Treatment of cancer with antibody drug conjugate (ADC) binding to 191p4d12 protein

Abstract

To provide a methods for the prevention or treatment of cancer.SOLUTION: Provided herein is a method including a step of administering an antibody drug conjugate (ADC) that bind to 191P4D12 protein to a subject.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 886,270, filed on August 13, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0002] 1. Field Provided herein are methods of treating cancer with an antibody - drug conjugate (ADC) that binds to the 191P4D12 protein.

Background Art

[0003] 2. Background Cancer is the leading cause of death in people aged 35 to 65 in the United States and the second most common cause of death worldwide. In 2019, it was estimated that there were approximately 1.7 million new cancer cases and approximately 610,000 cancer - related deaths in the United States (National Cancer Institute. 2019. Cancer Stat Facts: Cancer of Any Site. https: / / seer.cancer.gov / statfacts / html / all.html. Accessed June 5, 2019). Worldwide, the estimated number of new cancer cases in 2018 was 18.1 million, and approximately 9.6 million deaths in 2018 were due to cancer (World Health Organization. Press Release. Sept 2018. https: / / www.who.int / cancer / PRGlobocanFinal.pdf. Accessed June 5, 2019). Currently, most deaths are occurring in patients with metastatic cancer. In fact, over the past 20 years, due to advances in treatment including surgery, radiation therapy, and adjuvant chemotherapy, most patients with localized cancer have been cured. Patients in whom cancer has presented as a metastatic disease or has recurred have had little benefit from conventional therapies with respect to overall survival (OS) and have rarely been cured.

[0004] New treatment strategies for metastatic cancer include targeting molecular pathways important for cancer cell survival and novel cytotoxic compounds. The benefits of these new drugs are reflected in long-term survival; however, the outcome for most patients with distant metastases remains poor and new therapies are needed.

[0005] 191P4D12 (also known as nectin-4) is a type I transmembrane protein and a member of the family of related immunoglobulin-like adhesion molecules involved in cell-cell adhesion. 191P4D12 belongs to the nectin family of adhesion molecules. 191P4D12 is composed of an extracellular domain (ECD) containing three Ig-like subdomains, a transmembrane helix, and an intracellular region (Takai Y et al., Annu Rev Cell Dev Biol 2008;24:309-42). Nectins are thought to mediate Ca2+-independent cell-cell adhesion by both homophilic and heterophilic trans interactions at adherens junctions that can recruit cadherins and regulate cytoskeletal reorganization (Rikitake & Takai, Cell Mol Life Sci. 2008;65(2):253-63). The sequence identity of 191P4D12 to other nectin family members is low, in the range of 25% - 30% in the ECD (Reymond N et al, J Biol Chem 2001;43205-15). Nectin-promoted adhesion supports several biological processes such as immune regulation, host-pathogen interactions, and immune evasion (Sakisaka T et al., Current Opinion in Cell Biology 2007;19:593-602).

[0006] Breast cancer Worldwide, there will be approximately 2.1 million newly diagnosed cases of breast cancer in women in 2018, accounting for nearly 1 in 4 cancer cases in women. This disease is the most frequently diagnosed cancer in the majority of countries and is also the leading cause of cancer-related death in women. After a metastatic diagnosis, the prognosis is poor and the 5-year survival rate is approximately 15%.

[0007] The selection of an appropriate treatment for metastatic breast cancer is complex due to the many treatment options and the biological heterogeneity of the disease. Potential treatment options are influenced by the estrogen and progesterone receptors and the status of the human epidermal growth factor receptor 2 (HER2) of the tumor. The treatment options for subjects presenting with metastatic breast cancer can also be affected by which adjuvant therapy was used, how soon after adjuvant therapy the subject relapses, and the site of the metastases.

[0008] Hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer Hormone receptor-positive (HR+) / HER2-negative breast cancer is the most common breast cancer subtype (>70%) and occurs mainly in postmenopausal women. Initial treatment of women with metastatic disease consists mainly of endocrine therapy. This is usually administered alone, in combination with a CDK4 / 6 inhibitor, or as dual endocrine blockade. Systemic chemotherapy is recommended for women with endocrine resistance or symptomatic visceral disease.

[0009] Several cytotoxic chemotherapeutic agents, including anthracyclines, taxanes, gemcitabine, capecitabine, vinorelbine, eribulin, and ixabepilone, have shown activity in metastatic breast cancer. The response rates with these agents vary depending on the type of previous treatment and breast cancer subtype. In general, anthracycline-based combination therapies and taxanes such as paclitaxel and docetaxel are considered to be the most effective (Piccart M, Clin Breast Cancer 2008;100-13). Considering the extensive use of anthracyclines in adjuvant therapy and the increased risk of cardiotoxicity, the use of anthracyclines in the metastatic setting is limited. Taxanes are the agents most commonly used, particularly in the context of primary treatment, in patients with locally advanced or metastatic disease (Greene&Hennessy, J Oncol Pharm Pract 2015;201-12). Continuous single-agent therapy is recommended over combination due to low toxicity and limited survival benefit. Responses to commonly used single-agent chemotherapy in patients with HR+ / HER2-negative breast cancer have been mainly limited to subgroup analyses, which ranged from 11% to 36% (Robson M et al, N Engl J Med.2017;377(18):1792-3; Kaufman PA et al, J Clin Onco.2015;33(6):594-601; Cortes J et al, Lancet.2011;377:914-23). In general, responses tend to be lower in patients with a treatment history and have been reported to range from 10% to 13% (Perez EA et al, J Clin Oncol.2007;25:3407-14; Jones S et al, J Clin Oncol.1995;13(10):2567-74).

[0010] Triple-negative breast cancer Triple-negative breast cancer (TNBC) is defined by the absence of immunostaining for estrogen receptor (ER), progesterone receptor (PR), and HER2. Overall, approximately 15% to 20% of breast cancers are classified as TNBC. TNBC is associated with invasive tumor biology, visceral metastases, and poor prognosis (Plasilova ML et al, Medicine (Baltimore) 2016;95(35):e4614).

[0011] Taxane-based regimens are considered the standard of care in first-line therapy for patients with metastatic breast cancer, including TNBC. More recently, the FDA granted accelerated approval to atezolizumab in combination with nab-paclitaxel for the treatment of patients with unresectable locally advanced or metastatic TNBC that expresses programmed death ligand 1 (PD-L1) (median progression-free survival [PFS] 7.5 months vs 5.0 months; objective response rate [ORR] 56% vs 46%) (Schmid P et al, N Engl J Med. 2019;380(10):987-988). There is no standard approach for second-line or later therapy, and the chemotherapy options are the same as those for other subtypes. Single-agent cytotoxic chemotherapy agents are generally less preferred than combination chemotherapy due to lack of survival benefit and increased toxicity, except in the setting of invasive disease and visceral lesions (Cardoso F et al, Ann Oncol. 2017;28(2):208-217; National Comprehensive Cancer Network, 2017, Non-small cell lung cancer, NCCN clinical practice guidelines in oncology (NCCN guidelines), http: / / www.nccn.org / professionals / physician_gls / pdf / nscl.pdf. Accessed June 5, 2019). Standard chemotherapy in patients with prior treatment history is associated with low response rates (10% to 15%) and short progression-free survival (2 to 3 months) (Hurvitz & Mead, Curr Opin Obstet Gynecol. 2016;28(1):59-69).

[0012] Non-small cell lung cancer Lung cancer (both small cell and non-small cell) is the leading cause of cancer death in the United States (American Cancer Society. Key Statistics for Lung Cancer. 8 Jan 2019a. https: / / www.cancer.org / cancer / non-small-cell-lung-cancer / about / key-statistics.html. Accessed 5 Jun 2019). Most patients diagnosed with lung cancer are 65 years of age or older, and the average age at diagnosis is approximately 70 years.

[0013] Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers (Tan & Huq, Non-Small Cell Lung Cancer (NSCLC), April 13, 2019, https: / / emedicine.medscape.com / article / 279960-overview, Accessed 5 Jun 2019; American Cancer Society: What is non-small cell lung cancer, 16 May 2016, https: / / www.cancer.org / cancer / non-small-cell-lung-cancer / about / what-is-non-small-cell-lung-cancer.html, Accessed 5 Jun 2019) and can be further subdivided into histological types of squamous (approximately 30% of NSCLC cases) and non-squamous (approximately 40% of NSCLC cases) (American Cancer Society. Non-Small Cell Lung Cancer. 2019b. http: / / www.cancer.org / Cancer / LungCancer-Non-SmallCell / DetailedGuide / lung-cancer--non-small-cell--non-small-cell-lung-cancer. Accessed 5 Jun 2019).

[0014] Squamous non-small cell lung cancer Squamous NSCLC is a distinct histological subtype of NSCLC that is difficult to treat as a result of specific patient and disease characteristics, including older age, metastatic (including malignant or metastasized malignant) disease at diagnosis, comorbidities, and central tumor location (Socinski M et al, Cell Lung Cancer 2018;165-183). These characteristics are associated with treatment outcomes in metastatic (including malignant or metastasized malignant) squamous NSCLC, resulting in a median survival duration approximately 30% shorter than that of patients with other NSCLC subtypes.

[0015] Treatment options for metastatic (including malignant or metastasized malignant) squamous NSCLC, particularly for first-line treatment, are limited and consequently affect survival outcomes (National Comprehensive Cancer Network. 2017, Non-small cell lung cancer, NCCN clinical practice guidelines in oncology (NCCN guidelines), http: / / www.nccn.org / professionals / physician_gls / pdf / nscl.pdf, accessed June 5, 2019; Novello S et al, Ann Oncol 2016;27(Supple 5):v1-v27; Masters GA et al, J Clin Oncol 2015;33(30):3488-3515). Given the recent approvals of targeted therapies and immunotherapies for metastatic (including malignant or metastasized malignant) NSCLC and the continued drive towards personalized lung cancer treatment, it is also necessary to evaluate the effectiveness of these new treatments for metastatic (including malignant or metastasized malignant) squamous NSCLC.

[0016] Non-squamous non-small cell lung cancer Non-squamous NSCLC is a heterogeneous disease with multiple treatment options depending on patient factors including stage classification, presence of metastases, and presence of comorbidities among other considerations. Thus, current treatment options include surgical resection, chemotherapy, radiation, immunotherapy, and targeted therapy. Currently, first-line therapy for metastatic (including malignant or metastatic malignant) non-squamous NSCLC patients without targetable genetic abnormalities is doublet chemotherapy including a platinum agent. Except for bevacizumab, and despite extensive studies of multiple targeted and cytotoxic agents, addition of a third agent to doublet chemotherapy including a platinum agent has not been shown to improve progression-free or OS over doublet chemotherapy including a platinum agent alone in randomized trials (Reck M et al, Ann Oncol 2010;1804-09; Sandler A et al, N Engl J Med 2006;355:2542-50).

[0017] Head and neck cancer Head and neck cancer is a group of cancers that begin in the mouth, nose, throat, larynx, sinuses, or salivary glands (National Cancer Institute, Head and Neck Cancers, 29 Mar 2017, https: / / www.cancer.gov / types / head-and-neck / head-neck-fact-sheet, accessed 5 Jun 2019). Worldwide, more than 5.5 million people are affected by head and neck cancer (2.4 million in the mouth, 1.7 million in the pharynx, and 1.4 million in the larynx), causing more than 379,000 deaths (GBD.2016a. Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990–2015: a systematic analysis for the Global Burden of Disease Study 2015, accessed 5 Jun 2019, https: / / www.thelancet.com / journals / lancet / article / PIIS0140-6736(16)31678-6 / fulltext; GBD.2016b. Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980–2015: a systematic analysis for the Global Burden of Disease Study 2015, https: / / www.sciencedirect.com / science / article / pii / S0140673616310121, accessed 5 Jun 2019).Worldwide, approximately 600,000 cases of head and neck cancer will occur this year, and only 40% - 60% of patients will survive for five years (Rene Leemans C, et al. The molecular biology of head and neck cancer, Nature Reviews Cancer, 16 Dec 2011, accessed 5 Jun 2019, https: / / www.nature.com / articles / nrc2982).

[0018] The most important risk factors are smoking and alcohol consumption, which seem to have a synergistic effect (Decker & Goldstein, N Engl J Med. 1982;1151 - 1155). A subgroup of head and neck cancers, particularly a subgroup of oropharyngeal cancers, is caused by infection with high - risk types of human papillomavirus (HPV) (Rene Leemans C et al, The molecular biology of head and neck cancer, Nature Reviews Cancer, 16 Dec 2011, accessed 5 Jun 2019, https: / / www.nature.com / articles / nrc2982).

[0019] Treatment is mainly determined by the stage at presentation and may include a combination of surgery, radiotherapy, chemotherapy, and targeted therapy (National Cancer Institute, 2019, Cancer Stat Facts: Cancer of Any Site, https: / / seer.cancer.gov / statfacts / html / all.html, accessed 5 Jun 2019). However, patients often develop local - regional recurrence, distant metastases, and second primary tumors, so survival rates have not improved significantly over the past few decades. The available information regarding the molecular carcinogenesis mechanisms of head and neck cancer, as well as the genetic and biological heterogeneity of this disease, is limited and hinders the development of new treatment strategies.

[0020] Gastric cancer or esophageal cancer In 2019, an estimated 17,650 adult patients will be diagnosed with gastric cancer in the United States, and approximately 16,080 will die from this disease (American Cancer Society, Survival Rates for Esophageal Cancer, 31 Jan 2019c, https: / / www.cancer.org / cancer / esophagus-cancer / detection-diagnosis-staging / survival-rates.html, accessed 6 June 2019). In 2019, an estimated 27,510 adults will be diagnosed with esophageal cancer in the United States, and approximately 11,140 will die from this disease (American Cancer Society, Key Statistics About Stomach Cancer, 09 Jan 2019 d, https: / / www.cancer.org / cancer / stomach-cancer / about / key-statistics.html, accessed 6 June 2019). The proportion of adenocarcinoma of the esophagus and gastric cardia is increasing, while the proportion of squamous cell carcinoma of the esophagus and non-cardia adenocarcinoma of the stomach is decreasing, suggesting different etiologies (Crew & Neugut, World J Gastroenterol. 2016;354-362).

[0021] Chemotherapy can provide a significant reduction in symptoms for patients with unresectable disease, locally advanced disease or metastatic disease. Single agents (cisplatin, doxorubicin and mitomycin) that result in a partial response (PR) rate are considered to be the most active in gastrointestinal (GI) cancers (Preusser P et al, Oncology 1998;99-102). Combination regimens using these agents result in higher response rates (30% - 50%) compared to single-agent therapy, but are associated with a greater degree of toxicity and result in a similar OS (in the range of 6 - 10 months) (Preusser P et al, Oncology 1998;99-102). Therefore, the identification of new agents is essential when an extension of the patient's survival period is to be achieved.

[0022] There is a strong need for additional treatment methods for cancer. These include the use of antibodies and antibody-drug conjugates as treatment modalities.

Summary of the Invention

[0023] 3. Summary In one aspect, provided herein is a method for preventing or treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or antigen-binding fragment thereof comprises a heavy-chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy-chain variable region shown in SEQ ID NO:22, and a light-chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light-chain variable region shown in SEQ ID NO:23, and the subject has hormone receptor-positive and human epidermal growth factor receptor 2-negative (HR+ / HER2-) breast cancer.

[0024] In some embodiments of the methods provided herein, the HR+ / HER2- breast cancer is estrogen receptor (ER)-positive and / or progesterone receptor (PR)-positive and HER2-negative.

[0025] In some embodiments of the methods provided herein, the subject has locally advanced cancer or metastatic cancer.

[0026] In some embodiments of the methods provided herein, the subject has previously received at least one type of endocrine therapy and a cyclin-dependent kinase (CDK) 4 / 6 inhibitor in a metastatic or locally advanced setting.

[0027] In some embodiments of the methods provided herein, the subject has previously received treatment with a taxane or anthracycline.

[0028] In some aspects of the methods provided herein, the subject has a deleterious germline mutation in breast cancer susceptibility gene (BRCA)1 or BRCA2 and has been previously treated with a poly ADP ribose polymerase (PARP) inhibitor.

[0029] 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 conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, and the subject has ER-negative, PR-negative, and HER2-negative (ER- / PR- / HER2-) breast cancer.

[0030] In some aspects of the methods provided herein, e.g., without limitation, in some aspects of the method described in the paragraph immediately preceding this paragraph (paragraph

[0036] ), the subject has locally advanced cancer or metastatic cancer.

[0031] In some aspects of the methods provided herein, the subject has previously received at least two types of systemic therapy.

[0032] In some aspects of the methods provided herein, the subject has previously received treatment with a taxane.

[0033] In some aspects of the methods provided herein, e.g., without limitation, in some aspects of the method described in the paragraphs from four paragraphs preceding this paragraph to the paragraph immediately preceding this paragraph (paragraphs

[0036] -

[0039] ), the subject has a deleterious germline mutation in breast cancer susceptibility gene (BRCA)1 or BRCA2 and has been previously treated with a poly ADP ribose polymerase (PARP) inhibitor.

[0034] 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 that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, and the subject has squamous non-small cell lung cancer (NSCLC).

[0035] In some embodiments of the methods provided herein, for example, without limitation, in some aspects of the method described in the paragraph immediately preceding this paragraph (paragraph

[0041] ), the subject has locally advanced cancer or metastatic cancer.

[0036] In some embodiments of the methods provided herein, the subject has progressed or relapsed after platinum-based therapy.

[0037] In some embodiments of the methods provided herein, the subject has progressed or relapsed within 12 months after platinum-based therapy.

[0038] In some embodiments of the methods provided herein, the subject has previously been treated with an inhibitor of programmed cell death protein-1 (PD-1) or an inhibitor of programmed cell death-ligand 1 (PD-L1), optionally the inhibitor of PD-1 is nivolumab, and optionally the inhibitor of PD-L1 is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0039] 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 that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, and the subject has non-squamous NSCLC.

[0040] In some embodiments of the methods provided herein, the subject has wild-type epidermal growth factor receptor (EGFR) and wild-type anaplastic lymphoma kinase (ALK).

[0041] In some embodiments of the methods provided herein, for example, without limitation, the methods described in the paragraphs one before to two before this paragraph (paragraphs

[0046] -

[0047] ), the subject has locally advanced cancer or metastatic cancer.

[0042] In some embodiments of the methods provided herein, for example, without limitation, the methods described in the paragraphs one before to three before this paragraph (paragraphs

[0046] -

[0048] ), the subject has progressed or relapsed after platinum-based therapy.

[0043] In some embodiments of the methods provided herein, for example, without limitation, the methods described in the paragraphs one before to four before this paragraph (paragraphs

[0046] -

[0049] ), the subject has progressed or relapsed within 12 months after platinum-based therapy.

[0044] In some aspects of the methods provided herein, such as, without limitation, some aspects of the methods described in the paragraphs five paragraphs before to one paragraph before this paragraph (paragraphs

[0046] to

[0050] ), the subject has previously received treatment with an inhibitor of programmed cell death protein-1 (PD-1) or an inhibitor of programmed cell death-ligand 1 (PD-L1), optionally the inhibitor of PD-1 is nivolumab, and optionally the inhibitor of PD-L1 is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0045] 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 that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, and the subject has locally advanced or metastatic head and neck cancer.

[0046] In some aspects of the methods provided herein, such as, without limitation, some aspects of the methods described in the paragraph one paragraph before this paragraph (paragraph

[0052] ), the subject has progressed or relapsed after platinum-based therapy.

[0047] In some aspects of the methods provided herein, the subject has progressed or relapsed within 6 months after platinum-based therapy.

[0048] In some embodiments of the methods provided herein, for example, without limitation, the methods described in the paragraphs one to three paragraphs before this paragraph (paragraphs

[0052] to

[0054] ), the subject has previously received treatment with an inhibitor of programmed cell death protein-1 (PD-1) or an inhibitor of programmed cell death-ligand 1 (PD-L1), optionally the inhibitor of PD-1 is nivolumab, and optionally the inhibitor of PD-L1 is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0049] 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 conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, and the subject has gastric cancer or esophageal cancer.

[0050] In some embodiments of the methods provided herein, for example, without limitation, in some embodiments of the method described in the paragraph one paragraph before this paragraph (paragraph

[0056] ), the subject has locally advanced cancer or metastatic cancer.

[0051] In some embodiments of the methods provided herein, the subject has progressed or relapsed after platinum-based therapy or chemotherapy comprising fluoropyrimidine.

[0052] In some embodiments of the methods provided herein, the subject has progressed or relapsed within 6 months after platinum-based therapy or chemotherapy comprising fluoropyrimidine.

[0053] In some embodiments of the methods provided herein, the gastric cancer or esophageal cancer is a HER2-positive cancer and the subject has previously received HER2-directed therapy.

[0054] In some embodiments of the methods provided herein, 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.

[0055] In some embodiments of the methods provided herein, 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.

[0056] In some embodiments of the methods provided herein, 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.

[0057] In some embodiments of the methods provided herein, the antibody comprises a heavy chain comprising the amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO:7 and a light chain comprising the amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO:8.

[0058] In some embodiments of the methods provided herein, the antigen-binding fragment is a Fab, F(ab')2, Fv or scFv fragment.

[0059] In some embodiments of the methods provided herein, the antibody is a fully human antibody.

[0060] In some embodiments of the methods provided herein, the antibody or its antigen-binding fragment is recombinantly produced.

[0061] In some embodiments of the methods provided herein, the antibody-drug conjugate has the following structure: TIFF2025106332000002.tif36160, where L- represents an antibody or its antigen-binding fragment and p is from 1 to 10.

[0062] In some embodiments of the methods provided herein, p is from 2 to 8.

[0063] In some embodiments of the methods provided herein, p is from 3 to 5.

[0064] In some embodiments of the methods provided herein, the antibody or antigen-binding fragment is linked to each unit of monomethyl auristatin E (MMAE) via a linker.

[0065] In some embodiments of the methods provided herein, the linker is an enzymatically cleavable linker and forms a bond with a sulfur atom of the antibody or its antigen-binding fragment.

[0066] In some embodiments of the methods provided herein, the linker has the formula -Aa-Ww-Yy-, wherein -A- is an extension unit, a is 0 or 1, -W- is an amino acid unit, w is an integer in the range of 0 to 12, -Y- is a spacer unit, and y is 0, 1, or 2.

[0067] In some embodiments of the methods provided herein, the elongation unit has the structure of formula (1) below, the amino acid unit is valine citrulline, and the spacer unit is a PAB group containing the structure of formula (2) below. TIFF2025106332000003.tif69128

[0068] In some embodiments of the methods provided herein, the elongation unit forms a bond with the sulfur atom of an antibody or an antigen-binding fragment thereof, and the spacer unit is linked to MMAE via a carbamate group.

[0069] In some embodiments of the methods provided herein, the antibody-drug conjugate comprises 1 to 10 units of MMAE per antibody or antigen-binding fragment thereof.

[0070] In some embodiments of the methods provided herein, the antibody-drug conjugate comprises 2 to 8 units of MMAE per antibody or antigen-binding fragment thereof.

[0071] In some embodiments of the methods provided herein, the antibody-drug conjugate comprises 3 to 5 units of MMAE per antibody or antigen-binding fragment thereof.

[0072] In some embodiments of the methods provided herein, the antibody-drug conjugate is formulated into a pharmaceutical composition comprising a pharmaceutically acceptable excipient comprising L-histidine, polysorbate-20 (TWEEN-20), and at least one of trehalose dihydrate and sucrose.

[0073] In some embodiments of the methods provided herein, the pharmaceutical composition comprises the antibody-drug conjugate at a concentration of 1 to 20 mg / mL, 5 to 15 mg / mL, or 8 to 12 mg / mL.

[0074] In some embodiments of the methods provided herein, the antibody-drug conjugate is at a concentration of about 10 mg / mL.

[0075] In some embodiments of the methods provided herein, L-histidine is present in the range of 5 to 50 mM, 10 to 40 mM, 15 to 35 mM, 15 to 30 mM, or 15 to 25 mM.

[0076] In some embodiments of the methods provided herein, L-histidine is present at about 20 mM.

[0077] In some embodiments of the methods provided herein, the concentration of TWEEN-20 is in the range of 0.001 to 0.1% (v / v), 0.0025 to 0.075% (v / v), 0.005 to 0.05% (v / v), or 0.01 to 0.03% (v / v).

[0078] In some embodiments of the methods provided herein, the concentration of TWEEN-20 is about 0.02% (v / v).

[0079] In some embodiments of the methods provided herein, the pharmaceutical composition comprises trehalose dihydrate.

[0080] In some embodiments of the methods provided herein, trehalose dihydrate is present in the range of 1 to 20% (w / v), 2 to 15% (w / v), 3 to 10% (w / v), or 4 to 6% (w / v).

