Methods for treating cancer with antibody drug conjugates (ADCs) that bind to 191P4D12 protein

Abstract

Methods for the treatment of cancer using antibody drug conjugates (ADCs) that bind to 191P4D12 protein are provided herein. TIFF2022544227000068.tif157169

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 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 antibody drug conjugates (ADCs) that bind to 191P4D12 proteins. [Background technology]

[0003] 2. Background Cancer is the leading cause of death for people aged 35-65 in the United States and the second leading cause of death worldwide. In 2019, it was estimated that there were approximately 1.7 million new cancer cases and approximately 610,000 cancer 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). Globally, there were an estimated 18.1 million new cancer cases in 2018, and approximately 9.6 million deaths were due to cancer in 2018 (World Health Organization. Press Release. Sept 2018. https: / / www.who.int / cancer / PRGlobocanFinal.pdf. Accessed June 5, 2019). Currently, most deaths occur in patients with metastatic cancer. Indeed, over the past two decades, advances in treatment, including surgery, radiation therapy, and adjuvant chemotherapy, have cured most patients with localized cancer. Patients whose cancer has manifested as metastatic disease or recurred have only garnered modest benefits from conventional treatments in terms of overall survival (OS) and have rarely been cured.

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

[0005] 191P4D12 (also known as nectin-4) is a type I transmembrane protein and a member of a 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 through both homophilic and heterophilic trans-interactions at adherens junctions, which can recruit cadherins and regulate cytoskeletal rearrangements (Rikitake&Takai,Cell Mol Life Sci.2008;65(2):253-63). The sequence identity of 191P4D12 to other nectin family members is low, ranging from 25% to 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 Globally, approximately 2.1 million new cases of breast cancer will be diagnosed in women in 2018, accounting for nearly 1 in 4 cancer cases in women. The disease is the most frequently diagnosed cancer in the majority of countries and the leading cause of cancer-related deaths in women. After a metastatic diagnosis, the prognosis is poor, with a 5-year survival rate of approximately 15%.

[0007] The selection of appropriate treatment for metastatic breast cancer is complicated 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 human epidermal growth factor receptor 2 (HER2) status of the tumor. Treatment options for subjects presenting with metastatic breast cancer may also be influenced by which adjuvant therapy was used, how soon after adjuvant therapy the subject recurs, and the site of metastasis.

[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 primarily in postmenopausal women. Initial treatment for women with metastatic disease consists primarily of endocrine therapy, which is usually administered alone, in combination with CDK4 / 6 inhibitors, or as dual endocrine blockade. Systemic chemotherapy is recommended for women with endocrine refractory or symptomatic visceral disease.

[0009] Several cytotoxic chemotherapy agents have shown activity in metastatic breast cancer, including anthracyclines, taxanes, gemcitabine, capecitabine, vinorelbine, eribulin, and ixabepilone. Response rates with these agents vary depending on the type of prior therapy and breast cancer subtype. In general, anthracycline-based combination therapy and taxanes such as paclitaxel and docetaxel are considered to be the most effective (Piccart M, Clin Breast Cancer 2008;100-13). Given the widespread use of anthracyclines in the adjuvant setting and the increased risk of cardiac toxicity, the use of anthracyclines in the metastatic setting is limited. Taxanes are the most commonly used agents for patients with locally advanced or metastatic disease, especially in the first-line setting (Greene & Hennessy, J Oncol Pharm Pract 2015;201-12). Sequential monotherapy is recommended over combinations due to low toxicity and limited survival benefit. Responses to commonly used single-agent chemotherapy in patients with HR+ / HER2-negative breast cancer were largely confined to subgroup analyses, and these 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 tended to be lower in previously treated patients, reported to be in the 10% to 13% range (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%-20% of breast cancers are classified as TNBC. TNBC is associated with aggressive tumor biology, visceral metastasis, and poor prognosis (Plasilova ML et al, Medicine (Baltimore) 2016;95(35):e4614).

[0011] Taxane-based regimens are considered the standard of care in the first-line treatment of patients with metastatic breast cancer, including TNBC. More recently, the FDA granted accelerated approval of atezolizumab in combination with nab-paclitaxel for the treatment of patients with unresectable locally advanced or metastatic TNBC whose tumors express programmed death-ligand 1 (PD-L1) (median progression-free survival [PFS] 7.5 vs. 5.0 months; objective response rate (ORR) 56% vs. 46%) (Schmid P et al, N Engl J Med. 2019;380(10):987-988). No standard approach exists for second-line or subsequent treatment, and chemotherapy options are the same as those for other subtypes. Single-agent cytotoxic chemotherapy agents are generally preferred over combination chemotherapy due to lack of survival benefit and increased toxicity, except in the setting of invasive disease and visceral involvement (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 previously treated patients is associated with low response rates (10%-15%) and short progression-free survival (2-3 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 June 5, 2019). Most patients diagnosed with lung cancer are 65 years of age or older, with the average age at diagnosis being 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 June 5, 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 June 5, 2019) and can be subdivided into squamous (approximately 30% of NSCLC cases) and non-squamous (approximately 40% of NSCLC cases) histologies (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 June 5, 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 certain patient and disease characteristics, including older age, metastatic (including malignant or metastatic malignant) disease at diagnosis, comorbidities, and tumor central location (Socinski M et al, Cell Lung Cancer 2018;165-183). These characteristics are associated with poor treatment outcomes in metastatic (including malignant or metastatic malignant) squamous NSCLC, resulting in a median survival that is approximately 30% shorter than patients with other NSCLC subtypes.

