragment is a Fab, F (ab') 2, fv, or scFv.

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

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

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

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

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

69. The method of claim 67 or 68, wherein said linker is of the formula: -Aa-Ww-Yy-; wherein-A-is an extension subunit, a is 0 or 1; -W-is an amino acid unit, W is an integer ranging from 0 to 12; and-Y-is a spacer unit, Y is 0, 1 or 2.

70. The method of claim 69, wherein the extension subunit has the structure of formula (1); the amino acid unit is valine-citrulline; and the spacer unit is a PAB group comprising the structure of formula (2):

71. the method of claim 69 or 70, wherein the extension subunit forms a bond with a sulfur atom of the antibody or antigen binding fragment thereof; and wherein the spacer unit is linked to MMAE via a carbamate group.

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

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

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

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

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

77. The process of claim 76 wherein p is 2 to 8.

78. The method of claim 76 or 77, wherein p is 3 to 5.

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

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

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

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

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

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

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

86. The method of any one of claims 1-85, wherein the ADC is administered by Intravenous (IV) injection or infusion.

87. The method of any one of claims 1-86, wherein the ADC is administered by IV injection or infusion three times every four week period.

88. The method of any one of claims 1-87, wherein the ADC is administered by IV injection or infusion on days 1, 8, and 15 of a four week cycle.

89. The method of any one of claims 1-88, wherein the ADC is administered by IV injection or infusion of about 30 minutes three times every four week period.

90. The method of any one of claims 1-89, wherein the ADC is administered by IV injection or infusion for about 30 minutes on days 1, 8, and 15 of every four week cycle.

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

92. The method of any one of claims 1-91, wherein the ADC is formulated in a pharmaceutical composition comprising about 20mM L-histidine, about 0.02% (w/v) TWEEN-20, about 5.5% (w/v) trehalose dihydrate and hydrochloride, and wherein the pH of the pharmaceutical composition at 25 ℃ is about 6.0.

93. The method of any one of claims 1-91, wherein the ADC is formulated in a pharmaceutical composition comprising about 9mM histidine, about 11mM histidine monohydrochloride, about 0.02% (w/v) TWEEN-20 and about 5.5% (w/v) trehalose dihydrate, and wherein the pH of the pharmaceutical composition at 25 ℃ is about 6.0.

94. The method of any one of claims 1 to 93, wherein the ADC is an enrolment Shan Kangwei statin (EV) or a biological analogue thereof, wherein the EV is administered at a dose of about 1.25mg/kg of the subject's body weight, and wherein the dose is administered by IV injection or infusion for about 30 minutes on days 1, 8 and 15 of each four week cycle.

95. The method of any one of claims 1-94, wherein the population of subjects has a complete response after the treatment.

96. The method of any one of claims 1-94, wherein the population of subjects has a partial response after the treatment.

97. The method of any one of claims 1-94, wherein the population of subjects has a complete response or a partial response after the treatment.

98. The method of any one of claims 1-94, wherein the population of subjects has stable disease after the treatment.