[0081] In some embodiments of the methods provided herein, trehalose dihydrate is present at about 5.5% (w / v).

[0082] In some embodiments of the methods provided herein, trehalose dihydrate is present in the range of 50 mM to 300 mM, 75 mM to 250 mM, 100 mM to 200 mM, or 130 mM to 150 mM.

[0083] In some embodiments of the methods provided herein, trehalose dihydrate is present at about 146 mM.

[0084] In some embodiments of the methods provided herein, the pharmaceutical composition comprises sucrose.

[0085] In some embodiments of the methods provided herein, sucrose is present in the range of 1-20% (w / v), 2-15% (w / v), 3-10% (w / v), or 4-6% (w / v).

[0086] In some embodiments of the methods provided herein, sucrose is present at about 5.5% (w / v).

[0087] In some embodiments of the methods provided herein, sucrose is present in the range of 50 mM - 300 mM, 75 mM - 250 mM, 100 mM - 200 mM, or 130 mM - 150 mM.

[0088] In some embodiments of the methods provided herein, sucrose is present at about 146 mM.

[0089] In some embodiments of the methods provided herein, the pharmaceutical composition has a pH in the range of 5.5 - 6.5 or 5.7 - 6.3.

[0090] In some embodiments of the methods provided herein, the pharmaceutical composition has a pH of about 6.0.

[0091] In some embodiments of the methods provided herein, the pH is measured at room temperature, 15°C - 27°C, about 4°C, or about 25°C.

[0092] In some embodiments of the methods provided herein, the pharmaceutical composition contains hydrochloric acid (HCl) or succinic acid.

[0093] In some embodiments of the methods provided herein, the pharmaceutical composition contains about 20 mM L - histidine, about 0.02% (w / v) TWEEN - 20, about 5.5% (w / v) trehalose dihydrate, and HCl, and the pH is about 6.0 at 25°C.

[0094] In some aspects of the methods provided herein, the pharmaceutical composition comprises about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and succinic acid, and the pH is about 6.0 at 25°C.

[0095] In some aspects of the methods provided herein, the pharmaceutical composition comprises about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) sucrose, and HCl, and the pH is about 6.0 at 25°C.

[0096] In some aspects of the methods provided herein, the pharmaceutical composition comprises about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) sucrose, and succinic acid, and the pH is about 6.0 at 25°C.

[0097] In some aspects of the methods provided herein, the antibody-drug conjugate is administered at a dose of 1-10 mg / kg of subject body weight, 1-5 mg / kg of subject body weight, 1-2.5 mg / kg of subject body weight, or 1-1.25 mg / kg of subject body weight.

[0098] In some aspects of the methods provided herein, the antibody-drug conjugate is administered at a dose of about 1 mg / kg of subject body weight.

[0099] In some aspects of the methods provided herein, the antibody-drug conjugate is administered at a dose of about 1.25 mg / kg of subject body weight.

[0100] In some aspects of the methods provided herein, the antibody-drug conjugate is administered by intravenous (IV) injection or infusion.

[0101] In some aspects of the methods provided herein, the antibody-drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes in a cycle of twice every 3 weeks.

[0102] In some embodiments of the methods provided herein, the antibody-drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes on days 1 and 8 of each 3-week cycle.

[0103] In some embodiments of the methods provided herein, the antibody-drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes in a cycle of three times every 4 weeks.

[0104] In some embodiments of the methods provided herein, the antibody-drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes on days 1, 8, and 15 of each 4-week cycle. [Invention 1001] A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody or an antigen-binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or the antigen-binding fragment thereof comprises a heavy-chain variable region comprising complementarity-determining regions (CDRs) including the amino acid sequences of the CDRs of the heavy-chain variable region shown in SEQ ID NO:22, and a light-chain variable region comprising CDRs including the amino acid sequences of the CDRs of the light-chain variable region shown in SEQ ID NO:23, and the subject has hormone receptor-positive and human epidermal growth factor receptor 2-negative (HR+ / HER2-) breast cancer. Method. [Invention 1002] The method of Invention 1001, wherein the HR+ / HER2- breast cancer is estrogen receptor (ER)-positive and / or progesterone receptor (PR)-positive and HER2-negative. [Invention 1003] The method of Invention 1001 or Invention 1002, wherein the subject has locally advanced cancer or metastatic cancer. [Invention 1004] The method according to any one of 1001 to 1003 of the present invention, wherein the subject has previously received at least one type of endocrine therapy and a cyclin-dependent kinase (CDK) 4 / 6 inhibitor in a metastatic or locally advanced situation. [The present invention 1005] The method according to any one of 1001 to 1004 of the present invention, wherein the subject has previously received treatment with a taxane or an anthracycline. [The present invention 1006] The method according to any one of 1001 to 1005 of the present invention, wherein the subject has a harmful germline mutation in breast cancer susceptibility gene (BRCA) 1 or BRCA2 and has previously been treated with a poly ADP ribose polymerase (PARP) inhibitor. [The present invention 1007] A method for preventing or treating cancer in a subject, comprising the step of administering to the subject an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody or an antigen-binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region 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 the subject has ER-negative, PR-negative, and HER2-negative (ER- / PR- / HER2-) breast cancer (triple-negative breast cancer - TNBC). Method. [The present invention 1008] The method according to 1007 of the present invention, wherein the subject has locally advanced cancer or metastatic cancer. [The present invention 1009] The method according to 1007 or 1008 of the present invention, wherein the subject has previously received at least two types of systemic therapy. [The present invention 1010] The method according to 1009 of the present invention, wherein the subject has previously received treatment with a taxane. [The present invention 1011] A method according to any one of claims 1007 to 1010 of the present invention, wherein the subject has a harmful germline mutation in breast cancer susceptibility gene (BRCA) 1 or BRCA2 and has been previously treated with a poly ADP ribose polymerase (PARP) inhibitor. [Inventive step 1012] A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region 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 the subject has squamous non-small cell lung cancer (NSCLC). Method. [Inventive step 1013] The method according to claim 1012 of the present invention, wherein the subject has locally advanced cancer or metastatic cancer. [Inventive step 1014] The method according to claim 1012 or claim 1013 of the present invention, wherein the subject has progressed or relapsed after platinum-based therapy. [Inventive step 1015] The method according to claim 1014 of the present invention, wherein the subject has progressed or relapsed within 12 months after platinum-based therapy. [Inventive step 1016] The method according to any one of claims 1012 to 1015 of the present invention, wherein the subject has previously been treated with an inhibitor of programmed cell death protein-1 (PD-1) or an inhibitor of programmed cell death-ligand 1 (PD-L1), optionally the inhibitor of PD-1 is nivolumab, and optionally the inhibitor of PD-L1 is selected from the group consisting of atezolizumab, avelumab, and durvalumab. [Inventive step 1017] A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, The antibody-drug conjugate comprises an antibody that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region 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 the subject has non-squamous NSCLC, Method. [Invention 1018] The method of Invention 1017, wherein the subject has wild-type epidermal growth factor receptor (EGFR) and wild-type anaplastic lymphoma kinase (ALK). [Invention 1019] The method of Invention 1017 or Invention 1018, wherein the subject has locally advanced cancer or metastatic cancer. [Invention 1020] The method according to any one of Inventions 1017 to 1019, wherein the subject has progressed or relapsed after platinum-based therapy. [Invention 1021] The method of Invention 1020, wherein the subject has progressed or relapsed within 12 months after platinum-based therapy. [Invention 1022] The method according to any one of Inventions 1017 to 1021, wherein the subject has previously been treated with an inhibitor of programmed cell death protein-1 (PD-1) or an inhibitor of programmed cell death-ligand 1 (PD-L1), optionally the inhibitor of PD-1 is nivolumab, and optionally the inhibitor of PD-L1 is selected from the group consisting of atezolizumab, avelumab, and durvalumab. [Invention 1023] A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, The antibody-drug conjugate comprises an antibody that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), wherein the antibody or antigen-binding fragment thereof comprises a heavy-chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy-chain variable region shown in SEQ ID NO:22, and a light-chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light-chain variable region shown in SEQ ID NO:23, and the subject has locally advanced or metastatic head and neck cancer, Method. [Inventive concept 1024] The method of inventive concept 1023, wherein the subject has progressed or relapsed after platinum-based therapy. [Inventive concept 1025] The method of inventive concept 1024, wherein the subject has progressed or relapsed within 6 months after platinum-based therapy. [Inventive concept 1026] The method according to any one of inventive concepts 1023 to 1025, wherein the subject has previously been treated with an inhibitor of programmed cell death protein-1 (PD-1) or an inhibitor of programmed cell death-ligand 1 (PD-L1), optionally the inhibitor of PD-1 is nivolumab, and optionally the inhibitor of PD-L1 is selected from the group consisting of atezolizumab, avelumab, and durvalumab. [Inventive concept 1027] A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, the antibody-drug conjugate comprises an antibody that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), wherein the antibody or antigen-binding fragment thereof comprises a heavy-chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy-chain variable region shown in SEQ ID NO:22, and a light-chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light-chain variable region shown in SEQ ID NO:23, and the subject has gastric cancer or esophageal cancer, Method. [Invention 1028] The method of Invention 1027, wherein the subject has locally advanced cancer or metastatic cancer. [Invention 1029] The method of Invention 1027 or Invention 1028, wherein the subject has progressed or relapsed after chemotherapy including platinum-based therapy and / or fluoropyrimidine. [Invention 1030] The method of Invention 1029, wherein the subject has progressed or relapsed within 6 months after chemotherapy including platinum-based therapy or fluoropyrimidine. [Invention 1031] The method according to any one of Inventions 1027 to 1030, wherein the gastric cancer or esophageal cancer is a HER2-positive cancer and the subject has previously received HER2-directed therapy. [Invention 1032] The antibody or antigen-binding fragment thereof comprises CDR H1 comprising the amino acid sequence of SEQ ID NO:9, CDR H2 comprising the amino acid sequence of SEQ ID NO:10, CDR H3 comprising the amino acid sequence of SEQ ID NO:11; CDR L1 comprising the amino acid sequence of SEQ ID NO:12, CDR L2 comprising the amino acid sequence of SEQ ID NO:13, and CDR L3 comprising the amino acid sequence of SEQ ID NO:14, or The antibody or antigen-binding fragment thereof comprises CDR H1 comprising the amino acid sequence of SEQ ID NO:16, CDR H2 comprising the amino acid sequence of SEQ ID NO:17, CDR H3 comprising the amino acid sequence of SEQ ID NO:18; CDR L1 comprising the amino acid sequence of SEQ ID NO:19, CDR L2 comprising the amino acid sequence of SEQ ID NO:20, and CDR L3 comprising the amino acid sequence of SEQ ID NO:21, The method according to any one of Inventions 1001 to 1031. [Invention 1033] The method according to any one of Inventions 1001 to 1031, 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. [Invention 1034] Any method of the present invention from 1001 to 1031, wherein the antibody comprises a heavy chain comprising an amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO:7, and a light chain comprising an amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO:8. [The present invention 1035] Any method of the present invention from 1001 to 1034, wherein the antigen-binding fragment is a Fab, F(ab')2, Fv or scFv fragment. [The present invention 1036] Any method of the present invention from 1001 to 1035, wherein the antibody is a fully human antibody. [The present invention 1037] Any method of the present invention from 1001 to 1036, wherein the antibody or its antigen-binding fragment is recombinantly produced. [The present invention 1038] The antibody-drug conjugate has the following structure: TIFF2025106332000004.tif36160, where L- represents an antibody or its antigen-binding fragment, and p is from 1 to 10. Any method of the present invention from 1001 to 1037. [The present invention 1039] Any method of the present invention 1038, wherein p is from 2 to 8. [The present invention 1040] Any method of the present invention 1038, wherein p is from 3 to 5. [The present invention 1041] Any method of the present invention from 1001 to 1040, wherein the antibody or antigen-binding fragment is linked to each unit of monomethyl auristatin E (MMAE) via a linker. [The present invention 1042] Any method of the present invention 1041, wherein the linker is an enzyme-cleavable linker and forms a bond with the sulfur atom of the antibody or its antigen-binding fragment. [The present invention 1043] The linker is -A a -W w -Y y- having the formula, wherein -A- is an extension unit, a is 0 or 1, -W- is an amino acid unit, w is an integer in the range of 0 to 12, -Y- is a spacer unit, and y is 0, 1, or 2, the method of the present invention 1041. [The present invention 1044] The method of the present invention 1043, wherein the extension unit has the structure of the following formula (1), the amino acid unit is valine citrulline; and the spacer unit is a PAB group containing the structure of the following formula (2): TIFF2025106332000005.tif78128. [The present invention 1045] The method of the present invention 1043, wherein the extension unit forms a bond with the sulfur atom of an antibody or its antigen-binding fragment, and the spacer unit is linked to MMAE via a carbamate group. [The present invention 1046] The method according to any one of the present inventions 1001 to 1037, wherein the antibody-drug conjugate contains 1 to 10 units of MMAE per antibody or its antigen-binding fragment. [The present invention 1047] The method according to any one of the present inventions 1001 to 1037, wherein the antibody-drug conjugate contains 2 to 8 units of MMAE per antibody or its antigen-binding fragment. [The present invention 1048] The method according to any one of the present inventions 1001 to 1037, wherein the antibody-drug conjugate contains 3 to 5 units of MMAE per antibody or its antigen-binding fragment. [The present invention 1049] The method according to any one of the present inventions 1001 to 1048, wherein the antibody-drug conjugate is administered at a dose of 1 to 10 mg / kg of the subject body weight, 1 to 5 mg / kg of the subject body weight, 1 to 2.5 mg / kg of the subject body weight, or 1 to 1.25 mg / kg of the subject body weight. [The present invention 1050] The method of the present invention 1049, wherein the antibody-drug conjugate is administered at a dose of about 1 mg / kg of the subject body weight. [The present invention 1051] The method of the present invention 1049, wherein the antibody-drug conjugate is administered at a dose of about 1.25 mg / kg of the subject body weight. [The present invention 1052] Any method of the present invention from 1001 to 1051, wherein the antibody-drug conjugate is administered by intravenous (IV) injection or infusion. [The present invention 1053] Any method of the present invention from 1001 to 1051, wherein the antibody-drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes in a cycle of twice every three weeks. [The present invention 1054] Any method of the present invention from 1001 to 1051, wherein the antibody-drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes on the first day and the eighth day of each three-week cycle. [The present invention 1055] Any method of the present invention from 1001 to 1051, wherein the antibody-drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes in a cycle of three times every four weeks. [The present invention 1056] Any method of the present invention from 1001 to 1051, wherein the antibody-drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes on the first day, the eighth day, and the fifteenth day of each four-week cycle.

Brief Description of the Drawings

[0105] 4. Brief Description of the Drawings

Figure 1A-1

Figure 1A-2

Figure 1B

Figure 1C

Figure 1D

Figure 1E

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Figure 4-2

Mode for Carrying Out the Invention

[0106] 5. Detailed Description Before further describing the present disclosure, it should be understood that the present disclosure is not limited to the specific embodiments described herein, and that the terms used herein are for the purpose of describing only specific embodiments and are not intended to be limiting.

[0107] 5.1 Definitions The techniques and procedures described or referenced herein include those commonly understood and / or generally employed by those skilled in the art using conventional methodologies, such as widely used methods described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2d ed. 2010).

[0108] Unless otherwise specifically defined herein, technical and scientific terms used in this description have the meanings commonly understood by those skilled in the art. For the purpose of interpreting this specification, the following explanations of terms apply, and where appropriate, terms used in the singular form also include the plural form and vice versa. In the event of a conflict between the explanations of the terms described and the documents incorporated herein by reference, the explanations of the terms described below shall prevail.

[0109] The terms "antibody", "immunoglobulin", or "Ig" are used interchangeably herein and are used in the broadest sense, and specifically include, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies) as described below, antibody compositions having polyepitope specificity or monoepitope specificity, polyclonal antibodies or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies as long as they exhibit the desired biological activity), single-chain antibodies, and fragments thereof. Antibodies can be human, humanized, chimeric and / or affinity matured antibodies, as well as antibodies from other species, such as mice and rabbits. The term "antibody" is intended to include the polypeptide products of B cells within immunoglobulins that can bind to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids and the carboxy-terminal portion of each chain containing a constant region. See, for example, Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997). In a specific embodiment, the specific molecular antigen can be bound by the antibodies provided herein, which include polypeptides or epitopes. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotype (anti-Id) antibodies, and any functional fragments (e.g., antigen-binding fragments) of the above, where a functional fragment refers to a portion of a heavy or light chain polypeptide of an antibody that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fv (scFv) (including, for example, single-specificity, bispecificity, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fv (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetra-bodies, and minibodies.In particular, the antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules that include antigen-binding sites that bind to an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibody can be an agonist antibody or an antagonist antibody.

[0110] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of 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 and are directed against a single antigenic site. In contrast to polyclonal antibody preparations, which can include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0111] An "antigen" is a structure to which an antibody can selectively bind. The target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other natural or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell and is present, for example, on or within the cell, such as on or within a cancer cell.

[0112] An "intact" antibody includes an antigen-binding site as well as the CL and at least the heavy chain constant regions CH1, CH2, and CH3. The constant regions can include human constant regions or amino acid sequence variants thereof. In certain embodiments, the intact antibody has one or more effector functions.

[0113] The terms "antigen-binding fragment", "antigen-binding domain", "antigen-binding region" and similar terms refer to a portion of an antibody (e.g., a CDR) that interacts with an antigen and confers specificity and affinity for the antigen to the binding substance. As used herein, "antigen-binding fragment" includes "antibody fragments" that include a portion of an intact antibody, e.g., the antigen-binding region or variable region of an intact antibody. Examples of antibody fragments include, without limitation, Fab, Fab', F(ab')2, and Fv fragments; diabodies and di-diabodies (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. 90:6444-48; Lu et al., 2005, J. Biol. Chem. 280:19665-72; Hudson et al., 2003, Nat. Med. 9:129-34; International Publication No. 93 / 11161; and U.S. Pat. Nos. 5,837,242 and 6,492,123); single-chain antibody molecules (see, e.g., U.S. Pat. Nos. 4,946,778; 5,260,203; 5,482,858; and 5,476,786); dual variable domain antibodies (see, e.g., U.S. Pat. No. 7,612,181); single variable domain antibodies (sdAb) (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.

[0114] The term "bind" or "binding" refers to intermolecular interactions, including, for example, forming a complex. The interactions can be non-covalent interactions including, for example, hydrogen bond interactions, ionic bond interactions, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the association of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of all non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule such as an antigen is the affinity of the antibody or functional fragment for that epitope. The ratio of the dissociation rate (k off ) to the association rate (k on ) of a binding molecule (e.g., an antibody) to a monovalent antigen (k off / k on ) is the dissociation constant K D , which is inversely proportional to the affinity. The lower the K D value, the higher the affinity of the antibody. The value of K D is different for different complexes of antibody and antigen and depends on both k on and k off . The dissociation constant K D of the antibodies provided herein can be determined using any method provided herein or any other method well known to those of skill in the art. The affinity at one binding site does not necessarily reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants such as a multivalent antigen contacts an antibody containing multiple binding sites, the interaction between the antibody and the antigen at one site increases the probability of reaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity.

[0115] In connection with the antibodies or antigen-binding fragments thereof described herein, terms such as "binds to", "specifically binds to", and similar terms are also used interchangeably herein and refer to a binding molecule of an antigen-binding domain that specifically binds to an antigen such as a polypeptide. An antibody or antigen-binding fragment that binds to or specifically binds to an antigen may be cross-reactive with a related antigen. In certain embodiments, an antibody or antigen-binding fragment that binds to or specifically binds to an antigen does not cross-react with other antigens. An antibody or antigen-binding fragment that binds to or specifically binds to an antigen can be identified, for example, by immunoassay, Octet®, Biacore®, or other techniques known to those of skill in the art. In some embodiments, an antibody or antigen-binding fragment binds to an antigen with a higher affinity than any cross-reactive antigen determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), and thus binds to or specifically binds to the antigen. Typically, a specific or selective reaction is at least twice the background signal or noise, and may exceed 10-fold the background. For considerations regarding binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In certain embodiments, the degree of binding of an antibody or antigen-binding fragment to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its specific target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. Terms such as "specific binding", "specifically binds to", or "is specific for" mean binding that measurably differs from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule as compared to the binding of a control molecule, which is a molecule of a similar structure that generally does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by the excess of unlabeled target.Antibodies or antigen-binding fragments that bind to an antigen include those that can bind to the antigen with sufficient affinity such that the binding molecule is useful, for example, as a diagnostic agent when targeting the antigen. In certain embodiments, the antibody or antigen-binding fragment that binds to the antigen has a dissociation constant (K D ) of less than 1000 nM, less than 800 nM, less than 500 nM, less than 250 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM, or 1000 nM, 800 nM, 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In certain embodiments, the antibody or antigen-binding fragment binds to an epitope of an antigen that is conserved between antigens from different species (e.g., between the human species and the cynomolgus monkey species).

[0116] "Binding affinity" generally refers to the strength of the sum of non-covalent interaction between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, whereas high-affinity antibodies generally bind to antigens more rapidly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of the present disclosure. Specific exemplary embodiments include the following. In one embodiment, "K D " or "K DThe "value" can be measured by assays known in the art, such as binding assays. K D K can be measured, for example, by RIA performed using the Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865 - 81). K D Or K D The value can also be measured by, for example, Octet® using the Octet® QK384 system, or by Biacore® using Biacore® TM - 2000 or Biacore® TM - 3000, by using a biolayer interferometry (BLI) or surface plasmon resonance (SPR) assay. The "on - rate" or "rate of association" or "association rate" or "kon" can also be determined, for example, using the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques as described above, using an Octet® QK384, Biacore® TM - 2000, or Biacore® TM - 3000 system.

[0117] In certain embodiments, the antibody or antigen - binding fragment can include a "chimeric" sequence in which a portion of the heavy chain and / or light chain is identical or homologous to the 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(s) is identical or homologous to the corresponding sequence in an antibody derived from a different species or belonging to a different antibody class or subclass, and fragments of such antibodies can be included as long as they exhibit the desired biological activity (see U.S. Patent No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851 - 55).

[0118] In certain embodiments, the antibody or antigen-binding fragment can comprise a portion of a "humanized" form of a non-human (e.g., murine) antibody that comprises a chimeric antibody in which the native CDR residues are replaced with residues from the corresponding CDRs (e.g., donor antibody) of a non-human species such as mouse, rat, rabbit, or non-human primate, wherein the chimeric antibody comprises a human immunoglobulin (e.g., recipient antibody). Optionally, one or more FR region residues of the human immunoglobulin are replaced with the corresponding non-human residues. Further, the humanized antibody can comprise residues not found in the recipient antibody or donor antibody. These modifications are made to further refine antibody performance. The heavy or light chain of the humanized antibody can comprise substantially all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to the CDRs of the non-human immunoglobulin and all or substantially all of the FRs are the FRs of the human immunoglobulin sequence. In certain embodiments, the humanized antibody comprises at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region (Fc) of a human immunoglobulin. For further details, see Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-29; Presta, 1992, Curr.Op.Struct.Biol.2:593-96; Carter et al., 1992, Proc.Natl.Acad.Sci.USA 89:4285-89; U.S. Patent Nos. 6,800,738; 6,719,971; 6,639,055; 6,407,213; and 6,054,297.

[0119] In certain embodiments, an antibody or antigen-binding fragment can include a portion of a "fully human antibody" or "human antibody", and these terms are used interchangeably herein to refer to an antibody that includes human variable regions and, for example, human constant regions. In specific embodiments, these terms refer to an antibody that includes variable and constant regions of human origin. A "fully human" antibody can, in certain embodiments, also encompass an antibody that binds to a polypeptide and is encoded by a nucleic acid sequence that is a somatic variant of a naturally occurring human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies that include variable and constant regions corresponding to the human germline immunoglobulin sequences described by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is one that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or is produced using any of the techniques for making human antibodies. This definition of a human antibody specifically excludes humanized antibodies that include non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581) and yeast display libraries (Chao et al., 2006, Nature Protocols 1:755-68). The methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boerner et al., 1991, J. Immunol. 147(1):86-95; and van Dijk and van de Winkel, 2001, Curr. Opin. Pharmacol. 5:368-74 are also available for the preparation of human monoclonal antibodies.Human antibodies can be prepared by administering an antigen to a transgenic animal, such as a mouse, that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous locus has been inactivated (see, e.g., Jakobovits, 1995, Curr. Opin. Biotechnol. 6(5):561-66; Bruggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li et al., 2006, Proc. Natl. Acad. Sci. USA 103:3557-62 regarding human antibodies generated via human B cell hybridoma technology.

[0120] In certain embodiments, the antibody or antigen-binding fragment can comprise a portion of a "recombinant human antibody," which term refers to a human antibody prepared, expressed, made, or isolated by recombinant means, e.g., an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a recombinant combinatorial human antibody library, an antibody isolated from an animal that is transgenic and / or translchromosomal for human immunoglobulin genes (e.g., see Taylor, L.D. et al. (1992) Nucl. Acids Res. 20:6287-6295), or an antibody prepared, expressed, made, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, if transgenic animals for human Ig sequences are used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, but are sequences that do not naturally occur within the human antibody germline repertoire in vivo.

[0121] In certain embodiments, the antibody or antigen-binding fragment can include a portion of a “monoclonal antibody,” and as used herein, this term refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody typically recognizes a single epitope on an antigen. In specific embodiments, the “monoclonal antibody” as used herein is an antibody produced by a single hybridoma or other cell. The term “monoclonal” is not limited to a particular method for making the antibody. For example, monoclonal antibodies useful in the present disclosure can be prepared by the hybridoma method first described by Kohler et al., 1975, Nature 256:495, or can be made using recombinant DNA methods in bacteria or eukaryotic or plant cells (see, e.g., U.S. Patent No. 4,816,567). “Monoclonal antibodies” can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., 1991, Nature 352:624-28 and Marks et al., 1991, J. Mol. Biol. 222:581-97. Other methods for preparing clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art. See, e.g., Short Protocols in Molecular Biology (Ausubel et al. eds., 5th ed. 2002).

[0122] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the four-chain unit generally weighs about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, and the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H chain and L chain also have regularly spaced intra-chain disulfide cross-bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ isotype and ε isotype. Each L chain has a variable domain (VL) at the N-terminus and a constant domain (CL) at the other terminus. VL aligns with VH, and CL aligns with the first constant domain (CH1) of the heavy chain. Certain amino acid residues are thought to form the contact surface between the light chain variable domain and the heavy chain variable domain. The pairing of VH and VL forms a single antigen-binding site. For the structure and properties of various classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds., 5 th ed. 2001).

[0123] The term "Fab" or "Fab region" refers to the antibody region that binds to an antigen. Conventional IgG typically contains two Fab regions, each present in one of the two arms of the Y-shaped IgG structure. Each Fab region is typically composed of one variable region and one constant region of each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. VH, CH1, VL, and CL within the Fab region can be arranged in various ways to confer antigen-binding ability according to the present disclosure. For example, similar to the Fab region of conventional IgG, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on separate polypeptides. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and can be oriented in different orders as will be described in more detail in the following sections.

[0124] The terms "variable region", "variable domain", "V region" or "V domain" generally refer to a part of the light or heavy chain of an antibody that is located at the amino terminus of the chain, has a length of about 120 to about 130 amino acids in the heavy chain and about 100 to about 110 amino acids in the light chain, and is used for the binding and specificity of each specific antibody to a 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 the specific segments of the variable regions vary greatly in sequence among antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for a particular antigen. However, the variability is not uniformly distributed over the 110 amino acid span of the variable region. Instead, the V region consists of stretches of lower variability (e.g., relatively invariant), called framework regions (FRs), of about 15 to about 30 amino acids, separated by shorter regions of greater variability (e.g., extreme variability), called "hypervariable regions", each of which is about 9 to 12 amino acids long. The variable regions of the heavy and light chains each contain four FRs that predominantly adopt a β-sheet conformation, connected by three hypervariable regions that form loops connecting the β-sheet structures and in some cases forming part of the β-sheet structure. The hypervariable regions of each chain are held together in proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed. 1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions such as the involvement of the antibody in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions vary greatly in sequence among different antibodies. In a specific embodiment, the variable region is a human variable region.

[0125] The term "Kabat variable region residue numbering" or "amino acid position numbering as in Kabat", and variations thereof, refers to the numbering system used for the heavy chain variable region or light chain variable region of the antibody repertoire in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of the FR or CDR or insertion into the FR or CDR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insert (residue 52a according to Kabat) after residue 52 and three inserted residues (e.g., residues 82a, 82b, and 82c according to Kabat, etc.) after residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment in the region of homology between the antibody's sequence and the "standard" Kabat numbering sequence. The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is generally used when referring to residues within the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems are described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0126] As used in reference to an antibody, the term "heavy chain" refers to a polypeptide chain of about 50-70 kDa, the amino-terminal portion of which contains a variable region of about 120-130 or more amino acids and the carboxy-terminal portion of which contains a constant region. The constant region can be one of five different types (e.g., isotypes) designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ) based on the amino acid sequence of the heavy chain constant region. Different heavy chains vary in size, with α, δ, and γ containing about 450 amino acids and μ and ε containing about 550 amino acids. When combined with light chains, these different types of heavy chains give rise to the five well-known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, including the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4.

[0127] As used in reference to an antibody, the term "light chain" refers to a polypeptide chain of about 25 kDa, the amino-terminal portion of which contains a variable region of about 100 to about 110 or more amino acids and the carboxy-terminal portion of which contains a constant region. The approximate length of the light chain is from 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types designated kappa (κ) or lambda (λ).

[0128] As used herein, the terms "hypervariable region", "HVR", "complementary determining region", and "CDR" are used interchangeably. A "CDR" refers to one of three hypervariable regions (H1, H2, or H3) within the non-framework regions of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or one of three hypervariable regions (L1, L2, or L3) within the non-framework regions of the VL β-sheet framework of an antibody. Thus, a CDR is a variable region sequence that is interspersed within the framework region sequences.

[0129] The CDR regions are well-known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra). Alternatively, Chothia refers to the positions of structural loops (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering rules varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dubel eds., 2d ed. 2010)). The "contact" hypervariable regions are based on the analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). IMGT is an integrated information system specialized for human and other vertebrate immunoglobulins (IG), T cell receptors (TCR), and major histocompatibility complex (MHC). In this specification, CDRs are referred to with respect to both the amino acid sequence and the position within the light or heavy chain. The "position" of the CDRs within the structure of the immunoglobulin variable domain is conserved across species and is present within structures called loops, so that CDRs and framework residues can be readily identified by using a numbering system that aligns the variable domain sequences according to structural features.This information can be used when transplanting and substituting CDR residues from one species of immunoglobulin into an acceptor framework, typically from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, 2001, J. Mol. Biol. 309: 657-70. For example, the correspondence between numbering systems including Kabat numbering and the IMGT-specific numbering system is well-known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). The residues from each of these hypervariable regions or CDRs are shown below.

[0130] (Table 1) TIFF2025106332000006.tif77137

[0131] The boundaries of a given CDR can vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms "CDR" and "complementary determining region" of a given antibody or region thereof, such as the variable region, and the individual CDRs of an antibody or region thereof (e.g., "CDR-H1, CDR-H2") are to be understood to encompass the complementary determining regions defined by any of the known schemes described hereinabove. In some cases, a scheme for identifying one or more specific CDRs, such as CDRs defined by the Kabat, Chothia, or Contact methods, is specified. In other cases, a specific amino acid sequence of the CDR is given.

[0132] The hypervariable regions can include "extended hypervariable regions" such as: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0133] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains that do not directly participate in the binding of an antibody to an antigen but exhibit various effector functions such as interaction with Fc receptors. This term refers to the portion of the immunoglobulin molecule that contains a more conserved amino acid sequence compared to the other parts of the immunoglobulin that contain the antigen-binding site, the variable region. The constant region can include the CH1, CH2, and CH3 regions of the heavy chain as well as the CL region of the light chain.

[0134] The term "framework" or "FR" refers to the variable region residues adjacent to the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are variable domain residues other than hypervariable region residues or CDR residues.

[0135] The term "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, a native sequence Fc region, a recombinant Fc region, and a variant Fc region. The boundaries of the Fc region of an immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is often defined as extending from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during antibody production or purification, or by recombinant manipulation of the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include an antibody population in which all K447 residues have been removed, an antibody population in which the K447 residues have not been removed, and an antibody population that includes a mixture of antibodies having and not having the K447 residue. A "functional Fc region" has the "effector functions" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors), etc. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or antibody variable domain) and can be evaluated using various assays known to those of skill in the art. A "variant Fc region" includes an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification (e.g., substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid substitution, for example, about 1 to about 10 amino acid substitutions, or about 1 to about 5 amino acid substitutions, in the native sequence Fc region or the Fc region of a parental polypeptide. The variant Fc region as used herein can have at least about 80% homology, or at least about 90% homology, or for example, at least about 95% homology with the native sequence Fc region and / or the Fc region of a parental polypeptide.

[0136] As used herein, "epitope" is a term in the art and refers to a local region of an antigen to which a binding molecule (e.g., an antibody) can specifically bind. An epitope can be a linear epitope, a conformational epitope, a non-linear epitope, or a discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be consecutive amino acids of the polypeptide (a "linear" epitope), or the epitope can include amino acids from two or more non-consecutive regions of the polypeptide (a "conformational", "non-linear", or "discontinuous" epitope). In general, those skilled in the art will understand that linear epitopes may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, the binding molecule binds to a group of amino acids regardless of whether the natural three-dimensional protein structure is folded. In other embodiments, the binding molecule requires amino acid residues that constitute the epitope to assume a particular conformation (e.g., bend, twist, rotation, or fold) in order to recognize and bind the epitope.

[0137] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified, either naturally or by intervention, such as by the formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Polypeptides that include one or more analogs of amino acids, including but not limited to non-natural amino acids, and other modifications known in the art are also included in the definition. Since the polypeptides of the present disclosure can be based on antibodies or other members of the immunoglobulin superfamily, in certain embodiments, it is understood that "polypeptide" can exist as a single chain or as two or more associated chains.

[0138] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or State government or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognized pharmacopeia for use in animals and more particularly in humans.

[0139] "Excipient" means a pharmaceutically acceptable material, composition, or vehicle such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, absorption enhancers, antioxidants, binders, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, extenders, fillers, flavorants, humectants, lubricants, perfumes, preservatives, propellants, release agents, sterilants, sweeteners, solubilizers, wetting agents, and encapsulating materials or additives such as mixtures thereof. The term "excipient" can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete) or vehicle.

[0140] In one aspect, each component is "pharmaceutically acceptable" in the sense that it is compatible with the other components of the pharmaceutical formulation and is suitable for use in contact with human and animal tissues or organs without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some aspects, the pharmaceutically acceptable excipient is non-toxic to the cells or mammals to which it is exposed at the dosage and concentration used. In some aspects, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

[0141] The abbreviation "MMAE" refers to monomethyl auristatin E.

[0142] Unless otherwise specified, the term "alkyl" refers to saturated straight-chain or branched-chain hydrocarbons containing from about 1 to about 20 carbon atoms (as well as all combinations and sub-combinations of ranges and specific numbers of carbon atoms therein), with from about 1 to about 8 carbon atoms being preferred. Examples of alkyl groups are methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.The alkyl group, whether alone or as part of another group, may be substituted with one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including but not limited to -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2, and -CN, where each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, and the -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl groups may be further substituted with one or more groups, including but not limited to -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2, and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0143] Unless otherwise specified, the terms "alkenyl" and "alkynyl" refer to straight-chain and branched carbon chains containing from about 2 to about 20 carbon atoms (as well as all combinations and sub-combinations of ranges and specific numbers of carbon atoms therein), with from about 2 to about 8 carbon atoms being preferred. An alkenyl chain has at least one double bond in the chain, and an alkynyl chain has at least one triple bond in the chain. Examples of alkenyl groups include, but are not limited to, ethylene or vinyl, allyl, -1-butenyl, -2-butenyl, -isobutenylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, and -2,3-dimethyl-2-butenyl. Examples of alkynyl groups include, but are not limited to, acetylene, propargyl, acetylenyl, propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl and -3-methyl-1-butynyl.An alkenyl group and an alkynyl group, either alone or as part of another group, may be substituted with one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including but not limited to -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, where each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, and the -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl groups may be further substituted with one or more substituents, including but not limited to -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0144] Unless otherwise specified, the term "alkylene" refers to a saturated branched or straight-chain hydrocarbon radical containing from about 1 to about 20 carbon atoms (as well as all combinations and sub-combinations of ranges and specific numbers of carbon atoms therein), preferably from about 1 to about 8 carbon atoms, and having two monovalent radical centers derived by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Typical alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decalene, 1,4-cyclohexylene, etc.The alkylene group, whether alone or as part of another group, may be substituted with one or more groups, preferably 1 to 3 groups (and any additional substituents selected from halogen), including, but not limited to, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2 and -CN, wherein each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, and the -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl groups may be further substituted with one or more substituents including, but not limited to, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, wherein each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0145] Unless otherwise specified, the term "alkenylene" refers to an optionally substituted alkylene group containing at least one carbon-carbon double bond. Exemplary alkenylene groups include, for example, ethenylene (-CH=CH-) and propenylene (-CH=CHCH2-).

[0146] Unless otherwise specified, the term "alkynylene" refers to an optionally substituted alkylene group containing at least one carbon-carbon triple bond. Exemplary alkynylene groups include, for example, acetylene (-C≡C-), propargyl (-CH2C≡C-), and 4-pentynyl (-CH2CH2CH2C≡CH-).

[0147] Unless otherwise specified, the term "aryl" refers to a monovalent aromatic hydrocarbon radical of 6 to 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein) derived by removing one hydrogen atom from one carbon atom of the parent aromatic ring system. Some aryl groups are represented as "Ar" in exemplary structures. Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, phenyl, naphthalene, anthracene, biphenyl, and the like.

[0148] The aryl group, either alone or as part of another group, may be substituted with one or more, preferably 1 to 5, more preferably 1 to 2 groups including, but not limited to, -halogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, -NO2, -N3, -NH2, -NH(R'), -N(R')2 and -CN, wherein each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, and the -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), and -aryl groups may be further substituted with one or more substituents including, but not limited to, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, wherein each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0149] Unless otherwise specified, the term "arylene" refers to an optionally substituted aryl group that is divalent (i.e., derived by removing two hydrogen atoms from the same or two different carbon atoms of the parent aromatic ring system), and can be in the ortho, meta, or para configuration as shown in the following structures having phenyl as an exemplary aryl group. TIFF2025106332000007.tif22128Typical "-(C1-C8 alkylene) aryl", "-(C2-C8 alkenylene) aryl", and "-(C2-C8 alkynylene) aryl" groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and the like.

[0150] Unless otherwise specified, the term "heterocyclic ring" refers to a monocyclic, bicyclic, or polycyclic ring system having 3 to 14 ring atoms (also referred to as ring members), and at least one ring atom of at least one ring is a heteroatom selected from N, O, P, or S (as well as all combinations and partial combinations of ranges and specific numbers of carbon atoms and heteroatoms therein). The heterocyclic ring can have 1 to 4 ring heteroatoms independently selected from N, O, P, or S. One or more N, C, or S atoms in the heterocyclic ring can be oxidized. The monocyclic heterocyclic ring preferably has 3 to 7 ring members (e.g., 2 to 6 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P, or S), and the bicyclic heterocyclic ring preferably has 5 to 10 ring members (e.g., 4 to 9 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P, or S). The ring containing heteroatoms can be aromatic or non-aromatic. Unless otherwise specified, the heterocyclic ring is attached to its pendant group by any heteroatom or carbon atom that results in a stable structure. The heterocyclic ring is described in Paquette, "Principles of Modern Heterocyclic Chemistry" (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 - present), particularly Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. 82:5566 (1960).Examples of the "heterocyclic ring" group include, by way of example and without limitation, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidinyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiadiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4H-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phthalazinyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxyindolyl, benzoxazolinyl, and isatinoyl. Preferred "heterocyclic ring" groups include, but are not limited to, benzofuranyl, benzothiophenyl, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, and tetrazolyl.The complex ring group, either alone or as part of another group, may be substituted with one or more groups, preferably 1 to 2 groups, including but not limited to -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, -N3, -NH2, -NH(R'), -N(R')2 and -CN, where each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, and the -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, and -aryl groups may be further substituted with one or more substituents including but not limited to -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2 and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or aryl.

[0151] By way of example and not limitation, the carbon-bonded heterocycle can be bonded at the following positions: the 2-, 3-, 4-, 5-, or 6-position of pyridine; the 3-, 4-, 5-, or 6-position of pyridazine; the 2-, 4-, 5-, or 6-position of pyrimidine; the 2-, 3-, 5-, or 6-position of pyrazine; the 2-, 3-, 4-, or 5-position of furan, tetrahydrofuran, thiophene, thienyl, pyrrole, or tetrahydropyrrole; the 2-, 4-, or 5-position of oxazole, imidazole, or thiazole; the 3-, 4-, or 5-position of isoxazole, pyrazole, or isothiazole; the 2- or 3-position of aziridine; the 2-, 3-, or 4-position of azetidine; the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of quinoline; or the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of isoquinoline. Even more typically, the carbon-bonded heterocycle includes 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

[0152] By way of example and not limitation, the nitrogen-bonded heterocycle can be bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, or 1H-indazole; the 2-position of isoindole or isoindoline; the 4-position of morpholine; and the 9-position of carbazole or β-carboline. Even more typically, the nitrogen-bonded heterocycle includes 1-aziridyl, 1-azetyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0153] Unless otherwise specified, the term "carbocyclic ring" refers to a saturated or unsaturated non-aromatic monocyclic, bicyclic, or polycyclic ring system having from 3 to 14 ring atoms (and all combinations and sub-combinations of the ranges and specific numbers of carbon atoms therein), and all ring atoms are carbon atoms. The monocyclic carbocyclic ring preferably has from 3 to 6 ring atoms, more preferably 5 or 6 ring atoms. The bicyclic carbocyclic ring preferably has, for example, 7 to 12 ring atoms arranged as a bicyclo[4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged as a bicyclo[5,6] or [6,6] system. The term "carbocyclic ring" includes, for example, a monocyclic carbocyclic ring fused to an aryl ring (e.g., a monocyclic carbocyclic ring fused to a benzene ring). The carbocyclic ring preferably has from 3 to 8 carbocyclic ring atoms.The carbon ring group, either alone or as part of another group, may be substituted with one or more groups, preferably one or two groups (and any additional substituents selected from halogen), including but not limited to, for example, -halogen, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH2, -C(O)NHR', -C(O)N(R')2, -NHC(O)R', -SR', -SO3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2, and -CN, where each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, and the -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), and -aryl groups may be further substituted with one or more substituents including but not limited to, for example, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, -halogen, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH2, -C(O)NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SO3R'', -S(O)2R'', -S(O)R'', -OH, -N3, -NH2, -NH(R''), -N(R'')2, and -CN, where each R'' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl.

[0154] Examples of monocyclic carbocyclic substituents include - cyclopropyl, - cyclobutyl, - cyclopentyl, - 1 - cyclopenta - 1 - enyl, - 1 - cyclopent - 2 - enyl, - 1 - cyclopent - 3 - enyl, cyclohexyl, - 1 - cyclohex - 1 - enyl, - 1 - cyclohex - 2 - enyl, - 1 - cyclohex - 3 - enyl, - cycloheptyl, - cyclooctyl, - 1,3 - cyclohexadienyl, - 1,4 - cyclohexadienyl, - 1,3 - cycloheptadienyl, - 1,3,5 - cycloheptatrienyl, and - cyclooctadienyl.

[0155] "Carbocyclo", whether used alone or as part of another group, refers to a divalent (i.e., derived by removing two hydrogen atoms from the same or two different carbon atoms of the parent carbocyclic system) optionally substituted carbocyclic group as defined above.

[0156] Unless otherwise indicated in the context, a hyphen (-) designates the point of attachment to a pendant molecule. Thus, the terms "-(C1 - C8 alkylene)aryl" or "-C1 - C8 alkylene(aryl)" refer to a C1 - C8 alkylene radical as defined herein, where the alkylene radical is attached to the pendant molecule at any of the carbon atoms of the alkylene radical, and one of the hydrogen atoms attached to the carbon atoms of the alkylene radical is replaced by an aryl radical as defined herein.

[0157] When a particular group is "substituted", the group can have one or more substituents, preferably from 1 to 5 substituents, more preferably from 1 to 3 substituents, and most preferably from 1 to 2 substituents, selected independently from the list of substituents. However, the group can generally have any number of substituents selected from halogen. Multiple substituted groups are also shown as such. The definition of any substituent or variable at a particular position within a molecule is intended to be independent of its definition elsewhere within that molecule. It is understood that the substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and can be readily synthesized by techniques known in the art and the methods described herein.

[0158] As used herein, a protecting group refers to a group that temporarily or permanently and selectively blocks one reactive site in a polyfunctional compound. Suitable hydroxy protecting groups for use in the present invention are pharmaceutically acceptable and may or may not need to be cleaved from the parent compound after administration to a subject for the compound to be active. Cleavage is by normal metabolic processes in the body. Hydroxy protecting groups are well known in the art and are hereby incorporated by reference in their entirety for all purposes from Protective Groups in Organic Synthesis by T.W.Greene and P.G.M.Wuts(John Wiley&sons,3 rdReference is made to the (Edition), and includes, for example, ethers (e.g., alkyl ethers and silyl ethers including dialkylsilyl ethers, trialkylsilyl ethers, dialkylalkoxysilyl ethers), esters, carbonates, carbamates, sulfonates, and phosphate protecting groups. Examples of hydroxy protecting groups include methyl ether; methoxymethyl ether, methylthiomethyl ether, (phenyldimethylsilyl)methoxymethyl ether, benzyloxymethyl ether, p-methoxybenzyloxymethyl ether, p-nitrobenzyloxymethyl ether, o-nitrobenzyloxymethyl ether, (4-methoxyphenoxy)methyl ether, guaiacolmethyl ether, t-butoxymethyl ether, 4-pentenyl oxymethyl ether, siloxymethyl ether, 2-methoxyethoxymethyl ether, 2,2,2-trichloroethoxymethyl ether, bis(2-chloroethoxy)methyl ether, 2-(trimethylsilyl)ethoxymethyl ether, menthoxymethyl ether, tetrahydropyranyl ether, 1-methoxycyclohexyl ether, 4-methoxytetrahydrothiopyranyl ether, 4-methoxytetrahydrothiopyranyl ether S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl ether, 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl ether, 1,4-dioxan-2-yl ether, tetrahydrofuranyl ether, tetrahydrothiofuranyl ether;Replacement ethyl ethers, such as 1-ethoxyethyl ether, 1-(2-chloroethoxy)ethyl ether, 1-[2-(trimethylsilyl)ethoxy]ethyl ether, 1-methyl-1-methoxyethyl ether, 1-methyl-1-benzyloxyethyl ether, 1-methyl-1-benzyloxy-2-fluoroethyl ether, 1-methyl-1-phenoxyethyl ether, 2-trimethylsilyl ether, t-butyl ether, allyl ether, propargyl ether, p-chlorophenyl ether, p-methoxyphenyl ether, benzyl ether, p-methoxybenzyl ether 3,4-dimethoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether, tripropylsilyl ether, dimethylisopropylsilyl ether, diethylisopropylsilyl ether, dimethylhexylsilyl ether, t-butyldimethylsilyl ether, diphenylmethylsilyl ether, benzoylformate ester, acetate ester, chloroacetate ester, dichloroacetate ester, trichloroacetate ester, trifluoroacetate ester, methoxyacetate ester, triphenylmethoxyacetate ester, phenylacetate ester, benzoate ester, alkyl methyl carbonate, alkyl 9-fluorenylmethyl carbonate, alkyl ethyl carbonate, alkyl 2,2,2,-trichloroethyl carbonate, 1,1,-dimethyl-2,2,2-trichloroethyl carbonate, alkyl sulfonate, methanesulfonate, benzyl sulfonate, tosylate, methylene acetal, ethylidene acetal, and t-butyl methylidene ketal, but are not limited thereto. Preferred protecting groups are of the formula -R; a ,Si(R a )(R a )(R a ), -C(O)R a , -C(O)OR a , -C(O)NH(R a ), -S(O)2R a , -S(O)2OH, P(O)(OH)2,, and -P(O)(OH)OR a , where R a is C1-C20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, -C1-C 20 Alkylene (carbocyclic), -C2-C 20 Alkenylene (carbocyclic), -C2-C 20 Alkynylene (carbocyclic), -C6-C 10 Aryl, -C1-C 20 Alkylene (aryl), -C2-C 20 Alkenylene (aryl), -C2-C 20 Alkynylene (aryl), -C1-C 20 Alkylene (heterocyclic), -C2-C 20 Alkenylene (heterocyclic), or -C2-C 20 Alkynylene (heterocyclic), wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic, and heterocyclic radicals may be substituted, either alone or as part of another group.

[0159] The term "chemotherapeutic agent" refers to all chemical compounds effective in inhibiting tumor growth. Non-limiting examples of chemotherapeutic agents include alkylating agents such as nitrogen mustards, ethyleneimine compounds and alkyl sulfonates; antimetabolites such as folic acid, purine or pyrimidine antagonists; mitotic inhibitors such as antitubulin agents like vinca alkaloids, auristatins and derivatives of podophyllotoxin; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication such as DNA minor groove binders; and growth factor receptor antagonists. Further, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biological molecules and small molecules.

[0160] The term "compound" refers to and includes the chemical compound itself, as well as, whether or not explicitly stated and unless it is clear from the context that the following should be excluded: amorphous and crystalline forms of the compound, including polymorphs, which may be part of a mixture or may be isolated; free acid and free base forms of the compound, typically in the form shown in the structures provided herein; isomers of the compound, which refer to optical isomers and tautomers, where optical isomers include enantiomers and diastereomers, chiral and achiral isomers, and these optical isomers include isolated optical isomers and mixtures of optical isomers including racemic and non-racemic mixtures; where the isomers may be in isolated form or may be a mixture with one or more other isomers; isotopes of the compound, including deuterium-containing compounds and tritium-containing compounds, as well as compounds containing radioisotopes including therapeutically and diagnostically effective radioisotopes; multimeric forms of the compound, including forms such as dimers, trimers, etc.; salts of the compound, including acid addition salts and base addition salts, salts having organic and inorganic counterions, and zwitterionic forms, preferably pharmaceutically acceptable salts, where if the compound is associated with two or more counterions, the two or more counterions may be the same or different; and solvates of the compound, including inorganic solvates including organic solvates and hydrates, including hemisolvates, monosolvates, disolvates, etc., where if the compound is associated with two or more solvent molecules, the two or more solvent molecules may be the same or different. In some instances, references made herein to the compounds of the invention include explicit reference to one or more of the above forms, e.g., salts and / or solvates; however, this reference is for emphasis only and should not be construed as excluding other of the above-specified forms.

[0161] As used herein, the term "conservative substitution" is known to those of skill in the art and generally refers to an amino acid substitution that can be made without changing the biological activity of the resulting molecule. Those of skill in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially change biological activity (see, e.g., Watson et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin / Cummings Pub. Co., p. 224 (4th Edition 1987)). Such exemplary substitutions are preferably made according to the substitutions shown in Tables 2 and 3. For example, such changes include substituting any of isoleucine (I), valine (V), and leucine (L) with any other of these hydrophobic amino acids; substituting glutamic acid (E) with aspartic acid (D) and vice versa; substituting asparagine (N) with glutamine (Q) and vice versa; and substituting threonine (T) with serine (S) and vice versa. Other substitutions can also be considered conservative depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can often be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can often be exchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are often interchangeable at positions where the important feature of the amino acid residue is its charge and the different pK's of these two amino acid residues are not important. Still other changes can be considered "conservative" in certain environments (see, e.g., Table 3 herein; pages 13-15 of ''Biochemistry'' 2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-11886). Other substitutions are tolerated and can be determined empirically or according to known conservative substitutions.

[0162] (Table 2) Abbreviations of Amino Acids TIFF2025106332000008.tif112157

[0163] (Table 3) Amino Acid Substitution or Similarity Matrix Conforms to the GCG software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the higher the probability that a substitution will be found in the related native protein. TIFF2025106332000009.tif135140

[0164] The terms "identity" or "identical" are intended to mean sequence similarity between two polynucleotides or between two polypeptides. Similarity can be determined by comparing the positions in each sequence that can be aligned for purposes of comparison. If a given position in the two polypeptide sequences is not identical, the similarity or conservation at that position can be determined by, for example, evaluating the amino acid similarity at that position according to Table 3. The degree of similarity between sequences is a function of the number of matching or identical positions shared by the sequences. Alignment of two sequences to determine the percent sequence similarity can be performed using, for example, software programs known in the art such as those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999). Preferably, default parameters are used for the alignment, examples of which are shown below. One alignment program well known in the art that can be used is BLAST set to default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; descriptions = 50 sequences; sort order = high score; database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information.

[0165] The term "homolog" of a given amino acid sequence or nucleic acid sequence is intended to indicate the corresponding sequence of a "homolog" having substantial identity or homology to the given amino acid sequence or nucleic acid sequence.

[0166] The determination of the percent identity between two sequences, such as amino acid sequences or nucleic acid sequences, can be accomplished using mathematical algorithms. Preferred non-limiting examples of the mathematical algorithms utilized 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 into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. The BLAST nucleotide search can be performed using, for example, the NBLAST nucleotide program parameters set with score = 100 and wordlength = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. The BLAST protein search can be performed using, for example, the XBLAST program parameters set with score 50 and wordlength = 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI BLAST can be used to perform iterative searches to detect remote relationships between molecules (ibid.). When using the BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of each program (e.g., for XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) at world wide web, ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17.Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0167] The percent identity between two sequences can be determined using techniques similar to those above, with or without allowing gaps. In calculating the percent identity, typically only exact matches are counted.

[0168] The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity and function of cells and / or causes cell destruction. This term is intended to include toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including radioisotopes, chemotherapeutic agents, and fragments and / or variants. Examples of cytotoxic agents include auristatin (e.g., auristatin E, auristatin F, MMAE and MMAF), aureomycin, maytansinoid, ricin, ricin A chain, combrestatin, duocarmycin, dostarlimab, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracinedione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α-sarcin, gelonin, mitogelin, restrictocin, phenomycin, enomycin, curcin, crotonin, calicheamicin, Saponaria officinalis inhibitor, and glucocorticoids and other chemotherapeutic agents, and At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 or Bi213 , P 32 and Lu 177 Radioisotopes of Lu including, but not limited to, those containing P and Lu are mentioned. The antibody can also be conjugated to an anti-cancer prodrug activating enzyme capable of converting the prodrug to its active form.

[0169] As used herein, the terms "effective amount" or "therapeutically effective amount" refer to an amount of a binding molecule (e.g., an antibody) or pharmaceutical composition provided herein that is sufficient to produce a desired result.

[0170] The terms "subject" and "patient" may be used interchangeably. As used herein, in certain embodiments, a subject is a mammal such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human). In certain embodiments, the subject is a human. In one embodiment, the subject is a mammal, such as a human, diagnosed with a condition or disorder. In another embodiment, the subject is a mammal, such as a human, at risk of developing a condition or disorder.

[0171] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance existing outside the body to a patient by, for example, mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

[0172] As used herein, the terms "treating," "treatment," and "treat" refer to a decrease or improvement in the progression, severity, and / or duration of a disease or condition resulting from the administration of one or more therapies. Treatment can be determined by assessing whether there has been a decrease, alleviation, and / or reduction of one or more symptoms associated with the underlying disease such that improvement is observed in the patient, even though the patient may still be afflicted with the underlying disease. The term "treat" includes both the management and improvement of a disease. The terms "managing," "management," and "manage" refer to the beneficial effects obtained from a therapy that does not necessarily result in a cure of the disease in a subject.

[0173] The terms "prevent," "prevention," and "preventing" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or one or more associated symptoms (e.g., cancer).

[0174] The term "cancer" or "cancer cell" as used herein refers to a tissue or cell found in a neoplasm that has characteristics distinguishing it from normal tissue or tissue cells. Such characteristics include, but are not limited to, degree of anaplasia, irregularity of shape, indistinctness of cell borders, nuclear size, changes in nuclear or cytoplasmic structure, other phenotypic changes, the presence of cell proteins indicative of cancer or a pre-cancerous state, increased number of mitoses, and the ability to metastasize. Words related to "cancer" include carcinoma, sarcoma, tumor, epithelioma, leukemia, lymphoma, polyp, and scirrhus, transformation, neoplasm, and the like.

[0175] 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 than a given value or range.

[0176] As used in this disclosure and the claims, the singular forms "a", "an", and "the" include the plural unless the context clearly dictates otherwise.

[0177] When aspects are described herein using the term "comprising", it is to be understood that other similar aspects are always provided in relation to "consisting of" and / or "consisting essentially of". Also, when aspects are described herein using the phrase "consisting essentially of", it is to be understood that other similar aspects are always provided in relation to "consisting of".

[0178] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used herein in phrases such as "A, B, and / or C" is intended to include each of the following aspects: 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.

[0179] The term "variant" refers to a molecule that shows a variation from a described type or reference, such as a protein having one or more different amino acid residues at the corresponding position(s) of a specifically described protein (e.g., the 191P4D12 protein shown in FIG. 1). An analog is an example of a variant protein. Splice isoforms and single nucleotide polymorphisms (SNPs) are further examples of variants.

[0180] The "191P4D12 protein" and / or "191P4D12-related protein" of the present invention includes those specifically identified herein (see Figure 1), as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated / generated and characterized without undue experimentation according to the methods outlined herein or methods readily available in the art. Also included are fusion proteins that combine a portion or fragment of different 191P4D12 proteins, and fusion proteins of 191P4D12 proteins and heterologous polypeptides. Such 191P4D12 proteins are collectively referred to as 191P4D12-related proteins, the proteins of the present invention, or 191P4D12. The term "191P4D12-related protein" refers to a polypeptide fragment or 191P4D12 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids, or at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 330, at least 335, at least 339 or more amino acids.

[0181] 5.2 Method for treating cancer Methods are provided herein for treating various cancers including breast cancer (e.g., HER+ / HER2- breast cancer and triple negative breast cancer (ER- / PR- / HER2-) that is ER negative, PR negative and HER2 negative), lung cancer (e.g., squamous cell lung cancer, non-squamous cell lung cancer, squamous cell non-small cell lung cancer, and non-squamous cell non-small cell lung cancer), head and neck cancer, and gastric or esophageal cancer, using an antibody-drug conjugate (ADC) that binds to 191P4D12. In some embodiments, the ADC is enfortumab vedotin (also known as anti-191P4D12-ADC, Ha22-2(2,4)6.1vcMMAE, ASG-22CE, or AGS-22M6E).

[0182] 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 conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, and the subject has hormone receptor positive and human epidermal growth factor receptor 2 negative (HR+ / HER2-) breast cancer.

[0183] 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 conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, and the subject has ER-negative, PR-negative, and HER2-negative (ER− / PR− / HER2−) breast cancer.

[0184] In another 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 conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, and the subject has squamous non-small cell lung cancer (NSCLC).

[0185] In a further 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 conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, and the subject has non-squamous NSCLC.

[0186] 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 conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, and the subject has locally advanced or metastatic head and neck cancer.

[0187] In certain 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 that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, and the subject has gastric cancer or esophageal cancer.

[0188] In all of the methods provided herein, specifically in the methods described in the previous seven paragraphs (paragraphs

[0188] -

[0194] ), the therapeutic agents that can be used are described in Section 5.3, the selection of patients for treatment is described herein and exemplified in Sections 5.2 and 6, the dosing regimens and pharmaceutical compositions for administering the therapeutic agents are described in Sections 5.4 and 6 below, the biomarkers that can be used to identify, select patients for, determine the outcome of, and / or otherwise benchmark these methods are described herein and exemplified in Sections 5.2 and 6, and the therapeutic outcome of the methods provided herein can be an improvement in the biomarkers described herein, such as those described and exemplified in Sections 5.2 and 6. Thus, one of ordinary skill in the art will understand that the methods provided herein include all permutations and combinations of patients, therapeutic agents, dosing regimens, biomarkers, and therapeutic outcomes as described above and below.

[0189] In certain aspects, the methods provided herein are used for the treatment of breast cancer in a subject. In some aspects, the breast cancer is hormone receptor positive and human epidermal growth factor receptor 2 negative (HR+ / HER2−) breast cancer. In some aspects, the breast cancer is estrogen receptor (ER) positive and / or progesterone receptor (PR) positive, and HER2 negative. In some aspects, the breast cancer is ER positive, PR positive, and HER2 negative. In some aspects, the breast cancer is ER positive and HER2 negative. In some aspects, the breast cancer is PR positive and HER2 negative. In some aspects, for example, breast cancers including HR+ / HER2− breast cancer, ER positive, PR positive and HER2 negative breast cancer, ER positive and HER2 negative breast cancer, PR positive and HER2 negative breast cancer are confirmed histologically, cytologically, or both histologically and cytologically. In some aspects, the histological, cytological, or both histological and cytological confirmation is performed according to the American Society of Clinical Oncology / American Society of Pathology (ASCO / CAP) guidelines based on the most recently analyzed tissue.

[0190] In some embodiments, hormone receptor positive and human epidermal growth factor receptor 2 negative (HR+ / HER2−) breast cancer is locally advanced or metastatic breast cancer. In some embodiments, ER positive and / or progesterone receptor (PR) positive, and HER2 negative breast cancer is locally advanced or metastatic breast cancer. In some embodiments, ER positive, PR positive, and HER2 negative breast cancer is locally advanced or metastatic breast cancer. In some embodiments, ER positive and HER2 negative breast cancer is locally advanced or metastatic breast cancer. In some embodiments, PR positive and HER2 negative breast cancer is locally advanced or metastatic breast cancer. In some embodiments, locally advanced or metastatic breast cancer, including, for example, HR+ / HER2− breast cancer, ER positive, PR positive, and HER2 negative breast cancer, ER positive and HER2 negative breast cancer, and PR positive and HER2 negative breast cancer, is confirmed histologically, cytologically, or both histologically and cytologically. In some embodiments, such histological, cytological, or both histological and cytological confirmation is performed according to American Society of Clinical Oncology / American Society of Pathology (ASCO / CAP) guidelines based on the most recently analyzed tissue.

[0191] In some embodiments, a subject having breast cancer and being treated by the methods provided herein is receiving one or more endocrine therapies and cyclin-dependent kinase (CDK) 4 / 6 inhibitors in a metastatic or locally advanced setting. In some embodiments, a subject having breast cancer and being treated by the methods provided herein has received prior treatment with a taxane or anthracycline in any setting. In some embodiments, a subject having breast cancer and being treated by the methods provided herein has a deleterious germline mutation in breast cancer susceptibility gene (BRCA) 1 or 2 and must be treated with a poly(ADP-ribose) polymerase (PARP) inhibitor.

[0192] In some specific embodiments, the subject to be treated by the methods provided herein has histologically or cytologically confirmed HR+ / HER2− breast cancer that is defined as ER positive and / or progesterone receptor (PR) positive and HER2 negative according to the American Society of Clinical Oncology / American Society of Pathology (ASCO / CAP) guidelines based on the most recently analyzed tissue, has locally advanced or metastatic disease, has received one or more endocrine therapies and cyclin-dependent kinase (CDK) 4 / 6 inhibitors in a metastatic or locally advanced setting, has received prior treatment with a taxane or anthracycline in any setting, and / or has a deleterious germline mutation in breast cancer susceptibility gene (BRCA) 1 or 2 and must not have been treated with a poly ADP ribose polymerase (PARP) inhibitor.

[0193] In certain embodiments, the methods provided herein are used for the treatment of triple-negative breast cancer (TNBC) in a subject. In some embodiments, the TNBC is histologically and / or cytologically confirmed TNBC. In some embodiments, the TNBC is determined according to TNBC histology (ER negative / PR negative / HER2 negative) by the ASCO / CAP guidelines based on the most recently analyzed tissue. In some embodiments, the TNBC is locally advanced or metastatic. In some embodiments, the subject having TNBC and being treated by the methods provided herein has received two or more systemic therapies. In some embodiments, the subject having TNBC and being treated by the methods provided herein has received two or more systemic therapies including a taxane in any setting. In some embodiments, the subject having TNBC and being treated by the methods provided herein has a deleterious germline mutation in BRCA1, BRCA2, or both BRCA1 and BRCA2. In some embodiments, the subject having TNBC and being treated by the methods provided herein has been treated with a PARP inhibitor. In some embodiments, the subject to be treated by the methods provided herein for TNBC has any permutation or combination of the features described in this paragraph.

[0194] In some specific embodiments, the subject to be treated by the methods provided herein has histologically or cytologically confirmed triple-negative breast cancer (TNBC) defined as definite TNBC histology (ER-negative / PR-negative / HER2-negative) according to the ASCO / CAP guidelines based on the most recently analyzed tissue, has locally advanced or metastatic disease, has received two or more systemic therapies including a taxane in any setting, has a deleterious germline mutation in BRCA1 or BRCA2 or both, and / or has been treated with a PARP inhibitor.

[0195] In certain embodiments, the methods provided herein are used for the treatment of squamous non-small cell lung cancer (NSCLC) in a subject. In some embodiments, the squamous NSCLC is histologically and / or cytologically confirmed squamous NSCLC. In some embodiments, the squamous NSCLC is locally advanced or metastatic. In some embodiments, the subject having squamous NSCLC and being treated by the methods provided herein has progressed or recurred after platinum-based therapy, for example, if recurrence occurs within 12 months after completion of platinum-based therapy including platinum therapy administered as adjuvant therapy. In some embodiments, the subject having squamous NSCLC and being treated by the methods provided herein has received prior treatment with anti-programmed cell death protein-1 (PD-1) or anti-programmed cell death-ligand 1 (PD-L1) if eligible based on the subject's tumor PD-1 or PD-L1 expression and local treatment guidelines.

[0196] In some specific embodiments, the subject treated by the methods provided herein has histologically or cytologically confirmed squamous NSCLC, has locally advanced or metastatic disease, has progressed or recurred after platinum-based therapy, counted as a regimen, and / or has received prior treatment with anti-programmed cell death protein-1 (PD-1) or anti-programmed cell death-ligand 1 (PD-L1) if eligible based on the subject's tumor PD-1 or PD-L1 expression and local treatment guidelines, for example, recurrence occurs within 12 months after completion of platinum-based therapy including platinum therapy administered as adjuvant therapy.

[0197] In certain embodiments, the methods provided herein are used for the treatment of non-squamous NSCLC in a subject. In some embodiments, the squamous NSCLC is histologically and / or cytologically confirmed squamous NSCLC. In some embodiments, the squamous NSCLC is epidermal growth factor receptor (EGFR) wild-type and anaplastic lymphoma kinase (ALK) wild-type. In some embodiments, the squamous NSCLC is EGFR wild-type and ALK wild-type according to local laboratory standards. In some embodiments, the non-squamous NSCLC is locally advanced or metastatic. In some embodiments, the subject having squamous NSCLC and treated by the methods provided herein has progressed or recurred after platinum-based therapy, for example, recurrence occurs within 12 months after completion of platinum-based therapy including platinum therapy administered as adjuvant therapy in a metastatic or locally advanced setting. In some embodiments, the subject having squamous NSCLC and treated by the methods provided herein has received anti-PD-1 or anti-PD-L1 therapy if eligible based on the subject's tumor PD-1 or PD-L1 expression and local treatment guidelines.

[0198] In some specific embodiments, the subject treated by the methods provided herein has histologically or cytologically confirmed non-squamous NSCLC that is EGFR wild-type and ALK wild-type according to local laboratory standards, has locally advanced or metastatic disease, and has recurred within 12 months after completion of platinum-based therapy in a metastatic or locally advanced setting, including platinum therapy administered as adjuvant therapy, has progressed or recurred after platinum-based therapy, and is receiving anti-PD-1 or anti-PD-L1 therapy if eligible based on the subject's tumor PD-1 or PD-L1 expression and local treatment guidelines.

[0199] In certain embodiments, the methods provided herein are used for the treatment of head and neck cancer in a subject. In some embodiments, the head and neck cancer is a histologically and / or cytologically confirmed head and neck cancer. In some embodiments, the head and neck cancer is locally advanced or metastatic. In some embodiments, the subject having head and neck cancer and treated by the methods provided herein has progressed or recurred after a platinum-containing regimen in a metastatic or locally advanced setting, and this platinum-containing regimen does not include a platinum regimen administered as part of a curative setting of multimodality therapy unless the subject recurs or progresses within 6 months after completion. In some embodiments, the subject having head and neck cancer and treated by the methods provided herein is receiving anti-PD-1 or anti-PD-L1 therapy if eligible based on the subject's tumor PD-1 or PD-L1 expression and local treatment guidelines.

[0200] In some specific embodiments, the subject treated by the methods provided herein has histologically or cytologically confirmed head and neck cancer, has locally advanced or metastatic disease, has progressed or recurred after a platinum-containing regimen in a metastatic or locally advanced setting, which does not include a platinum regimen administered as part of a curative setting of multimodality therapy unless the subject recurs or progresses within 6 months after completion, and is receiving anti-PD-1 or anti-PD-L1 therapy if eligible based on the subject's tumor PD-1 or PD-L1 expression and local treatment guidelines.

[0201] In certain embodiments, the methods provided herein are used for the treatment of gastric cancer or esophageal cancer in a subject. In some embodiments, the gastric cancer or esophageal cancer is histologically and / or cytologically confirmed gastric cancer or esophageal cancer. In some embodiments, the gastric cancer or esophageal cancer is locally advanced or metastatic. In some embodiments, a subject having head and neck cancer and being treated by the methods provided herein has progressed or recurred after a chemotherapy regimen comprising fluoropyrimidine and platinum for metastatic or locally advanced disease, and this chemotherapy regimen does not include a neoadjuvant or adjuvant regimen unless the subject recurs or progresses within 6 months after completion. In some embodiments, a subject having head and neck cancer and being treated by the methods provided herein has received HER2-directed therapy if having HER2-positive cancer. In some embodiments, a subject having head and neck cancer and being treated by the methods provided herein has HER2-positive cancer and has received HER2-directed therapy.

[0202] In some specific embodiments, a subject being treated by the methods provided herein has histologically or cytologically confirmed gastric cancer or esophageal cancer, has a locally advanced or metastatic disease, has progressed or recurred after a chemotherapy regimen comprising fluoropyrimidine and platinum for metastatic or locally advanced disease, and this chemotherapy regimen does not include a neoadjuvant or adjuvant regimen unless the subject recurs or progresses within 6 months after completion, has HER2-positive cancer, and has received HER2-directed therapy. In another specific embodiment, a subject being treated by the methods provided herein has histologically or cytologically confirmed gastric cancer or esophageal cancer, has a locally advanced or metastatic disease, has progressed or recurred after a chemotherapy regimen comprising fluoropyrimidine and platinum for metastatic or locally advanced disease, and this chemotherapy regimen does not include a neoadjuvant or adjuvant regimen unless the subject recurs or progresses within 6 months after completion.

[0203] In certain aspects, the methods provided herein are used to treat a subject having a cancer that expresses 191P4D12 RNA, expresses 191P4D12 protein, or expresses both 191P4D12 RNA and 191P4D12 protein. In certain aspects, the methods provided herein are used to treat a subject having a cancer that expresses both 191P4D12 RNA and 191P4D12 protein, including, for example, squamous NSCLC, non-squamous NSCLC, gastric (GEJ) cancer, esophageal cancer, HNSCC, NSCLC adenocarcinoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), and breast cancer (including HR+ / HER2-breast cancer and TNBC). In some aspects, 191P4D12 RNA expression in cancer is determined by polynucleotide hybridization, sequencing (evaluating the relative abundance of sequences), and / or PCR (including RT-PCR). In some aspects, 191P4D12 protein expression in cancer is determined by IHC, analysis in fluorescence-activated cell sorting (FACS), and / or Western blotting. In some aspects, 191P4D12 protein expression in cancer is determined by two methods of IHC.

[0204] In certain aspects, the methods provided herein are used to treat a subject having a cancer that expresses 191P4D12 RNA, expresses 191P4D12 protein, or expresses both 191P4D12 RNA and 191P4D12 protein, and the cancer is sensitive to cytotoxic agents (such as Vinca and MMAE) that block microtubule polymerization. In certain aspects, the methods provided herein are used to treat a subject having a cancer that expresses both 191P4D12 RNA and 191P4D12 protein and is sensitive to cytotoxic agents (such as Vinca and MMAE) that block microtubule polymerization, including, for example, squamous NSCLC, non-squamous NSCLC, gastric (GEJ) cancer, esophageal cancer, HNSCC, NSCLC adenocarcinoma, head and neck cancer (e.g., head and neck squamous cell carcinoma), and breast cancer (including HR+ / HER2-breast cancer and TNBC).

[0205] In some embodiments, the subject that can be treated by the methods provided herein is, for example, a subject having hormone receptor positive and human epidermal growth factor receptor 2 negative (HR+ / HER2−) breast cancer, a subject having ER negative, PR negative and HER2 negative (ER− / PR− / HER2−) breast cancer, a subject having NSCLC, a subject having non-squamous NSCLC, a subject having head cancer, a subject having neck cancer, a subject having head and neck cancer, a subject having gastric cancer, a subject having esophageal cancer, and / or a subject having gastric cancer or esophageal cancer, i.e., a subject having a solid tumor.

[0206] In certain embodiments, the subjects that can be treated by the methods provided herein further include subjects having locally advanced solid tumors, metastatic solid tumors (including metastatic malignancies), and locally advanced and metastatic solid tumors. In some embodiments, the solid tumors that can be treated by the methods provided herein are locally advanced HR+ / HER2− breast cancer, locally advanced ER− / PR− / HER2− breast cancer, locally advanced NSCLC, locally advanced non-squamous NSCLC, locally advanced head cancer, locally advanced neck cancer, locally advanced head and neck cancer, locally advanced gastric cancer, locally advanced esophageal cancer, and / or locally advanced gastroesophageal cancer. In other embodiments, the solid tumors that can be treated by the methods provided herein are metastatic (including malignant or metastatic malignant) HR+ / HER2− breast cancer, metastatic (including malignant or metastatic malignant) ER− / PR− / HER2− breast cancer, metastatic (including malignant or metastatic malignant) NSCLC, metastatic (including malignant or metastatic malignant) non-squamous NSCLC, metastatic (including malignant or metastatic malignant) head cancer, metastatic (including malignant or metastatic malignant) neck cancer, metastatic (including malignant or metastatic malignant) head and neck cancer, metastatic (including malignant or metastatic malignant) gastric cancer, metastatic (including malignant or metastatic malignant) esophageal cancer, and / or metastatic (including malignant or metastatic malignant) gastroesophageal cancer.

[0207] In some embodiments, locally advanced, metastatic (including metastatic malignant), and locally advanced and metastatic solid tumors are confirmed histologically, cytologically, or both histologically and cytologically.

[0208] In some embodiments, the subject to be treated by the methods provided herein has progressed or recurred after one or more other treatments for cancer. The one or more treatments after which the subject has subsequently progressed or recurred include, for example, endocrine therapy, cyclin-dependent kinase (CDK) 4 / 6 inhibitors (including metastatic or locally advanced settings), treatment with taxanes, treatment with anthracyclines, poly ADP ribose polymerase (PARP) inhibitors, platinum-based therapies, therapy with inhibitors of programmed cell death protein-1 (PD-1), inhibitors of programmed cell death-ligand 1 (PD-L1), chemotherapy including fluoropyrimidines, HER2-directed therapies, and / or one or more of the therapies provided in this paragraph and any permutation or combination of two or more of the therapies described herein.

[0209] In certain embodiments, the subject to be treated by the methods provided herein has previously received at least 2, 3, 4, 5, or 6 systemic therapies. Such systemic therapies can be any treatment that uses substances that move through the bloodstream, reach cells throughout the body, and have an effect. Such systemic therapies can be those described in the previous paragraph (paragraph

[0215] ). In one embodiment, such systemic therapy is a taxane.

[0210] In certain embodiments, a subject treatable by the methods provided herein has progressed or recurred within 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, or 24 months after one or any combination of other treatments, including, for example, without limitation, any of the treatments described in the paragraph two paragraphs before this paragraph (paragraph

[0215] ). In some particular embodiments, the subject has progressed or recurred within 6 months after a platinum-based therapy or chemotherapy comprising a fluoropyrimidine. In other particular embodiments, the subject has progressed or recurred within 6 months after a platinum-based therapy. In a further embodiment, the subject has progressed or recurred within 12 months after a platinum-based therapy.

[0211] In some embodiments, a subject treatable by the methods provided herein has already received one or more other treatments for cancer. The one or more treatments received by the subject include, for example, endocrine therapy, cyclin-dependent kinase (CDK) 4 / 6 inhibitors (including metastatic or locally advanced settings), treatment with a taxane, treatment with an anthracycline, poly ADP ribose polymerase (PARP) inhibitors, platinum-based therapy, therapy with an inhibitor of programmed cell death protein-1 (PD-1), an inhibitor of programmed cell death-ligand 1 (PD-L1), chemotherapy comprising a fluoropyrimidine, HER2-directed therapy, and / or one or more of any permutation or combination of two or more of the therapies provided in this paragraph and the therapies described herein.

[0212] In some embodiments, a subject that can be treated by the methods provided herein has undergone one or more other cancer treatments as described in the previous paragraph (paragraph

[0218] ) and has progressed or recurred after one or more other cancer treatments as described in the paragraph four paragraphs before this paragraph (paragraph

[0215] ), in any combination or permutation thereof.

[0213] In some embodiments, a subject that can be treated by the methods provided herein has certain phenotypic or genotypic characteristics. In one embodiment, the subject has HR+ / HER2− breast cancer that is also estrogen receptor (ER) positive and HER2 negative. In one embodiment, the subject has HR+ / HER2− breast cancer that is also progesterone receptor (PR) positive and HER2 negative. In one embodiment, the subject has HR+ / HER2− breast cancer that is also estrogen receptor (ER) positive, progesterone receptor (PR) positive, and HER2 negative. In one embodiment, the subject has a germline mutation that is deleterious to breast cancer susceptibility gene (BRCA) 1, BRCA2, or both BRCA1 and BRCA2. In one embodiment, the subject has ER− / PR− / HER2− breast cancer. In one embodiment, the subject has wild-type epidermal growth factor receptor (EGFR). In one embodiment, the subject has wild-type anaplastic lymphoma kinase (ALK). In one embodiment, the subject has both wild-type epidermal growth factor receptor (EGFR) and wild-type anaplastic lymphoma kinase (ALK). In some embodiments, the subject has any permutation and combination of the phenotypic or genotypic characteristics described herein.

[0214] In some embodiments, phenotypic or genotypic characteristics are determined histologically, cytologically, or both histologically and cytologically. In one embodiment, HR+ / HER2- breast cancer that is also estrogen receptor (ER) positive and HER2 negative is determined histologically, cytologically, or both histologically and cytologically. In one embodiment, HR+ / HER2- breast cancer that is also progesterone receptor (PR) positive and HER2 negative is determined histologically, cytologically, or both histologically and cytologically. In one embodiment, HR+ / HER2- breast cancer that is also estrogen receptor (ER) positive, progesterone receptor (PR) positive, and HER2 negative is determined histologically, cytologically, or both histologically and cytologically. In one embodiment, a deleterious germline mutation in breast cancer susceptibility gene (BRCA) 1, BRCA2, or both BRCA1 and BRCA2 is determined histologically, cytologically, or both histologically and cytologically. In one embodiment, ER-negative, PR-negative, and HER2-negative (ER- / PR- / HER2-) breast cancer is determined histologically, cytologically, or both histologically and cytologically. In one embodiment, wild-type epidermal growth factor receptor (EGFR) is determined histologically, cytologically, or both histologically and cytologically. In one embodiment, wild-type anaplastic lymphoma kinase (ALK) is determined histologically, cytologically, or both histologically and cytologically. In one embodiment, both wild-type epidermal growth factor receptor (EGFR) and wild-type anaplastic lymphoma kinase (ALK) are determined histologically, cytologically, or both histologically and cytologically.

[0215] In some embodiments of the methods provided herein, histological and / or cytological determination of phenotypic and / or genotypic characteristics is performed as described in the American Society of Clinical Oncology / American Society of Pathology (ASCO / CAP) guidelines based on the most recently analyzed tissue, which guidelines are incorporated herein by reference in their entirety.

[0216] In some embodiments, phenotypic or genotypic characteristics are determined by sequencing, including next-generation sequencing (e.g., NGS of Illumina, Inc.), DNA hybridization, and / or RNA hybridization. In one embodiment, HR+ / HER2- breast cancer that is also estrogen receptor (ER) positive and HER2 negative is determined by sequencing, including next-generation sequencing (e.g., NGS of Illumina, Inc.), DNA hybridization, and / or RNA hybridization. In one embodiment, HR+ / HER2- breast cancer that is also progesterone receptor (PR) positive and HER2 negative is determined by sequencing, including next-generation sequencing (e.g., NGS of Illumina, Inc.), DNA hybridization, and / or RNA hybridization. In one embodiment, HR+ / HER2- breast cancer that is also estrogen receptor (ER) positive, progesterone receptor (PR) positive, and HER2 negative is determined by sequencing, including next-generation sequencing (e.g., NGS of Illumina, Inc.), DNA hybridization, and / or RNA hybridization. In one embodiment, a deleterious germline mutation in breast cancer susceptibility gene (BRCA) 1, BRCA2, or both BRCA1 and BRCA2 is determined by sequencing, including next-generation sequencing (e.g., NGS of Illumina, Inc.), DNA hybridization, and / or RNA hybridization. In one embodiment, ER-negative, PR-negative, and HER2-negative (ER- / PR- / HER2-) breast cancer is determined by sequencing, including next-generation sequencing (e.g., NGS of Illumina, Inc.), DNA hybridization, and / or RNA hybridization. In one embodiment, wild-type epidermal growth factor receptor (EGFR) is determined by sequencing, including next-generation sequencing (e.g., NGS of Illumina, Inc.), DNA hybridization, and / or RNA hybridization.In one aspect, wild-type anaplastic lymphoma kinase (ALK) is determined by sequencing including next-generation sequencing (e.g., NGS of Illumina, Inc.), DNA hybridization, and / or RNA hybridization. In one aspect, both wild-type epidermal growth factor receptor (EGFR) and wild-type anaplastic lymphoma kinase (ALK) are determined by sequencing including next-generation sequencing (e.g., NGS of Illumina, Inc.), DNA hybridization, and / or RNA hybridization.

[0217] In some aspects, one or more other treatments for cancer that the subject has received or from which the subject's cancer has progressed or recurred are PD-1 inhibitors or PD-L1 inhibitors. In certain aspects, the PD-1 inhibitor is pembrolizumab or nivolumab. In other aspects, the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab. Other examples of PD-l / PD-L1 inhibitors include those described in U.S. Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and PCT International Publication Nos. 2003042402; 2008156712; 2010089411; 2010036959; 2011066342; 2011159877; 2011082400, and 2011161699, but are not limited thereto, and all of these are hereby incorporated by reference in their entirety.

[0218] In certain embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In one embodiment, the anti-PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558, or MDX1106), or pembrolizumab (also known as MK-3475, SCH 900475, or lambrolizumab). In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab is a human IgG4 anti-PD-1 monoclonal antibody and is commercially available under the trade name Opdivo™. In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 antibody and is commercially available under the trade name Keytruda™. In yet another embodiment, the anti-PD-1 antibody is CT-011, a humanized antibody. In yet another embodiment, the anti-PD-1 antibody is AMP-224, a fusion protein. In another embodiment, the PD-1 antibody is BGB-A317. BGB-A317 is a monoclonal antibody with a unique binding signature that has been specifically engineered for its ability to bind to Fcγ receptor I and has high affinity for PD-1 and excellent target specificity.

[0219] In a further embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one embodiment, the anti-PD-L1 antibody is MEDI4736 (durvalumab). In another embodiment, the anti-PD-L1 antibody is BMS-936559 (also known as MDX-1105-01). In yet another embodiment, the PD-L1 inhibitor is atezolizumab (also known as MPDL3280A and Tecentriq®).

[0220] In some embodiments, the subject that can be treated by the methods provided herein is a mammal. In some embodiments, the subject that can be treated by the methods provided herein is a human.

[0221] 5.3 Anti-191P4D12 Antibody-Drug Conjugate Generally, the methods provided herein utilize the anti-191P4D12 ADC described in U.S. Patent No. 8,637,642, which is hereby incorporated by reference in its entirety. The anti-191P4D12 antibody-drug conjugates provided herein comprise an antibody that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of a cytotoxic agent (or drug moiety). The cytotoxic agent (or drug moiety) can be covalently attached directly or via a linker unit (LU).

[0222] In some embodiments, the antibody-drug conjugate compound has the following formula or is a pharmaceutically acceptable salt or solvate thereof: L-(LU-D) p (I) Wherein, L is an antibody unit, e.g., an anti-191P4D12 antibody or an antigen-binding fragment thereof provided in Section 5.3.1 below, (LU-D) is a linker unit-drug unit moiety, where, LU- is a linker unit, D is a drug unit having antiproliferative or cytotoxic activity against target cells, p is an integer from 1 to 20.

[0223] In some embodiments, p ranges from 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from 2 to 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.

[0224] In some embodiments, the antibody-drug conjugate compound has the following formula or is a pharmaceutically acceptable salt or solvate thereof: L-(A a -W w -Y y -D) p (II) Wherein, L is an antibody unit, for example, the anti-191P4D12 antibody provided in Section 5.3.1 below or an antigen-binding fragment thereof; and -A a -W w -Y y - is a linker unit (LU), where -A- is an extension unit, a is 0 or 1, each -W- is independently an amino acid unit, w is an integer in the range of 0 to 12, -Y- is a self-immolative spacer unit, y is 0, 1 or 2, D is a drug unit having cytostatic or cytotoxic activity against target cells, p is an integer from 1 to 20.

[0225] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1 or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, p is in the range of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is in the range of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is 1, 2, 3, 4, 5 or 6. In some embodiments, p is 2 or 4. 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.

[0226] In the case of a composition comprising a plurality of antibodies or antigen-binding fragments thereof, the drug load is represented by p, which is the average number of drug molecules per antibody unit. The drug load can range from 1 to 20 drugs (D) per antibody. The average number of drugs per antibody in a preparation of the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the antibody-drug conjugate with respect to p can also be determined. In some cases, the separation, purification, and characterization of a homogeneous antibody-drug conjugate with a specific value of p from antibody-drug conjugates with other drug loads can be achieved by means such as reverse-phase HPLC or electrophoresis. In an exemplary embodiment, p is from 2 to 8.

[0227] 5.3.1 Anti-191P4D12 Antibody or Antigen-Binding Fragment In one aspect, an antibody or antigen-binding fragment thereof that binds to a 191P4D12-related protein is an antibody or antigen-binding fragment that specifically binds to the 191P4D12 protein comprising the amino acid sequence of SEQ ID NO:2 (see Figure 1A). The corresponding cDNA encoding the 191P4D12 protein has the sequence of SEQ ID NO:1 (see Figure 1A).

[0228] Antibodies that specifically bind to the 191P4D12 protein comprising the amino acid sequence of SEQ ID NO:2 include antibodies that can bind to other 191P4D12-related proteins. For example, an antibody that binds to the 191P4D12 protein comprising the amino acid sequence of SEQ ID NO:2 can bind to 191P4D12-related proteins such as 191P4D12 variants and homologs or analogs thereof.

[0229] In some aspects, the anti-191P4D12 antibodies provided herein are monoclonal antibodies.

[0230] 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.

[0231] In some embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising complementarity determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22, which is the amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 136th amino acid (serine) of SEQ ID NO:7, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, which is the amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) of SEQ ID NO:8. SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:7 and SEQ ID NO:8 are shown in FIGS. 1D and 1E and are as listed below. TIFF2025106332000010.tif172166

[0232] The CDR sequences can be determined according to well-known numbering systems. As described above, the CDR regions are well-known to those skilled in the art and are defined by well-known numbering systems. For example, the Kabat complementarity-determining regions (CDRs) are based on the variability of the sequences and are the most commonly used (see, e.g., Kabat et al., supra). Alternatively, Chothia refers to the positions of structural loops (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering rules varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Dubel eds., 2d ed. 2010)). The "contact" hypervariable regions are based on the analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). IMGT is an integrated information system specialized for human and other vertebrate immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complex (MHC). In this specification, CDRs are referred to with respect to both the amino acid sequences and the positions within the light or heavy chains.The "positions" of the CDRs within the structure of immunoglobulin variable domains are conserved across species and are present within structures called loops, such that CDRs and framework residues can be readily identified by using a numbering system that aligns variable domain sequences according to structural features. This information can be used when transplanting and substituting CDR residues from an immunoglobulin of one species into an acceptor framework, typically from a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, 2001, J. Mol. Biol. 309:657-70. Correspondences between numbering systems, including, for example, Kabat numbering and the IMGT unique numbering system, are well known to those of skill in the art (see, e.g., Kabat supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al. supra). Residues from each of these hypervariable regions or CDRs are shown in Table 1 above.

[0233] In some embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDRs comprising the amino acid sequences of the complementarity determining regions (CDRs) of the heavy chain variable region shown in SEQ ID NO:22 according to Kabat numbering, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23 according to Kabat numbering.

[0234] In some embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDRs comprising the amino acid sequences of the complementarity determining regions (CDRs) of the heavy chain variable region shown in SEQ ID NO:22 according to AbM numbering, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23 according to AbM numbering.

[0235] In other embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDRs comprising the amino acid sequences of the complementarity determining regions (CDRs) of the heavy chain variable region shown in SEQ ID NO: 22 according to Chothia numbering, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to Chothia numbering.

[0236] In other embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDRs comprising the amino acid sequences of the complementarity determining regions (CDRs) of the heavy chain variable region shown in SEQ ID NO: 22 according to Contact numbering, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to Contact numbering.

[0237] In yet other embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDRs comprising the amino acid sequences of the complementarity determining regions (CDRs) of the heavy chain variable region shown in SEQ ID NO: 22 according to IMGT numbering, and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO: 23 according to IMGT numbering.

[0238] As described above, CDR sequences according to different numbering systems can be readily determined using online tools such as those provided by, for example, 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 determined by ANARCI according to Kabat numbering are listed in Table 4 below.

[0239] (Table 4) TIFF2025106332000011.tif31166

[0240] As 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 the IMGT numbering determined by ANARCI are listed in Table 5 below.

[0241] (Table 5) TIFF2025106332000012.tif31166

[0242] 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:NO:12, CDR L2 comprising the amino acid sequence of SEQ ID NO:NO:13, and CDR L3 comprising the amino acid sequence of SEQ ID NO:NO:14.

[0243] 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:NO:19, CDR L2 comprising the amino acid sequence of SEQ ID NO:NO:20, and CDR L3 comprising the amino acid sequence of SEQ ID NO:NO:21.

[0244] 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.

[0245] In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO:7, and a light chain comprising an amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO:8.

[0246] In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to optimize the binding affinity and / or other biological properties of the antibody, including but not limited to specificity, thermal stability, expression level, effector function, glycosylation, reduction of immunogenicity, or solubility. Accordingly, it is contemplated that antibody variants can be prepared in addition to the antibodies described herein. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the coding DNA and / or by synthesis of the desired antibody or polypeptide. Those skilled in the art will recognize that amino acid changes can alter the post-translational processes of the antibody, such as changing the number or location of glycosylation sites or altering membrane anchoring properties.

[0247] In some embodiments, the antibodies provided herein are chemically modified, for example, by covalent attachment of any type of molecule to the antibody. Antibody derivatives can include, for example, antibodies chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting groups / blocking groups, proteolytic cleavage, conjugation to a cell ligand or other protein, etc. Any of a number of chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicaamycin, etc. Further, the antibody can include one or more non-classical amino acids.

[0248] A mutation can be a substitution, deletion, or insertion of one or more codons encoding a single - domain antibody or polypeptide that results in a change in the amino - acid sequence compared to the original antibody or polypeptide. Amino - acid substitutions can be, for example, conservative amino - acid substitutions, such as substitution of leucine with serine, which results from substituting one amino acid with another having similar structural and / or chemical properties. Mutations can be introduced into the nucleotide sequences encoding the molecules provided herein using standard techniques known to those of skill in the art, including, for example, site - directed mutagenesis and PCR - mediated mutagenesis that result in amino - acid substitutions. Insertions or deletions can range from about 1 to 5 amino acids. In certain embodiments, the substitution, deletion, or insertion comprises less than 25 amino - acid substitutions, less than 20 amino - acid substitutions, less than 15 amino - acid substitutions, less than 10 amino - acid substitutions, less than 5 amino - acid substitutions, less than 4 amino - acid substitutions, less than 3 amino - acid substitutions, or less than 2 amino - acid substitutions compared to the original molecule. In a specific embodiment, the substitution is a conservative amino - acid substitution at one or more predicted non - essential amino - acid residues. Permissible mutations can be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for the activity exhibited by the parental antibody.

[0249] Amino - acid insertions include amino - terminal and / or carboxyl - terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as in - sequence insertions of single or multiple amino - acid residues. Examples of terminal insertions include antibodies having an N - terminal methionyl residue.

[0250] Antibodies generated by conservative amino acid substitutions are included within the present disclosure. In conservative amino acid substitutions, an amino acid residue is substituted with an amino acid residue that contains a side chain with a similar charge. As noted above, families of amino acid residues that contain side chains with similar charges are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed, the activity of the protein can be determined, and conservative (e.g., within groups of amino acids having similar properties and / or side chains) substitutions can be made so as to maintain or not significantly change the properties.

[0251] Amino acids can be grouped according to the similarity of the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2d ed. 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H). Alternatively, the naturally occurring residues can be grouped into classes based on common side-chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0252] For example, any cysteine residue that does not participate in maintaining the proper conformation of the antibody can be substituted with another amino acid, such as alanine or serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking.

[0253] Modifications can be made using methods known in the art such as oligonucleotide-mediated (site-specific) mutagenesis, alanine scanning, and PCR mutagenesis. Site-specific mutagenesis (see, e.g., Carter, 1986, Biochem J. 237:1-7; and Zoller et al., 1982, Nucl. Acids Res. 10:6487-500), cassette mutagenesis (see, e.g., Wells et al., 1985, Gene 34:315-23), or other known techniques can be performed on the cloned DNA to produce anti-MSLN antibody variant DNA.

[0254] Covalent modification of antibodies is included within the scope of the present disclosure. Covalent modification includes reacting a target amino acid residue of the antibody with an organic derivatizing agent capable of reacting with a selected side chain or N-terminal or C-terminal residue of the antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0255] Other types of covalent modifications of antibodies included within the scope of the present disclosure include changing 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 conjugating the antibody to one of various non-proteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylene, by, for example, the methods described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.

[0256] In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 70% homology or identity with the heavy chain shown 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 with the heavy chain shown 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 with the heavy chain shown 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 with the heavy chain shown in SEQ ID NO:7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 90% homology or identity with the heavy chain shown 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 with the heavy chain shown in SEQ ID NO:7.

[0257] In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a light chain having greater than 70% homology or identity with the light chain shown 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 with the light chain shown 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 with the light chain shown 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 with the light chain shown 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 with the light chain shown 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 with the light chain shown in SEQ ID NO:8.

[0258] In some embodiments, the anti-191P4D12 antibody provided herein comprises heavy and light chain CDR regions that are identical to the heavy and light chain CDR regions of an antibody designated Ha22-2(2,4)6.1 produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267, or amino acid sequences homologous to the amino acid sequences of the heavy and light chain CDR regions of Ha22-2(2,4)6.1, wherein the antibody retains the desired functional properties of the anti-191P4D12 antibody designated Ha22-2(2,4)6.1 produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267.

[0259] In some embodiments, the 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 comprises CDRs that include the amino acid sequences of the heavy chain variable region CDRs shown in the antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267; (b) the light chain variable region comprises CDRs that include the amino acid sequences of the light chain variable region CDRs shown in the antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267.

[0260] In some embodiments, the anti-191P4D12 antibodies provided herein comprise heavy and light chain variable regions that are the heavy and light chain variable regions of an antibody designated Ha22-2(2,4)6.1 produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267 (see Figure 3), or amino acid sequences homologous to the amino acid sequences of the heavy and light chain variable regions of Ha22-2(2,4)6.1, wherein the antibody retains the desired functional properties of the anti-191P4D12 antibodies provided herein. As the constant regions of the antibodies of the invention, constant regions of any subclass can be selected. In one embodiment, a human IgG1 constant region can be used as the heavy chain constant region and a human Igκ constant region can be used as the light chain constant region.

[0261] In some embodiments, the anti-191P4D12 antibodies provided herein comprise heavy and light chains that are the heavy and light chains of an antibody designated Ha22-2(2,4)6.1 produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267, or heavy and light chains comprising amino acid sequences homologous to the amino acid sequences of the heavy and light chains of Ha22-2(2,4)6.1, wherein the antibody retains the desired functional properties of the anti-191P4D12 antibodies provided herein.

[0262] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise heavy chain variable regions and light chain variable regions, wherein (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous or identical to the amino acid sequence of the heavy chain variable region of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267; and (b) The light chain variable region comprises an amino acid sequence that is at least 80% homologous or identical to the amino acid sequence of the light chain variable region of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267.

[0263] In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 85% homologous or identical to the amino acid sequence of the heavy chain variable region of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267. In other embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 90% homologous or identical to the amino acid sequence of the heavy chain variable region of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267. In still other embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 95% homologous or identical to the amino acid sequence of the heavy chain variable region of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267. In other embodiments, the heavy chain variable region may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical to the amino acid sequence of the heavy chain variable region of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267.

[0264] In some embodiments, the light chain variable region comprises an amino acid sequence that is at least 85% identical or homologous to the amino acid sequence of the light chain variable region of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267. In other embodiments, the light chain variable region comprises an amino acid sequence that is at least 90% identical or homologous to the amino acid sequence of the light chain variable region of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267. In yet other embodiments, the light chain variable region comprises an amino acid sequence that is at least 95% identical or homologous to the amino acid sequence of the light chain variable region of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267. In other embodiments, the light chain variable region may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or homologous to the amino acid sequence of the light chain variable region of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267.

[0265] In other embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain and a light chain, wherein (a) the heavy chain comprises an amino acid sequence that is at least 80% identical or homologous to the amino acid sequence of the heavy chain of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267; and (b) the light chain comprises an amino acid sequence that is at least 80% identical or homologous to the amino acid sequence of the light chain of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267.

[0266] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 85% identical or the same as the heavy chain amino acid sequence of an antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267. In other embodiments, the heavy chain comprises an amino acid sequence that is at least 90% identical or the same as the heavy chain amino acid sequence of an antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267. In still other embodiments, the heavy chain comprises an amino acid sequence that is at least 95% identical or the same as the heavy chain amino acid sequence of an antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267. In other embodiments, the heavy chain may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or the same as the heavy chain amino acid sequence of an antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267.

[0267] In some embodiments, the light chain comprises an amino acid sequence that is at least 85% homologous or identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267. In other embodiments, the light chain comprises an amino acid sequence that is at least 90% homologous or identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267. In yet other embodiments, the light chain comprises an amino acid sequence that is at least 95% homologous or identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267. In other embodiments, the light chain may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) accession number PTA-11267.

[0268] The engineered antibodies provided herein include those having modifications to framework residues within VH and / or VL (e.g., to improve antibody properties). Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "revert" one or more framework residues to their germline sequences. More specifically, an antibody that has undergone somatic mutation may contain framework residues that are different from the germline sequences from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, somatic mutations can be "reverted" to the germline sequences, for example, by site-directed mutagenesis or PCR-mediated mutagenesis (e.g., reverting from leucine to methionine). Such "reverted" antibodies are also intended to be encompassed by the present invention.

[0269] 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 and thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in more detail in U.S. Patent Application Publication No. 2003 / 0153043 by Carr et al.

[0270] In addition to, or instead of, modifications made within the framework region or the CDR region, the antibodies of the present invention can be engineered to include modifications within the Fc region, typically to modify one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antibody-dependent cell cytotoxicity. Further, the anti-191P4D12 antibodies provided herein can also be chemically modified (e.g., one or more chemical moieties can be conjugated to the antibody) or modified to alter its glycosylation to modify one or more functional properties of the antibody. Each of these aspects is described in further detail below.

[0271] In one aspect, the hinge region of CH1 is modified such that the number of cysteine residues within the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 to Bodmer et al. The number of cysteine residues within the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the anti-191P4D12 antibody.

[0272] In another aspect, the Fc hinge region of the antibody is mutated to decrease the biological half-life of the anti-191P4D12 antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has reduced Staphylococcyl protein A (SpA) binding compared to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 to Ward et al.

[0273] In another aspect, the anti-191P4D12 antibody is modified to increase its biological half-life. Various approaches are possible. For example, mutations can be introduced as described in Ward U.S. Patent No. 6,277,375. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to include the salvage receptor binding epitope derived from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patents Nos. 5,869,046 and 6,121,022 by Presta et al.

[0274] In yet another aspect, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to modify one or more effector functions of the antibody. For example, one or more amino acids selected from amino acid-specific residues can be replaced with different amino acid residues such that the antibody has an altered affinity for an effector ligand while retaining the antigen-binding ability of the parental antibody. The effector ligand for which the affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in both U.S. Patents Nos. 5,624,821 and 5,648,260 by Winter et al.

[0275] The reactivity of the anti-191P4D12 antibody with the 191P4D12-related protein can be established by many well-known means including Western blot, immunoprecipitation, ELISA, and FACS analysis, using the 191P4D12-related protein, 191P4D12-expressing cells or their extracts as necessary. The 191P4D12 antibody or its fragment can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelating agents, or enzymes. Furthermore, bispecific antibodies specific for two or more 191P4D12 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53:2560-2565).

[0276] In yet another specific embodiment, the anti-191P4D12 antibody provided herein is an antibody comprising the heavy and light chains of an antibody designated Ha22-2(2,4)6.1. The heavy chain of Ha22-2(2,4)6.1 consists of an amino acid sequence ranging from the 20th E residue to the 466th K residue of SEQ ID NO:7, and the light chain of Ha22-2(2,4)6.1 consists of an amino acid sequence ranging from the 23rd D residue to the 236th C residue of the sequence of SEQ ID NO:8.

[0277] The hybridoma producing the antibody designated Ha22-2(2,4)6.1 was sent (via Federal Express) to the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108 on August 18, 2010, and was assigned the accession number PTA-11267.

[0278] 5.3.2 Cytotoxic agent (drug unit) In some embodiments, the ADC comprises an antibody or an antigen-binding fragment thereof conjugated to an auristatin which is dolastatin or a peptide analogue and derivative of dolastatin (U.S. Patent Nos. 5,635,483; 5,780,588). Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and inhibit nuclei and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anti-cancer activity (U.S. Patent No. 5,663,149) and anti-fungal activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug unit can be conjugated to the antibody via the N (amino) terminus or C (carboxyl) terminus of the peptidic drug unit (WO 02 / 088172).

[0279] Exemplary auristatin embodiments are disclosed in Senter et al, Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, published March 28, 2004, and include the N-terminal conjugated monomethyl auristatin drug units DE and DF described in U.S. Patent Application Publication No. 2005 / 0238649, the disclosure of which is hereby expressly incorporated by reference in its entirety.

[0280] In some embodiments, the auristatin is MMAE (where the wavy line indicates a covalent bond to the linker of the antibody-drug conjugate). TIFF2025106332000013.tif24140

[0281] In some embodiments, an exemplary embodiment comprising MMAE and a linker component (further described herein) has the following structure (where L represents the antibody and p ranges from 1 to 12). TIFF2025106332000014.tif36160

[0282] Typically, peptide-based drug units can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods well known in the field of peptide chemistry (see E. Schroder and K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press). Auristatin / dolastatin drug units can be prepared according to the methods of U.S. Patent No. 5,635,483; U.S. Patent No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G.R., et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784.

[0283] 5.3.3 Linker Typically, an antibody-drug conjugate includes a linker unit between a drug unit (e.g., MMAE) and an antibody unit (e.g., anti-191P4D12 antibody or an antigen-binding fragment thereof). In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In still other embodiments, the linker unit is not cleavable and the drug is released, for example, by antibody degradation.

[0284] In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome or endosome or caveola). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme including, but not limited to, lysosomal or endosomal proteases. In some embodiments, the peptidyl linker is at least 2 amino acids in length or at least 3 amino acids in length. Cleaving agents can include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug within the target cell (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typically, it is a peptidyl linker cleavable by an enzyme present in 191P4D12-expressing cells. For example, a peptidyl linker cleavable by cathepsin B, a thiol-dependent protease highly expressed in cancerous tissue, can be used (e.g., Phe-Leu or Gly-Phe-Leu-Gly linker (SEQ ID NO:15)). Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, which is hereby incorporated by reference in its entirety for all purposes. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker). One advantage of using intracellular proteolytic release of therapeutic agents is that the agent is typically attenuated when conjugated and the serum stability of the conjugate is typically high.

[0285] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, the pH-sensitive linker is hydrolyzable under acidic conditions. For example, acid-labile linkers that are hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) can be used (e.g., see U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions such as in blood, but are unstable at pH less than about pH 5.5 or less than pH 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether linked to a therapeutic agent via an acylhydrazone bond, etc.) (e.g., see U.S. Patent No. 5,622,929).

[0286] In yet other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). For example, various disulfide linkers are known in the art, including those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB, and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U.Press, 1987). See also U.S. Patent No. 4,880,935).

[0287] In yet other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0288] In yet other embodiments, the linker unit is not cleavable and the drug is released by antibody degradation (see U.S. Patent Application Publication No. 2005 / 0238649, which is hereby incorporated by reference in its entirety for all purposes).

[0289] Typically, the linker is substantially insensitive to the extracellular environment. As used herein, "substantially insensitive to the extracellular environment" in the context of a linker means that when the antibody-drug conjugate is present in the extracellular environment (e.g., in plasma), no more than about 20%, typically no more than about 15%, more typically no more than about 10%, even more typically no more than about 5%, about 3%, or about 1% of the linker in a sample of the antibody-drug conjugate is cleaved. Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating an antibody-drug conjugate compound with plasma for a predetermined time (e.g., 2 hours, 4 hours, 8 hours, 16 hours, or 24 hours) and then quantifying the amount of free drug present in the plasma.

[0290] In other non-exclusive aspects, the linker promotes cellular internalization. In certain aspects, the linker promotes cellular internalization when conjugated to a therapeutic agent (i.e., in the context of the linker-therapeutic agent moiety of the antibody-drug conjugate compounds described herein). In yet other aspects, the linker promotes cellular internalization when conjugated to both an auristatin compound and an anti-191P4D12 antibody or antigen-binding fragment thereof.

[0291] Various exemplary linkers that can be used with the compositions and methods of the invention are described in International Publication No. WO 2004 / 010957, U.S. Patent Application Publication No. 2006 / 0074008, U.S. Patent Application Publication No. 2005 / 0238649, and U.S. Patent Application Publication No. 2006 / 0024317 (each of which is hereby incorporated by reference in its entirety for all purposes).

[0292] A "linker unit" (LU) is a bifunctional compound that can be used to link a drug unit and an antibody unit to form an antibody-drug conjugate. In some aspects, the linker unit has the formula: -A a -W w -Y y - having, wherein, -A- is an elongation unit, a is 0 or 1, each -W- is independently an amino acid unit, w is an integer in the range of 0 to 12, -Y- is a self-sacrificing spacer unit, y is 0, 1 or 2.

[0293] 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.

[0294] 5.3.3.1 Elongation Unit The extension unit (A), when present, can link the antibody unit to the amino acid unit (-W-) if the amino acid unit (-W-) is present, to the spacer unit (-Y-) if the spacer unit (-Y-) is present, or to the drug unit (-D). Useful functional groups that can be present naturally or via chemical manipulation on the anti-191P4D12 antibody or its antigen-binding fragment (e.g., Ha22-2(2,4)6.1) include, but are not limited to, sulfhydryl, amino, hydroxyl, anomeric hydroxyl groups of carbohydrates, and carboxyl. Suitable functional groups are sulfhydryl and amino. In one example, a sulfhydryl group can be generated by reduction of an intramolecular disulfide bond of the anti-191P4D12 antibody or its antigen-binding fragment. In another aspect, a sulfhydryl group can be generated by reaction of an amino group of the lysine moiety of the anti-191P4D12 antibody or antigen-binding fragment with 2-iminothiolane (Traut's reagent) or other sulfhydryl-generating reagents. In certain embodiments, the anti-191P4D12 antibody or its antigen-binding fragment is a recombinant antibody and is engineered to carry one or more lysines. In certain other embodiments, the recombinant anti-191P4D12 antibody is engineered to carry additional sulfhydryl groups, such as additional cysteine.

[0295] In one aspect, the extension unit forms a bond with a sulfur atom of the antibody unit. The sulfur atom can be derived from a sulfhydryl group of the antibody. Representative extension units of this aspect are shown within the brackets of Formulas IIIa and IIIb below, where L-, -W-, -Y-, -D, w, and y are as defined above, and R 17 is -C1-C 10 alkylene-, -C1-C 10 alkenylene-, -C1-C 10 alkynylene-, carbocyclo-, -O-(C1-C8 alkylene)-, O-(C1-C8 alkenylene)-, -O-(C1-C8 alkynylene)-, -arylene-, -C1-C 10 alkylene-arylene-, -C2-C 10 alkenylene-arylene, -C2-C 10Alkynylene-arylene, -arylene-C1-C 10 Alkylene-, -arylene-C2-C 10 Alkenylene-, -arylene-C2-C 10 Alkynylene-, -C1-C 10 Alkylene-(carbocyclo)-, -C2-C 10 Alkenylene-(carbocyclo)-, -C2-C 10 Alkynylene-(carbocyclo)-, -(carbocyclo)-C1-C 10 Alkylene-, -(carbocyclo)-C2-C 10 Alkenylene-, -(carbocyclo)-C2-C 10 Alkynylene, -heterocyclo-, -C1-C 10 Alkylene-(heterocyclo)-, -C2-C 10 Alkenylene-(heterocyclo)-, -C2-C 10 Alkynylene-(heterocyclo)-, -(heterocyclo)-C1-C 10 Alkylene-, -(heterocyclo)-C2-C 10 Alkenylene-, -(heterocyclo)-C1-C 10 Alkynylene-, -(CH2CH2O) r -, or -(CH2CH2O) r -CH2-, where r is an integer in the range of 1 to 10, wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic, carbocyclo, heterocyclic, and arylene radicals may be substituted, either alone or as part of another group. In some embodiments, the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic, carbocyclo, heterocyclic, and arylene radicals are unsubstituted, either alone or as part of another group.

[0296] In some embodiments, R 17 is, -C1-C 10 Alkylene-, -carbocyclo-, -O-(C1-C8 alkylene)-, -arylene-, -C1-C 10Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(carbocyclo)-, -(carbocyclo)-C1-C 10 Alkylene-, -C3-C8 heterocyclo-, -C1-C 10 Alkylene-(heterocyclo)-, -(heterocyclo)-C1-C 10 Alkylene-, -(CH2CH2O) r -, and -(CH2CH2O) r -CH2-, where r is an integer in the range of 1 to 10, wherein the alkylene group is unsubstituted and the remaining groups may be substituted.

[0297] It should be understood that from all exemplary embodiments, even if not explicitly shown, 1 to 20 drug units (p = 1 to 20) can be linked to the antibody unit. TIFF2025106332000015.tif57128

[0298] An exemplary extension unit is R 17 is an extension unit of formula IIIa where it is -(CH2)5-. TIFF2025106332000016.tif24128

[0299] Another exemplary extension unit is R 17 is -(CH2CH2O) r -CH2-, and r is 2, which is an extension unit of formula IIIa. TIFF2025106332000017.tif24128

[0300] An exemplary extension unit is R 17 is arylene- or arylene-C1-C 10 alkylene-, which is an extension unit of formula IIIa. In some embodiments, the aryl group is an unsubstituted phenyl group.

[0301] Yet another exemplary extension unit is R 17 is an extension unit of formula IIIb where it is -(CH2)5-. TIFF2025106332000018.tif20128

[0302] In certain embodiments, the extension unit is linked to the antibody unit via a disulfide bond between a sulfur atom of the antibody unit and a sulfur atom of the extension unit. Representative extension units of this embodiment are depicted within the brackets of Formula IV, wherein R 17 , L-, -W-, -Y-, -D, w and y are as defined above. TIFF2025106332000019.tif18128

[0303] It should be noted that throughout this application, the S moiety in the following formulas refers to the sulfur atom of the antibody unit, unless otherwise indicated by context. TIFF2025106332000020.tif12128

[0304] In certain structural descriptions of the sulfur-bonded ADCs herein, the antibody is represented as "L". It can also be denoted as "Ab-S". Inclusion of "S" simply indicates the feature of the sulfur bond and does not imply that a particular sulfur atom has multiple linker-drug moieties. The left bracket of the structure using the designation "Ab-S" may also be placed to the left of the sulfur atom between Ab and S, which is an equivalent notation of the ADCs of the present invention described throughout this specification.

[0305] In yet other embodiments, the extension moiety includes a reactive site capable of forming a bond with a primary or secondary amino group of the antibody unit. Examples of these reactive sites include, but are not limited to, activated esters such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Representative extension units of this embodiment are depicted within the brackets of Formulas Va and Vb, wherein -R 17 -, L-, -W-, -Y-, -D, w and y are as defined above. TIFF2025106332000021.tif53128

[0306] In some embodiments, the extension moiety comprises a reactive site that is reactive with a modified carbohydrate (-CHO) group that may be present on the antibody unit. For example, the carbohydrate can be gently oxidized using a reagent such as sodium periodate, and the resulting (-CHO) units of the oxidized carbohydrate can be condensed with an extension moiety containing functional groups such as hydrazide, oxime, primary or secondary amines, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide as described in Kaneko et al., 1991, Bioconjugate Chem. 2:133-41. Representative extension units of this embodiment are depicted within the brackets of Formulas VIa, VIb, and VIc, wherein -R 17 -, L-, -W-, -Y-, -D, w and y are as defined above. TIFF2025106332000022.tif77128

[0307] 5.3.3.2 Amino acid unit The amino acid unit (-W-), when present, connects the extension unit to the spacer unit if a spacer unit is present, connects the extension unit to the drug unit if no spacer unit is present, and connects the antibody unit to the drug unit if neither an extension unit nor a spacer unit is present.

[0308] W w - can be, for example, a monopeptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. Each -W- unit independently has the formula shown below within the brackets, and w is an integer in the range of 0 to 12: TIFF2025106332000023.tif29128 wherein R 19are hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH2OH, -CH(OH)CH3, -CH2CH2SCH3, -CH2CONH2, -CH2COOH, -CH2CH2CONH2, -CH2CH2COOH, -(CH2)3NHC(=NH)NH2, -(CH2)3NH2, -(CH2)3NHCOCH3, -(CH2)3NHCHO, -(CH2)4NHC(=NH)NH2, -(CH2)4NH2, -(CH2)4NHCOCH3, -(CH2)4NHCHO, -(CH2)3NHCONH2, -(CH2)4NHCONH2, -CH2CH2CH(OH)CH2NH2, 2-pyridylmethyl-, 3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl, is TIFF2025106332000024.tif107148.

[0309] In some embodiments, the amino acid unit can be enzymatically cleaved by one or more enzymes including cancer or tumor-related proteases to release the drug unit (-D), and in one embodiment, the drug unit (-D) is protonated in vivo upon release to provide the drug (D).

[0310] In certain embodiments, the amino acid unit comprises natural amino acids. In other embodiments, the amino acid unit comprises non-natural amino acids. Exemplary Ww units are represented by the following Formulas VII-IX: TIFF2025106332000025.tif21128 wherein R 20 and R 21 are as follows: TIFF2025106332000026.tif120147TIFF2025106332000027.tif21128 wherein R 20 , R 21 and R 22 are as follows: TIFF2025106332000028.tif37134TIFF2025106332000029.tif21128 wherein R 20 , R 21 , R 22 and R23 is as follows: TIFF2025106332000030.tif28137。

[0311] Exemplary amino acid units include R 20 is benzyl and R 21 is -(CH2)4NH2; R 20 is isopropyl and R 21 is -(CH2)4NH2; or R 20 is isopropyl and R 21 is -(CH2)3NHCONH2, including but not limited to the units of formula VII above.

[0312] Another exemplary amino acid unit is a unit of formula VIII where R 20 is benzyl, R 21 is benzyl, R 22 is -(CH2)4NH2.

[0313] Useful -W w - units can be designed and their selectivity optimized for enzymatic cleavage by specific enzymes, such as tumor - related proteases. In one aspect, -W w - units are those whose cleavage is catalyzed by cathepsin B, C, and D, or plasmin proteases.

[0314] In one aspect, -W w - is a dipeptide, tripeptide, tetrapeptide or pentapeptide. When R 19 , R 20 , R 21 , R 22 or R 23 is other than hydrogen, the carbon atom to which R 19 , R 20 , R 21 , R 22 or R 23 is attached is chiral.

[0315] R 19 , R 20 , R21 , R 22 or R 23 Each carbon atom to which R is attached is, independently, in the (S) or (R) configuration.

[0316] In one specific embodiment, the amino acid unit is valine-citrulline (vc or Val-Cit). In another specific embodiment, the amino acid unit is phenylalanine-lysine (i.e., fk). In yet another specific embodiment, the amino acid unit is N-methylvaline-citrulline. In yet another specific embodiment, the amino acid unit is 5-aminovaleric acid, homophenylalanine lysine, tetraisoguinoline carboxylate lysine, cyclohexylalanine lysine, isonipecotic acid lysine, β-alanine lysine, glycine serine valine glutamine, and isonepecotic acid.

[0317] 5.3.3.3 Spacer unit The spacer unit (-Y-), when present, links the amino acid unit, if present, to the drug unit. Alternatively, the spacer unit links the extension unit to the drug unit if no amino acid unit is present. The spacer unit also links the drug unit to the antibody unit if neither the amino acid unit nor the extension unit is present.

[0318] Spacer units are of two general types: non-self-sacrificing or self-sacrificing. A non-self-sacrificing spacer unit is one in which some or all of the spacer unit remains attached to the drug unit after cleavage of the amino acid unit from the antibody-drug conjugate, particularly enzymatic cleavage. Examples of non-self-sacrificing spacer units include, but are not limited to, (glycine-glycine) spacer units and glycine spacer units (both depicted in Scheme 1) (below). When a conjugate containing a glycine-glycine spacer unit or a glycine spacer unit undergoes enzymatic cleavage by an enzyme (e.g., a tumor cell-associated protease, a cancer cell-associated protease, or a lymphocyte-associated protease), the glycine-glycine-drug unit or glycine-drug unit is cleaved from L-Aa-Ww-. In one aspect, an independent hydrolysis reaction occurs within the target cell, cleaving the glycine-drug unit bond and releasing the drug. TIFF2025106332000031.tif54131

[0319] In some aspects, the non-self-sacrificing spacer unit (-Y-) is -Gly-. In some aspects, the non-self-sacrificing spacer unit (-Y-) is -Gly-Gly-.

[0320] In one aspect, there is no spacer unit (y = 0 for -Y y -).

[0321] Alternatively, an antibody-drug conjugate containing a self-sacrificing spacer unit can release -D. As used herein, the term "self-sacrificing spacer" refers to a bifunctional chemical moiety that can covalently link two spaced-apart chemical moieties together to form a stable three-part molecule. This naturally separates from the second chemical moiety when the bond to the first moiety is cleaved.

[0322] In some aspects, -Y y - is such that the phenylene moiety is Q mis a p-aminobenzyl alcohol (PAB) unit replaced thereby (see Schemes 2 and 3), where Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro or -cyano, and m is an integer in the range of 0 to 4. The alkyl group, alkenyl group and alkynyl group may be either unsubstituted or substituted, either alone or as part of another group.

[0323] In some embodiments, -Y- is -W w linked to via the amino nitrogen atom of the PAB group and is a PAB group directly bonded to D via a carbonate, carbamate or ether group. Without being bound to a particular theory or mechanism, Scheme 2 shows a possible drug release mechanism of a PAB group directly bonded to D via a carbamate or carbonate group, as described by Toki et al., 2002, J. Org. Chem. 67:1866-1872. TIFF2025106332000032.tif105128

[0324] In Scheme 2, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro or -cyano, m is an integer in the range of 0 to 4, and p is in the range of 1 to about 20. The alkyl group, alkenyl group and alkynyl group may be either unsubstituted or substituted, either alone or as part of another group.

[0325] Without being bound to a particular theory or mechanism, Scheme 3 shows a possible drug release mechanism of a PAB group directly bonded to D via an ether or amine bond, and D contains an oxygen or nitrogen group that is part of the drug unit. TIFF2025106332000033.tif127128

[0326] In Scheme 3, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro or -cyano, m is an integer in the range of 0 to 4, and p ranges from 1 to about 20. The alkyl, alkenyl and alkynyl groups may be either unsubstituted or substituted, either alone or as part of another group.

[0327] Other examples of self-immolative spacers include aromatic compounds electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzyl acetals, but are not limited thereto. Substituted and unsubstituted 4-aminobutyramides (Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., 1972, J. Amer. Chem. Soc. 94:5815), and spacers that undergo cyclization upon hydrolysis of the amide bond, such as 2-aminophenylpropionic acid amide (Amsberry et al., 1990, J. Org. Chem. 55:5867), can be used. The elimination of amine-containing drugs substituted at the α-position of glycine (Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of a self-immolative spacer.

[0328] In one aspect, the spacer unit is a branched bis(hydroxymethyl)-styrene (BHMS) unit as shown in Scheme 4, which can be used to incorporate and release multiple drugs. TIFF2025106332000034.tif42128

[0329] In Scheme 4, Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro or -cyano, m is an integer in the range of 0 to 4, n is 0 or 1, and p is in the range of 1 to about 20. The alkyl group, alkenyl group and alkynyl group may be either unsubstituted or substituted, either alone or as part of another group.

[0330] In some embodiments, the -D units are the same. In yet another embodiment, the -D moieties are different.

[0331] In one aspect, the spacer unit (-Y y -) is represented by Formulae X to XII: TIFF2025106332000035.tif28128 wherein Q is -C1-C8 alkyl, -C1-C8 alkenyl, -C1-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C1-C8 alkenyl), -O-(C1-C8 alkynyl), -halogen, -nitro or -cyano, and m is an integer in the range of 0 to 4. The alkyl group, alkenyl group and alkynyl group may be either unsubstituted or substituted, either alone or as part of another group. TIFF2025106332000036.tif36128.

[0332] Embodiments of Formulae I and II comprising an antibody-drug conjugate compound can include: TIFF2025106332000037.tif19128 (wherein w and y are each 0, 1 or 2), TIFF2025106332000038.tif19128 (wherein w and y are each 0), TIFF2025106332000039.tif166128.

[0333] 5.3.3.4 Drug Loading Drug loading is represented by p and is the average number of drug units per antibody in the molecule. The drug loading can range from 1 to 20 drug units (D) per antibody. The ADCs provided herein include, for example, a collection of antibodies or antigen-binding fragments conjugated with drug units in the range of 1 to 20. The average number of drug units per antibody in a preparation of an ADC from a conjugation reaction can be characterized by conventional means such as mass spectrometry and ELISA assays. The quantitative distribution of the ADC with respect to p can also be determined. In some cases, the separation, purification, and characterization of a homogeneous ADC with a particular value of p from ADCs having other drug loadings can be achieved by means such as electrophoresis.

[0334] In certain embodiments, the drug payload of the ADCs provided herein ranges from 1 to 20. In certain embodiments, the drug payload of the ADCs provided herein ranges from 1 to 18. In certain embodiments, the drug payload of the ADCs provided herein ranges from 1 to 15. In certain embodiments, the drug payload of the ADCs provided herein ranges from 1 to 12. In certain embodiments, the drug payload of the ADCs provided herein ranges from 1 to 10. In certain embodiments, the drug payload of the ADCs provided herein ranges from 1 to 9. In certain embodiments, the drug payload of the ADCs provided herein ranges from 1 to 8. In certain embodiments, the drug payload of the ADCs provided herein ranges from 1 to 7. In certain embodiments, the drug payload of the ADCs provided herein ranges from 1 to 6. In certain embodiments, the drug payload of the ADCs provided herein ranges from 1 to 5. In certain embodiments, the drug payload of the ADCs provided herein ranges from 1 to 4. In certain embodiments, the drug payload of the ADCs provided herein ranges from 1 to 3. In certain embodiments, the drug payload of the ADCs provided herein ranges from 2 to 12. In certain embodiments, the drug payload of the ADCs provided herein ranges from 2 to 10. In certain embodiments, the drug payload of the ADCs provided herein ranges from 2 to 9. In certain embodiments, the drug payload of the ADCs provided herein ranges from 2 to 8. In certain embodiments, the drug payload of the ADCs provided herein ranges from 2 to 7. In certain embodiments, the drug payload of the ADCs provided herein ranges from 2 to 6. In certain embodiments, the drug payload of the ADCs provided herein ranges from 2 to 5. In certain embodiments, the drug payload of the ADCs provided herein ranges from 2 to 4. In certain embodiments, the drug payload of the ADCs provided herein ranges from 3 to 12. In certain embodiments, the drug payload of the ADCs provided herein ranges from 3 to 10. In certain embodiments, the drug payload of the ADCs provided herein ranges from 3 to 9. In certain embodiments, the drug payload of the ADCs provided herein ranges from 3 to 8. In certain embodiments, the drug payload of the ADCs provided herein ranges from 3 to 7. In certain embodiments, the drug payload of the ADCs provided herein ranges from 3 to 6.In certain embodiments, the drug payload of the ADCs provided herein ranges from 3 to 5. In certain embodiments, the drug payload of the ADCs provided herein ranges from 3 to 4.

[0335] In certain embodiments, the drug payload of the ADCs provided herein ranges from 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.

[0336] In certain embodiments, the drug payload of the ADCs provided herein is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or more. In certain embodiments, the drug payload 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.

[0337] In certain embodiments, during the conjugation reaction, fewer drug units are conjugated to the antibody than the theoretical maximum. The antibody may contain, for example, lysine residues that do not react with either the drug-linker intermediate or the linker reagent. Generally, the antibody does not contain many free and reactive cysteine thiol groups that can be linked to drug units; indeed, most cysteine thiol residues in the antibody exist as disulfide bridges. In certain embodiments, the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonyl ethylphosphine (TCEP) under partial or complete reducing conditions to generate reactive cysteine thiol groups. In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine. In some embodiments, the linker unit or the drug unit is conjugated via a lysine residue on the antibody unit. In some embodiments, the linker unit or the drug unit is conjugated via a cysteine residue on the antibody unit.

[0338] In some embodiments, the amino acid that binds to the linker unit or the drug unit is in the heavy chain of the antibody or an antigen-binding fragment thereof. In some embodiments, the amino acid that binds to the linker unit or the drug unit is in the light chain of the antibody or an antigen-binding fragment thereof. In some embodiments, the amino acid that binds to the linker unit or the drug unit is in the hinge region of the antibody or an antigen-binding fragment thereof. In some embodiments, the amino acid that binds to the linker unit or the drug unit is in the Fc region of the antibody or an antigen-binding fragment thereof. In other embodiments, the amino acid that binds to the linker unit or the drug unit is in the constant region of the antibody or an antigen-binding fragment thereof (e.g., CH1, CH2 or CH3 of the heavy chain, or CH1 of the light chain). In yet other embodiments, the amino acid that binds to the linker unit or the drug unit is in the VH framework region of the antibody or an antigen-binding fragment thereof. In yet other embodiments, the amino acid that binds to the linker unit or the drug unit is in the VL framework region of the antibody or an antigen-binding fragment thereof.

[0339] The loading (drug / antibody ratio) of the ADC can be controlled in various ways, for example, (i) by limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody, (ii) by limiting the reaction time or temperature of conjugation, (iii) by partial or limited reduction conditions for cysteine thiol modification, (iv) by engineering the amino acid sequence of the antibody by recombinant techniques such that the number and position of cysteine residues are modified for control of the number and / or position of linker-drug linkages (such as thioMab or thioFab prepared as disclosed herein and in International Publication No. WO 2006 / 034488, which is incorporated herein by reference in its entirety).

[0340] When multiple nucleophilic groups react with a drug-linker intermediate or a linker reagent and subsequently react with a drug unit reagent, it should be understood that the resulting product is a mixture of ADC compounds in which one or more drug units are distributed and bound to antibody units. The average number of drugs per antibody can be calculated from the mixture by a dual ELISA antibody assay specific for the antibody and specific for the drug. Individual ADC molecules can be identified in the mixture by mass spectrometry and separated by HPLC, for example, hydrophobic interaction chromatography (e.g., Hamblett, K.J., et al. "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate," Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27 - 31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, S.C., et al. "Controlling the location of drug attachment in antibody-drug conjugates," Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27 - 31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, homogeneous ADCs having a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.

[0341] Methods for preparing, screening, and characterizing antibody-drug conjugates are known to those of 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.

[0342] In some embodiments, the antibody-drug conjugate for the methods provided herein is prepared according to the method described in U.S. Patent No. 8,637,642 and is AGS-22M6E having the following formula: TIFF2025106332000040.tif36160wherein L is Ha22-2(2,4)6.1 and p is from 1 to 20.

[0343] In some embodiments, p ranges from 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 ranges from 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.

[0344] In some embodiments, the ADC used in the methods provided herein is enfortumab vedotin. Enfortumab vedotin is a fully human immunoglobulin G1 kappa (IgG1 Κ ) antibody conjugated to a microtubule disrupting agent (MMAE) via a protease-cleavable linker and is an ADC composed of (Challita-Eid PM et al, Cancer Res. 2016;76(10):3003-13). Enfortumab vedotin binds to the 191P4D12 protein on the cell surface, resulting in the internalization of the ADC-191P4D12 complex, which is then transported to the lysosomal compartment where MMAE is released via proteolytic cleavage of the linker, thereby inducing antitumor activity. The intracellular release of MMAE then disrupts tubulin polymerization, resulting in G2 / M cell cycle arrest and apoptotic cell death (Francisco JA et al, Blood. 2003 Aug 15;102(4):1458-65).

[0345] As described above and in U.S. Patent No. 8,637,642, AGS-22M6E is an ADC derived from a mouse hybridoma cell line. Enfortumab vedotin is a Chinese hamster ovary (CHO) cell line-derived equivalent of the AGS-22M6E ADC and is an exemplary product used for human therapy. Enfortumab vedotin has the same amino acid sequence, linker, and cytotoxic drug as AGS-22M6E. The equivalence between enfortumab vedotin and AGS-22M6E was confirmed through extensive analytical and biological property evaluation tests such as binding affinity for 191P4D12, in vitro cytotoxicity, and in vivo antitumor activity.

[0346] 5.4 Pharmaceutical Compositions In certain aspects of the methods provided herein, the ADCs used in the methods are provided as "pharmaceutical compositions." Such pharmaceutical compositions include the antibody-drug conjugates provided herein, and one or more pharmaceutically acceptable or physiologically acceptable excipients. In certain aspects, the antibody-drug conjugate is provided in combination with or separately from one or more additional agents. Compositions comprising such one or more additional agents, and one or more pharmaceutically acceptable or physiologically acceptable excipients are also provided. In certain aspects, the antibody-drug conjugate and additional agent(s) are present in therapeutically acceptable amounts. The pharmaceutical compositions can be used in accordance with the methods and uses provided herein. Thus, for example, the pharmaceutical compositions can be administered ex vivo or in vivo to a subject to perform the therapeutic methods and uses provided herein. The pharmaceutical compositions provided herein can be formulated to be compatible with the intended method or route of administration, and exemplary routes of administration are described herein.

[0347] In some aspects, pharmaceutical compositions of antibody-drug conjugates that modulate cancer or tumors are provided.

[0348] In certain aspects of the methods provided herein, a pharmaceutical composition comprising an ADC can further comprise other therapeutically active agents or compounds disclosed herein or known to those of skill in the art that can be used for the treatment or prevention of the various diseases and disorders (e.g., cancer) described herein. As noted above, the additional therapeutically active agent or compound may be present in a separate pharmaceutical composition(s).

[0349] Pharmaceutical compositions typically comprise at least one therapeutically effective amount of at least one of the antibody-drug conjugates provided herein, and one or more pharmaceutically acceptable formulation agents. In certain aspects, the pharmaceutical composition further comprises one or more additional agents described herein.

[0350] In one aspect, the pharmaceutical composition comprises the antibody-drug conjugate provided herein. In some aspects, the pharmaceutical composition comprises a therapeutically effective amount of the antibody-drug conjugate provided herein. In certain aspects, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0351] In some aspects, the antibody-drug conjugate in the pharmaceutical composition provided herein is selected from the antibody-drug conjugates described in Section 5.3 below.

[0352] In certain embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of 0.1 mg / mL to 100 mg / mL. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of 1 mg / mL to 20 mg / mL. In other embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of 5 mg / mL to 15 mg / mL. In other embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of 8 mg / mL to 12 mg / mL. In other embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of 9 mg / mL to 11 mg / mL. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of about 9.5 mg / mL. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of about 9.6 mg / mL. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of about 9.7 mg / mL. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of about 9.8 mg / mL. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of about 9.9 mg / mL. In still other embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of about 10 mg / mL. In still other embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of about 10.1 mg / mL. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of about 10.2 mg / mL. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of about 10.3 mg / mL. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of about 10.3 mg / mL. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of about 10.4 mg / mL. In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate at a concentration of about 10.5 mg / mL.

[0353] 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.

[0354] In some embodiments, the concentration of L - histidine useful in the pharmaceutical compositions provided herein ranges from 5 mM to 50 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein ranges from 10 mM to 40 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein ranges from 15 mM to 35 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein ranges from 15 mM to 30 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein ranges from 15 mM to 25 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein ranges from 15 mM to 35 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein is about 16 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein is about 17 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein is about 18 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein is about 19 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein is about 20 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein is about 21 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein is about 22 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein is about 23 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein is about 24 mM. In some embodiments, the concentration of L - histidine in the pharmaceutical compositions provided herein is about 25 mM.

[0355] In some embodiments, the concentration of TWEEN-20 useful in the pharmaceutical compositions provided herein ranges from 0.001% to 0.1% (v / v). In another embodiment, the concentration of TWEEN-20 ranges from 0.0025% to 0.075% (v / v). In one embodiment, the concentration of TWEEN-20 ranges from 0.005% to 0.05% (v / v). In another embodiment, the concentration of TWEEN-20 ranges from 0.0075% to 0.025% (v / v). In another embodiment, the concentration of TWEEN-20 ranges from 0.0075% to 0.05% (v / v). In another embodiment, the concentration of TWEEN-20 ranges from 0.01% to 0.03% (v / v). In a particular embodiment, the concentration of TWEEN-20 is about 0.01% (v / v). In a particular embodiment, the concentration of TWEEN-20 is about 0.015% (v / v). In a particular embodiment, the concentration of TWEEN-20 is about 0.016% (v / v). In a particular embodiment, the concentration of TWEEN-20 is about 0.017% (v / v). In a particular embodiment, the concentration of TWEEN-20 is about 0.018% (v / v). In a particular embodiment, the concentration of TWEEN-20 is about 0.019% (v / v). In a particular embodiment, the concentration of TWEEN-20 is about 0.02% (v / v). In a particular embodiment, the concentration of TWEEN-20 is about 0.021% (v / v). In a particular embodiment, the concentration of TWEEN-20 is about 0.022% (v / v). In a particular embodiment, the concentration of TWEEN-20 is about 0.023% (v / v). In a 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).

[0356] In one aspect, the concentration of trehalose dihydrate useful in the pharmaceutical compositions provided herein ranges from 1% to 20% (w / v). In another aspect, the concentration of trehalose dihydrate ranges from 2% to 15% (w / v). In one aspect, the concentration of trehalose dihydrate ranges from 3% to 10% (w / v). In another aspect, the concentration of trehalose dihydrate ranges from 4% to 9% (w / v). In another aspect, the concentration of trehalose dihydrate ranges from 4% to 8% (w / v). In another aspect, the concentration of trehalose dihydrate ranges from 4% to 7% (w / v). In another aspect, the concentration of trehalose dihydrate ranges from 4% to 6% (w / v). In another aspect, the concentration of trehalose dihydrate ranges from 4.5% to 6% (w / v). In another aspect, the concentration of trehalose dihydrate is about 4.6% (w / v). In another aspect, the concentration of trehalose dihydrate is about 4.7% (w / v). In another aspect, the concentration of trehalose dihydrate is about 4.8% (w / v). In another aspect, the concentration of trehalose dihydrate is about 4.9% (w / v). In another aspect, the concentration of trehalose dihydrate is about 5.0% (w / v). In another aspect, the concentration of trehalose dihydrate is about 5.1% (w / v). In another aspect, the concentration of trehalose dihydrate is about 5.2% (w / v). In another aspect, the concentration of trehalose dihydrate is about 5.3% (w / v). In another aspect, the concentration of trehalose dihydrate is about 5.4% (w / v). In another aspect, the concentration of trehalose dihydrate is about 5.5% (w / v). In another aspect, the concentration of trehalose dihydrate is about 5.6% (w / v). In another aspect, the concentration of trehalose dihydrate is about 5.7% (w / v). In another aspect, the concentration of trehalose dihydrate is about 5.8% (w / v). In another aspect, the concentration of trehalose dihydrate is about 5.9% (w / v). In another aspect, the concentration of trehalose dihydrate is about 6.0% (w / v). In another aspect, the concentration of trehalose dihydrate is about 6.1% (w / v). In another aspect, the concentration of trehalose dihydrate is about 6.2% (w / v). In another aspect, the concentration of trehalose dihydrate is about 6.3% (w / v). In another aspect, the concentration of trehalose dihydrate is about 6.4% (w / v).In another aspect, the concentration of trehalose dihydrate is about 6.5% (w / v).

[0357] In certain aspects, the molar concentration of trehalose dihydrate is from 50 mM to 300 mM. In other aspects, the molar concentration of trehalose dihydrate is from 75 mM to 250 mM. In some aspects, the molar concentration of trehalose dihydrate is from 100 mM to 200 mM. In other aspects, the molar concentration of trehalose dihydrate is from 130 mM to 150 mM. In some aspects, the molar concentration of trehalose dihydrate is from 135 mM to 150 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 135 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 136 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 137 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 138 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 139 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 140 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 141 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 142 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 143 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 144 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 145 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 146 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 150 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 151 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 151 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 152 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 153 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 154 mM. In certain aspects, the molar concentration of trehalose dihydrate is about 155 mM.

[0358] In one aspect, the concentration of sucrose useful in the pharmaceutical compositions provided herein ranges from 1% to 20% (w / v). In another aspect, the concentration of sucrose ranges from 2% to 15% (w / v). In one aspect, the concentration of sucrose ranges from 3% to 10% (w / v). In another aspect, the concentration of sucrose ranges from 4% to 9% (w / v). In another aspect, the concentration of sucrose ranges from 4% to 8% (w / v). In another aspect, the concentration of sucrose ranges from 4% to 7% (w / v). In another aspect, the concentration of sucrose ranges from 4% to 6% (w / v). In another aspect, the concentration of sucrose ranges from 4.5% to 6% (w / v). In another aspect, the concentration of sucrose is about 4.6% (w / v). In another aspect, the concentration of sucrose is about 4.7% (w / v). In another aspect, the concentration of sucrose is about 4.8% (w / v). In another aspect, the concentration of sucrose is about 4.9% (w / v). In another aspect, the concentration of sucrose is about 5.0% (w / v). In another aspect, the concentration of sucrose is about 5.1% (w / v). In another aspect, the concentration of sucrose is about 5.2% (w / v). In another aspect, the concentration of sucrose is about 5.3% (w / v). In another aspect, the concentration of sucrose is about 5.4% (w / v). In another aspect, the concentration of sucrose is about 5.5% (w / v). In another aspect, the concentration of sucrose is about 5.6% (w / v). In another aspect, the concentration of sucrose is about 5.7% (w / v). In another aspect, the concentration of sucrose is about 5.8% (w / v). In another aspect, the concentration of sucrose is about 5.9% (w / v). In another aspect, the concentration of sucrose is about 6.0% (w / v). In another aspect, the concentration of sucrose is about 6.1% (w / v). In another aspect, the concentration of sucrose is about 6.2% (w / v). In another aspect, the concentration of sucrose is about 6.3% (w / v). In another aspect, the concentration of sucrose is about 6.4% (w / v). In another aspect, the concentration of sucrose is about 6.5% (w / v).

[0359] In certain embodiments, the molar concentration of sucrose is from 50 mM to 300 mM. In other embodiments, the molar concentration of sucrose is from 75 mM to 250 mM. In some embodiments, the molar concentration of sucrose is from 100 mM to 200 mM. In other embodiments, the molar concentration of sucrose is from 130 mM to 150 mM. In some embodiments, the molar concentration of sucrose is from 135 mM to 150 mM. In certain embodiments, the molar concentration of sucrose is about 135 mM. In certain embodiments, the molar concentration of sucrose is about 136 mM. In certain embodiments, the molar concentration of sucrose is about 137 mM. In certain embodiments, the molar concentration of sucrose is about 138 mM. In certain embodiments, the molar concentration of sucrose is about 139 mM. In certain embodiments, the molar concentration of sucrose is about 140 mM. In certain embodiments, the molar concentration of sucrose is about 141 mM. In certain embodiments, the molar concentration of sucrose is about 142 mM. In certain embodiments, the molar concentration of sucrose is about 143 mM. In certain embodiments, the molar concentration of sucrose is about 144 mM. In certain embodiments, the molar concentration of sucrose is about 145 mM. In certain embodiments, the molar concentration of sucrose is about 146 mM. In certain embodiments, the molar concentration of sucrose is about 150 mM. In certain embodiments, the molar concentration of sucrose is about 151 mM. In certain embodiments, the molar concentration of sucrose is about 151 mM. In certain embodiments, the molar concentration of sucrose is about 152 mM. In certain embodiments, the molar concentration of sucrose is about 153 mM. In certain embodiments, the molar concentration of sucrose is about 154 mM. In certain embodiments, the molar concentration of sucrose is about 155 mM.

[0360] In some embodiments, the pharmaceutical compositions provided herein contain HCl. In other embodiments, the pharmaceutical compositions provided herein contain succinic acid.

[0361] 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.

[0362] In some embodiments, the pH is measured at room temperature. In other embodiments, the pH is measured at 15°C to 27°C. In still other embodiments, the pH is measured at 4°C. In still other embodiments, the pH is measured at 25°C.

[0363] In some embodiments, the pH is adjusted by HCl. In some embodiments, the pharmaceutical composition contains 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 contains 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 contains HCl and the pharmaceutical composition has a pH of about 5.7 at room temperature. In some more specific embodiments, the pharmaceutical composition contains HCl and the pharmaceutical composition has a pH of about 5.8 at room temperature. In some more specific embodiments, the pharmaceutical composition contains HCl and the pharmaceutical composition has a pH of about 5.9 at room temperature. In some more specific embodiments, the pharmaceutical composition contains HCl and the pharmaceutical composition has a pH of about 6.0 at room temperature. In some more specific embodiments, the pharmaceutical composition contains HCl and the pharmaceutical composition has a pH of about 6.1 at room temperature. In some more specific embodiments, the pharmaceutical composition contains HCl and the pharmaceutical composition has a pH of about 6.2 at room temperature. In some more specific embodiments, the pharmaceutical composition contains HCl and the pharmaceutical composition has a pH of about 6.3 at room temperature.

[0364] 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°C to 27°C. 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°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 5.7 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 5.8 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 5.9 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 6.0 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 6.1 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 6.2 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 6.3 at 15°C to 27°C.

[0365] 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.

[0366] 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°C to 27°C. 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°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 5.7 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 5.8 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 5.9 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 6.0 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 6.1 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 6.2 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 6.3 at 15°C to 27°C.

[0367] In some particular embodiments, the pharmaceutical compositions provided herein comprise at least one of about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, and about 5.5% (w / v) trehalose dihydrate or about 5% (w / v) sucrose. In some embodiments, the pharmaceutical compositions provided herein further comprise HCl or succinic acid. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25°C.

[0368] In some specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate and HCl. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25°C.

[0369] In some specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5% (w / v) sucrose, and HCl. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25 °C.

[0370] In other specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and succinic acid. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25 °C.

[0371] In some specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5% (w / v) sucrose, and succinic acid. In some embodiments, the pH is about 6.0 at room temperature. In some embodiments, the pH is about 6.0 at 25 °C.

[0372] In a specific embodiment, what is provided herein is (a) The following structure: An antibody-drug conjugate having TIFF2025106332000041.tif36160, where L represents an antibody or an antigen-binding fragment thereof, and p is from 1 to 10; and (b) A pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and HCl wherein the antibody-drug conjugate is at a concentration of about 10 mg / mL and the pH is about 6.0 at 25 °C.

[0373] In another specific embodiment, the pharmaceutical composition provided herein is (a) The following structure: An antibody-drug conjugate comprising TIFF2025106332000042.tif36160, wherein L- represents an antibody or an antigen-binding fragment thereof, and p is from 1 to 10; and (b) A pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and succinic acid comprising, wherein the antibody-drug conjugate is at a concentration of about 10 mg / mL and the pH is about 6.0 at 25°C.

[0374] In yet another specific embodiment, the pharmaceutical composition provided herein (a) Has the following structure: An antibody-drug conjugate comprising TIFF2025106332000043.tif36160, wherein L- represents an antibody or an antigen-binding fragment thereof, and p is from 1 to 10; and (b) A pharmaceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.0% (w / v) sucrose, and HCl comprising, wherein the antibody-drug conjugate is at a concentration of about 10 mg / mL and the pH is about 6.0 at 25°C.

[0375] Although specific numbers (and ranges thereof) are provided, in certain embodiments, numbers within, for example, 2%, 5%, 10%, 15% or 20% of said numbers (or ranges) are also contemplated. Other exemplary pharmaceutical compositions are provided in the experimental section below.

[0376] The primary solvent in the vehicle can be either essentially aqueous or non-aqueous. Further, the vehicle can include other pharmaceutically acceptable excipients for modifying or maintaining the pH, osmolality, viscosity, sterility or stability of the pharmaceutical composition. In certain embodiments, the pharmaceutically acceptable vehicle is an aqueous buffer. In other embodiments, the vehicle includes, for example, sodium chloride and / or sodium citrate.

[0377] The pharmaceutical compositions provided herein may include, as described herein, antibody-drug conjugates and / or other pharmaceutically acceptable formulation agents for modifying or maintaining the release rate of the additional agent. Such formulation agents include substances known to those of skill in the art in the preparation of sustained-release formulations. For further references on pharmaceutically and physiologically acceptable formulation agents, see, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, The Merck Index, 12th Ed. (1996, Merck Publishing Group, Whitehouse, NJ); and Pharmaceutical Principles of Solid Dosage Forms (1993, Technonic Publishing Co., Inc., Lancaster, Pa.). Additional pharmaceutical compositions suitable for administration are known in the art and applicable to the methods and compositions provided herein.

[0378] In some embodiments, the pharmaceutical compositions provided herein are in liquid form. In other embodiments, the pharmaceutical compositions provided herein are lyophilized.

[0379] The pharmaceutical compositions can be formulated to be compatible with their intended route of administration. Thus, the pharmaceutical compositions include excipients suitable for administration by routes including parenteral (e.g., subcutaneous (s.c.), intravenous, intramuscular, or intraperitoneal), intradermal, oral (e.g., ingestion), inhalation, intracavitary, intracranial, and transdermal (topical). Other exemplary routes of administration are described herein.

[0380] The pharmaceutical composition can be in the form of an aqueous or oily suspension for sterile injection. This suspension can be formulated using suitable dispersing or wetting agents and suspending agents disclosed herein or known to those skilled in the art. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as, for example, a solution in 1,3-butanediol. Acceptable diluents, solvents and dispersion media that can be used include water, Ringer's solution, isotonic sodium chloride solution, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS), ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Furthermore, sterile fixed oils have conventionally been used as solvents or suspending media. For this purpose, any bland fixed oil including synthetic monoglycerides or diglycerides can be used. In addition, fatty acids such as oleic acid are used in the preparation of injectables. Prolonged absorption of certain injectable formulations can be achieved by including agents that delay absorption (e.g., aluminum monostearate or gelatin).

[0381] In one aspect, the pharmaceutical compositions provided herein can be administered parenterally by injection, infusion, or implantation for local or systemic administration. Parenteral administration as used herein includes intravenous, intraarterial, intraperitoneal, intrathecal, intracerebroventricular, intraurethral, intrasternal, intracranial, intramuscular, intraarticular, and subcutaneous administration.

[0382] In one aspect, the pharmaceutical compositions provided herein can be formulated in any dosage form suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms, that are suitable as a solution or suspension in a liquid prior to injection. Such dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmacy (see, e.g., Remington, The Science and Practice of Pharmacy, supra).

[0383] In one aspect, a pharmaceutical composition intended for parenteral administration may contain one or more pharmaceutically acceptable excipients including, but not limited to, an aqueous vehicle, a water-miscible vehicle, a non-aqueous vehicle, an antibacterial or preservative against microbial growth, a stabilizer, a solubilizer, an isotonic agent, a buffer, an antioxidant, a local anesthetic, a suspending and dispersing agent, a wetting or emulsifying agent, a complexing agent, a sequestering or chelating agent, a cryoprotectant, a lyoprotectant, a thickening agent, a pH adjuster, and an inert gas.

[0384] In one aspect, 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 for injection, dextrose and lactated Ringer's injection. Non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil, and palm kernel oil. Water-miscible vehicles include, but are not limited to, ethanol, 1,3-butanediol, liquid polyethylene glycols (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N-methyl-2-pyrrolidone, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0385] In one aspect, suitable antibacterial or preservative agents include, but are not limited to, phenol, cresol, mercury, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl- and propyl-parabens, and sorbic acid. Suitable isotonic agents include, but are not limited to, sodium chloride, glycerin, and dextrose. Suitable buffering agents include, but are not limited to, phosphates and citrates. Suitable antioxidants are those described herein, including bisulfites and sodium metabisulfite. Suitable local anesthetics include, but are not limited to, procaine hydrochloride. Suitable suspending and dispersing agents are those described herein, including sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Suitable emulsifying agents include those described herein, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate. Suitable metal ion sequestering or chelating agents include, but are not limited to, EDTA. Suitable pH adjusters include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Suitable complexing agents include cyclodextrins, such as α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, and sulfobutyl ether 7-β-cyclodextrin (CAPTISOL®, CyDex, Lenexa, KS), but are not limited thereto.

[0386] In one aspect, the pharmaceutical compositions provided herein can be formulated for single or multiple administrations. Single-dose formulations are packaged in ampules, vials, or syringes. Multiple-dose parenteral formulations can include an antibacterial agent at a bacteriostatic or fungistatic concentration. All parenteral formulations must be sterile, as known and practiced in the art.

[0387] In one aspect, the pharmaceutical composition is provided as a ready-to-use sterile solution. In another aspect, the pharmaceutical composition is provided as a sterile dry soluble product, including a lyophilized powder and a subcutaneous tablet, which is reconstituted with a vehicle before use. In yet another aspect, the pharmaceutical composition is provided as a ready-to-use sterile suspension. In yet another aspect, the pharmaceutical composition is provided as a sterile dry insoluble product, which is reconstituted with a vehicle before use. In yet another aspect, the pharmaceutical composition is provided as a ready-to-use sterile emulsion.

[0388] In one aspect, the pharmaceutical compositions provided herein can be formulated as immediate or modified release dosage forms, including delayed release, sustained release, pulsatile release, controlled release, targeted release, and programmed release forms.

[0389] Dispersible powders and granules suitable for the preparation of aqueous suspensions by the addition of water provide the active ingredient mixed with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.

[0390] The pharmaceutical composition can also include excipients for protecting the composition from rapid degradation or elimination from the body, such as controlled release formulations including implants, liposomes, hydrogels, prodrugs, and microencapsulation delivery systems. For example, time delay materials such as glyceryl monostearate or glyceryl stearate can be used alone or in combination with waxes. Prolonged absorption of injectable pharmaceutical compositions can be achieved by including an agent that delays absorption, such as aluminum monostearate or gelatin. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

[0391] The pharmaceutical compositions provided herein can be stored at -80°C, 4°C, 25°C, or 37°C.

[0392] The lyophilized composition can be prepared by lyophilizing the liquid pharmaceutical composition 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 and can be reconstituted for administration as a solution, emulsion and other mixtures. They can also be reconstituted and formulated as solids or gels.

[0393] In some embodiments, the preparation of the lyophilized formulation provided herein includes batch processing of the formulated bulk solution for lyophilization, aseptic filtration, filling into vials, freezing of the vials in the lyophilization chamber, subsequent lyophilization, stoppering and capping.

[0394] A lyophilizer can be used for the preparation of the lyophilized formulation. For example, the VirTis Genesis Model EL pilot unit can be used. The unit incorporates a chamber with three shelves (the total available shelf area is about 0.4 square meters), an external condenser, and a mechanical vacuum pump system. Cascade mechanical refrigeration can cool the shelves to -70 °C or below and the external condenser to -90 °C or below. The shelf temperature and chamber pressure were automatically controlled to + / -0.5 °C and + / -2 microns (milliTorr), respectively. The unit was equipped with a capacitance manometer vacuum gauge, a Pirani vacuum gauge, a pressure transducer (for measuring 0 to 1 atmosphere), and a relative humidity sensor.

[0395] The lyophilized powder can be prepared by dissolving the antibody-drug conjugate or a pharmaceutically acceptable derivative thereof provided herein in a suitable solvent. In some embodiments, the lyophilized powder is sterilized. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial contains a single dose or multiple doses of the antibody-drug conjugate. The lyophilized powder can be stored under suitable conditions, for example, from about 4°C to room temperature.

[0396] Reconstitution of this lyophilized powder with water for injection provides a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable excipients. Such amounts can be determined empirically and adjusted according to specific needs.

[0397] Exemplary reconstitution procedures are shown below: (1) Attach an 18- or 20-gauge needle to a 5 mL or 3 mL syringe and fill the syringe with water for injection (WFI) grade water; (2) Using the syringe scale, measure the appropriate amount of WFI while ensuring there are no air bubbles in the syringe; (3) Insert the needle into the rubber stopper; (4) Dispense the entire contents of the syringe from the vial wall into the container, remove the syringe and needle, and place them in a sharps container; (4) Continuously swirl the vial and carefully solubilize the entire contents of the vial until completely reconstituted (e.g., for about 20 seconds to about 40 seconds), minimizing excessive agitation of the protein solution that can cause foaming.

[0398] 5.5 Method of Use of the Pharmaceutical Composition in Combination Therapy The method of inhibiting the growth of tumor cells by using the pharmaceutical composition provided herein in combination with chemotherapy or radiation or both comprises administering the pharmaceutical composition before, during, or after the initiation of chemotherapy or radiation therapy, and in any combination thereof (i.e., before and during the initiation of chemotherapy and / or radiation therapy, before and after the initiation, during and after the initiation, or before, during, and after the initiation). Depending on the treatment protocol and the needs of a particular patient, this method is carried out to provide the most effective treatment and ultimately extend the patient's lifespan.

[0399] Administration of chemotherapeutic agents can be accomplished by a variety of methods including systemic administration by parenteral and enteral routes. In one aspect, the chemotherapeutic agents are administered separately. Specific examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon α, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, gemcitabine, chlorambucil, taxol, and combinations thereof.

[0400] The radiation source used in combination with the pharmaceutical composition provided herein can be either external or internal to the patient being treated. When the source is external to the patient, the treatment is known as external beam radiation therapy (EBRT). When the radiation source is internal to the patient, the treatment is called brachytherapy (BT).

[0401] The above treatment regimen can be further combined with additional cancer therapeutic agents and / or regimens, such as additional chemotherapy, cancer vaccines, signal transduction inhibitors, agents useful for treating abnormal cell proliferation or cancer, antibodies that inhibit tumor growth by binding to IGF-1R (e.g., anti-CTLA-4 antibodies described in International Publication No. 2005 / 092380 (Pfizer)) or other ligands, and cytokines.

[0402] When a mammal undergoes additional chemotherapy, the above chemotherapeutic agents can be used. Further, growth factor inhibitors, biological response modifiers, antihormonal therapy, selective estrogen receptor modulators (SERMs), angiogenesis inhibitors, and antiandrogen agents can be used. For example, antihormonal agents, such as antiestrogen agents like Nolvadex (tamoxifen), or antiandrogen agents like Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)propionanilide) can be used.

[0403] In some embodiments, the pharmaceutical composition provided herein is used in combination with a second therapeutic agent, for example, to treat cancer.

[0404] In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor. As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that wholly or partially reduces, inhibits, impedes, or modulates one or more checkpoint proteins. Without being limited to a particular theory, checkpoint proteins regulate the activation or function of T cells. A number of checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 and its ligands PD-L1 and PD-L2 (Pardoll, Nature Reviews Cancer, 2012, 12, 252-264). These proteins appear to be involved in co-stimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins appear to regulate and maintain self-tolerance as well as the duration and magnitude of physiological immune responses. Immune checkpoint inhibitors include or are derived from antibodies.

[0405] In one embodiment, 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. Patent Nos. 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238, all of which are hereby incorporated by reference in their entirety. In one embodiment, the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP-675,206). In another embodiment, the anti-CTLA-4 antibody is ipilimumab (also known as MDX-010 or MDX-101). Ipilimumab is a fully human monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is commercially available under the trade name Yervoy(®).

[0406] In one aspect, the checkpoint inhibitor is a PD-1 / PD-L1 inhibitor. Examples of PD-1 / PD-L1 inhibitors include those described in U.S. Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, as well as PCT International Publication Nos. 2003042402; 2008156712; 2010089411; 2010036959; 2011066342; 2011159877; 2011082400; and 2011161699, but are not limited thereto, and all of these are hereby incorporated by reference in their entirety.

[0407] In one aspect, the checkpoint inhibitor is a PD-1 inhibitor. In one aspect, the PD-1 inhibitor is an anti-PD-1 antibody. In one aspect, the anti-PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558, or MDX1106), or pembrolizumab (also known as MK-3475, SCH 900475, or lambrolizumab). In one aspect, the anti-PD-1 antibody is nivolumab. Nivolumab is a human IgG4 anti-PD-1 monoclonal antibody and is commercially available under the trade name Opdivo™. In another aspect, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 antibody and is commercially available under the trade name Keytruda™. In yet another aspect, the anti-PD-1 antibody is CT-011, a humanized antibody. CT-011 administered alone was unable to show a response in the treatment of relapsed acute myeloid leukemia (AML). In yet another aspect, the anti-PD-1 antibody is AMP-224, a fusion protein. In another aspect, the PD-1 antibody is BGB-A317. BGB-A317 is a monoclonal antibody with a unique binding signature that has been specifically engineered for its ability to bind to Fcγ receptor I and has high affinity and excellent target specificity for PD-1.

[0408] In one aspect, the checkpoint inhibitor is a PD-L1 inhibitor. In one aspect, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one aspect, the anti-PD-L1 antibody is MEDI4736 (durvalumab). In another aspect, the anti-PD-L1 antibody is BMS-936559 (also known as MDX-1105-01). In yet another aspect, the PD-L1 inhibitor is atezolizumab (also known as MPDL3280A and Tecentriq®).

[0409] In one aspect, the checkpoint inhibitor is a PD-L2 inhibitor. In one aspect, the PD-L2 inhibitor is an anti-PD-L2 antibody. In one aspect, the anti-PD-L2 antibody is rHIgM12B7A.

[0410] In one aspect, the checkpoint inhibitor is a lymphocyte activation gene 3 (LAG-3) inhibitor. In one aspect, the LAG-3 inhibitor is IMP321, a soluble Ig fusion protein (Brignone et al., J. Immunol., 2007, 179, 4202-4211). In another aspect, the LAG-3 inhibitor is BMS-986016.

[0411] In one aspect, the checkpoint inhibitor is a B7 inhibitor. In one aspect, the B7 inhibitor is a B7-H3 inhibitor or a B7-H4 inhibitor. In one aspect, the B7-H3 inhibitor is MGA271, an anti-B7-H3 antibody (Loo et al., Clin. Cancer Res., 2012, 3834).

[0412] In one aspect, the checkpoint inhibitor is a TIM3 (T cell immunoglobulin domain and mucin domain 3) inhibitor (Fourcade et al., J. Exp. Med., 2010, 207, 2175-86; Sakuishi et al., J. Exp. Med., 2010, 207, 2187-94).

[0413] In one aspect, the checkpoint inhibitor is an OX40 (CD134) agonist. In one aspect, the checkpoint inhibitor is an anti-OX40 antibody. In one aspect, the anti-OX40 antibody is anti-OX-40. In another aspect, the anti-OX40 antibody is MEDI6469.

[0414] In one aspect, the checkpoint inhibitor is a GITR agonist. In one aspect, the checkpoint inhibitor is an anti-GITR antibody. In one aspect, the anti-GITR antibody is TRX518.

[0415] In one aspect, the checkpoint inhibitor is a CD137 agonist. In one aspect, the checkpoint inhibitor is an anti-CD137 antibody. In one aspect, the anti-CD137 antibody is urelumab. In another aspect, the anti-CD137 antibody is PF-05082566.

[0416] In one aspect, the checkpoint inhibitor is a CD40 agonist. In one aspect, the checkpoint inhibitor is an anti-CD40 antibody. In one aspect, the anti-CD40 antibody is CF-870,893.

[0417] In one aspect, the checkpoint inhibitor is recombinant human interleukin-15 (rhIL-15).

[0418] In one aspect, the checkpoint inhibitor is an IDO inhibitor. In one aspect, the IDO inhibitor is INCB024360. In another aspect, the IDO inhibitor is indoximod.

[0419] In ce...

Claims

**Claim 1** A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or the antigen-binding fragment thereof comprises a heavy-chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy-chain variable region shown in SEQ ID NO:22, and a light-chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light-chain variable region shown in SEQ ID NO:23, and the subject has hormone receptor-positive and human epidermal growth factor receptor 2-negative (HR+ / HER2-) breast cancer, the method. **Claim 2** The method according to claim 1, wherein the HR+ / HER2- breast cancer is estrogen receptor (ER)-positive and / or progesterone receptor (PR)-positive and HER2-negative. **Claim 3** The method according to claim 1 or claim 2, wherein the subject has locally advanced cancer or metastatic cancer. **Claim 4** The method according to any one of claims 1 to 3, wherein the subject has previously received at least one type of endocrine therapy and a cyclin-dependent kinase (CDK) 4 / 6 inhibitor in a metastatic or locally advanced setting. **Claim 5** The method according to any one of claims 1 to 4, wherein the subject has previously received treatment with a taxane or anthracycline. **Claim 6** The method according to any one of claims 1 to 5, wherein the subject has a deleterious germline mutation in the breast cancer susceptibility gene (BRCA) 1 or BRCA2 and has previously been treated with a poly ADP ribose polymerase (PARP) inhibitor. **Claim 7** A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or the antigen-binding fragment thereof comprises a heavy-chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy-chain variable region shown in SEQ ID NO:22, and a light-chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light-chain variable region shown in SEQ ID NO:23, and The subject has ER-negative, PR-negative, and HER2-negative (ER- / PR- / HER2-) breast cancer (triple-negative breast cancer - TNBC). Method.

8. The method according to claim 7, wherein the subject has locally advanced cancer or metastatic cancer.

9. The method according to claim 7 or claim 8, wherein the subject has previously received at least two types of systemic therapy.

10. The method according to claim 9, wherein the subject has previously received treatment with a taxane.

11. The method according to any one of claims 7 to 10, wherein the subject has a deleterious germline mutation in breast cancer susceptibility gene (BRCA) 1 or BRCA2 and has been previously treated with a poly ADP ribose polymerase (PARP) inhibitor.

12. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, the antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethyl auristatin E (MMAE), the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region comprising complementarity-determining regions (CDRs) comprising the amino acid sequences of the CDRs of the heavy chain variable region shown in SEQ ID NO:22 and a light chain variable region comprising CDRs comprising the amino acid sequences of the CDRs of the light chain variable region shown in SEQ ID NO:23, and the subject has squamous non-small cell lung cancer (NSCLC). Method.

13. The method according to claim 12, wherein the subject has locally advanced cancer or metastatic cancer.

14. The method according to claim 12 or claim 13, wherein the subject has progressed or relapsed after platinum-based therapy.

15. The method according to claim 14, wherein the subject has progressed or relapsed within 12 months after platinum-based therapy.

16. The method according to any one of claims 12 to 15, wherein the subject has previously been treated with an inhibitor of programmed cell death protein-1 (PD-1) or an inhibitor of programmed cell death-ligand 1 (PD-L1), optionally the inhibitor of PD-1 is nivolumab, and optionally the inhibitor of PD-L1 is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

17. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, The antibody-drug conjugate comprises an antibody that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region 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 the subject has non-squamous NSCLC, method.

18. The method according to claim 17, wherein the subject has wild-type epidermal growth factor receptor (EGFR) and wild-type anaplastic lymphoma kinase (ALK).

19. The method according to claim 17 or 18, wherein the subject has locally advanced or metastatic cancer.

20. The method according to any one of claims 17 to 19, wherein the subject has progressed or relapsed after platinum-based therapy.

21. The method according to claim 20, wherein the subject has progressed or relapsed within 12 months after platinum-based therapy.

22. The method according to any one of claims 17 to 21, wherein the subject has previously been treated with an inhibitor of programmed cell death protein-1 (PD-1) or an inhibitor of programmed cell death-ligand 1 (PD-L1), optionally the inhibitor of PD-1 is nivolumab, and optionally the inhibitor of PD-L1 is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

23. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, the antibody-drug conjugate comprises an antibody that binds to 191P4D12 or an antigen-binding fragment thereof conjugated to one or more units of monomethyl auristatin E (MMAE), wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region 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 the subject has locally advanced or metastatic head and neck cancer, method.

24. The method according to claim 23, wherein the subject has progressed or relapsed after platinum-based therapy.

25. The method according to claim 24, wherein the subject has progressed or relapsed within 6 months after platinum-based therapy.

26. The method according to any one of claims 23 to 25, wherein the subject has previously been treated with an inhibitor of programmed cell death protein-1 (PD-1) or an inhibitor of programmed cell death-ligand 1 (PD-L1), optionally the inhibitor of PD-1 is nivolumab, and optionally the inhibitor of PD-L1 is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

27. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of an antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody or an antigen-binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethyl auristatin E (MMAE), and the antibody or its antigen-binding fragment comprises a heavy-chain variable region comprising a complementarity-determining region (CDR) comprising the amino acid sequences of the CDRs 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 sequences of the CDRs of the light-chain variable region shown in SEQ ID NO:23, and the subject has gastric cancer or esophageal cancer, a method.

28. The method according to claim 27, wherein the subject has locally advanced cancer or metastatic cancer.

29. The method according to claim 27 or claim 28, wherein the subject has progressed or relapsed after platinum-based therapy and / or chemotherapy comprising fluoropyrimidine.

30. The method according to claim 29, wherein the subject has progressed or relapsed within 6 months after platinum-based therapy or chemotherapy comprising fluoropyrimidine.

31. The method according to any one of claims 27 to 30, wherein the gastric cancer or esophageal cancer is HER2-positive cancer and the subject has previously received HER2-directed therapy.

32. The antibody or its antigen-binding fragment 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 An 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, The method according to any one of claims 1 to 31.

33. The method according to any one of claims 1 to 31, 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.

34. The method according to any one of claims 1 to 31, wherein the antibody comprises a heavy chain comprising an amino acid sequence ranging from the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO:7 and a light chain comprising an amino acid sequence ranging from the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO:

8.

35. The method according to any one of claims 1 to 34, wherein the antigen-binding fragment is a Fab, F(ab') 2 , Fv or scFv fragment.

36. The method according to any one of claims 1 to 35, wherein the antibody is a fully human antibody.

37. The method according to any one of claims 1 to 36, wherein the antibody or antigen-binding fragment thereof is recombinantly produced.

38. The antibody-drug conjugate has the following structure: wherein L- represents an antibody or antigen-binding fragment thereof, and p is 1 to 10. The method according to any one of claims 1 to 37.

39. The method according to claim 38, wherein p is 2 to 8.

40. The method according to claim 38, wherein p is 3 to 5.

41. The method according to any one of claims 1 to 40, wherein the antibody or antigen-binding fragment is linked to each unit of monomethyl auristatin E (MMAE) via a linker.

42. The method according to claim 41, wherein the linker is an enzymatically cleavable linker that forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof.

43. The linker is -A a -W w -Y y having the formula of -A-a-W-w-Y-y-, wherein -A- is an elongation unit, a is 0 or 1, -W- is an amino acid unit, w is an integer in the range of 0 to 12, -Y- is a spacer unit, and y is 0, 1, or 2, the method according to claim 41.

44. The method according to claim 43, wherein the extension unit has the structure of formula (1) and the amino acid unit is valine citrulline; the spacer unit is a PAB group comprising the structure of formula (2). 。

45. The method according to claim 43, wherein the elongation unit forms a bond with a sulfur atom of the antibody or its antigen-binding fragment, and the spacer unit is linked to MMAE via a carbamate group.

46. The method according to any one of claims 1 to 37, wherein the antibody-drug conjugate comprises 1 to 10 units of MMAE per antibody or its antigen-binding fragment.

47. The method according to any one of claims 1 to 37, wherein the antibody-drug conjugate comprises 2 to 8 units of MMAE per antibody or its antigen-binding fragment.

48. The method according to any one of claims 1 to 37, wherein the antibody-drug conjugate comprises 3 to 5 units of MMAE per antibody or its antigen-binding fragment.

49. The method according to any one of claims 1 to 48, wherein the antibody-drug conjugate is administered at a dose of 1 to 10 mg / kg of subject body weight, 1 to 5 mg / kg of subject body weight, 1 to 2.5 mg / kg of subject body weight, or 1 to 1.25 mg / kg of subject body weight.

50. The method according to claim 49, wherein the antibody-drug conjugate is administered at a dose of about 1 mg / kg of subject body weight.

51. The method according to claim 49, wherein the antibody-drug conjugate is administered at a dose of about 1.25 mg / kg of subject body weight.

52. The method according to any one of claims 1 to 51, wherein the antibody-drug conjugate is administered by intravenous (IV) injection or infusion.

53. The method according to any one of claims 1 to 51, wherein the antibody-drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes in a cycle of twice every 3 weeks.

54. The method according to any one of claims 1 to 51, wherein the antibody-drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes on the 1st and 8th days of each 3-week cycle.

55. The method according to any one of claims 1 to 51, wherein the antibody-drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes in a cycle of three times every 4 weeks.

56. The method according to any one of claims 1 to 51, wherein the antibody-drug conjugate formulated into a pharmaceutical composition is administered by intravenous (IV) injection or infusion over about 30 minutes on the 1st, 8th, and 15th days of each 4-week cycle.

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