[0015] Treatment options are limited, especially for the first-line treatment of metastatic (including malignant or metastatic malignant) squamous NSCLC, which consequently affects 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 approval of targeted therapies and immunotherapies for metastatic (including malignant or metastatic malignant) NSCLC and the continued move towards individualization of lung cancer treatment, the efficacy of these new treatments for metastatic (including malignant or metastatic malignant) squamous NSCLC also needs to be evaluated.

[0016] Non-squamous non-small cell lung cancer Nonsquamous NSCLC is a heterogeneous disease with multiple treatment options depending on patient factors including staging, presence of metastases, and presence of comorbidities among other considerations. Thus, current treatment options include surgical resection, chemotherapy, radiation, immunotherapy, and targeted therapy. Currently, the first-line therapy for patients with metastatic (including malignant or metastatic malignant) nonsquamous NSCLC without targetable genetic abnormalities is platinum-based doublet chemotherapy. With the exception of bevacizumab, and despite extensive research of multiple targeted and cytotoxic agents, the addition of a third agent to platinum-based doublet chemotherapy has not been shown to improve time to progression or OS over platinum-based doublet chemotherapy 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 June 5, 2019). Worldwide, over 5.5 million people are affected by head and neck cancer (2.4 million mouth, 1.7 million throat, and 1.4 million larynx), causing over 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 June 5, 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, accessed June 5, 2019, https: / / www.thelancet.com / journals / lancet / article / PIIS0140-6736(16)31678-6 / fulltext). 2015, https: / / www.sciencedirect.com / science / article / pii / S0140673616310121, accessed June 5, 2019).Worldwide, approximately 600,000 cases of head and neck cancer will occur this year, and only 40%-60% of patients will survive five years (Rene Leemans C, et al. The molecular biology of head and neck cancer, Nature Reviews Cancer, 16 Dec 2011, accessed June 5, 2019, https: / / www.nature.com / articles / nrc2982).

[0018] The most important risk factors are smoking and alcohol consumption, which appear to have a synergistic effect (Decker & Goldstein, N Engl J Med. 1982; 1151-1155). A subgroup of head and neck cancers, especially oropharyngeal cancer, 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 June 5, 2019, https: / / www.nature.com / articles / nrc2982).

[0019] Treatment is primarily determined by the stage at presentation but may include a combination of surgery, radiation therapy, chemotherapy, and targeted therapy (National Cancer Institute, 2019, Cancer Stat Facts: Cancer of Any Site, https: / / seer.cancer.gov / statfacts / html / all.html, accessed June 5, 2019). However, survival has not improved significantly in recent decades, as patients often develop locoregional recurrence, distant metastases, and second primary tumors. Limited information is available regarding the molecular oncogenesis of head and neck cancer, as well as the genetic and biological heterogeneity of the disease, hindering the development of new therapeutic strategies.

[0020] Stomach or esophageal cancer In 2019, an estimated 17,650 adult patients in the United States will be diagnosed with gastric cancer and approximately 16,080 will die from the 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 June 6, 2019). In 2019, an estimated 27,510 adults in the United States will be diagnosed with esophageal cancer and approximately 11,140 will die from the disease (American Cancer Society, Key Statistics About Stomach Cancer, 09 Jan 2019d, https: / / www.cancer.org / cancer / stomach-cancer / about / key-statistics.html, accessed June 6, 2019). The rates of esophageal adenocarcinoma and gastric cardia adenocarcinoma are increasing, whereas the rates of esophageal squamous cell carcinoma and noncardia gastric adenocarcinoma are 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, locally advanced or metastatic disease. Single agents (cisplatin, doxorubicin and mitomycin) that result in partial response (PR) rates 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 monotherapy, but are associated with a greater degree of toxicity and result in a similar OS (range 6-10 months) (Preusser P et al, Oncology 1998;99-102). Identification of new agents is therefore essential if extended patient survival is to be achieved.

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

[0023] 3. Overview 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 monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23, 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 or metastatic cancer.

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

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

[0028] In some embodiments 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 monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23, 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, including but not limited to the methods described in the paragraph immediately preceding this one (paragraph

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

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

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

[0033] In some aspects of the methods provided herein, including, but not limited to, those described in the first to fourth paragraphs prior to this paragraph (paragraphs

[0036] to

[0039] ), the subject has a deleterious germline mutation in breast cancer susceptibility gene (BRCA) 1 or BRCA2 and has previously been 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 or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23, and the subject has squamous non-small cell lung cancer (NSCLC).

[0035] In some aspects of the methods provided herein, including, but not limited to, the methods described in the paragraph immediately preceding this one (paragraph

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

[0036] In some aspects 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 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 where the inhibitor of PD-1 is nivolumab, and optionally where 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 or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23, 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, including, but not limited to, the methods described in the paragraphs one to two prior to this paragraph (paragraphs

[0046] to

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

[0042] In some aspects of the methods provided herein, for example, but not limited to, the methods described in the paragraphs one to three prior to this paragraph (paragraphs

[0046] to

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

[0043] In some aspects of the methods provided herein, for example, but not limited to, the methods described in the paragraphs one to four prior to this paragraph (paragraphs

[0046] to

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

[0044] In some aspects of the methods provided herein, for example, but not limited to, the methods described in the first to fifth paragraphs prior to 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 where the PD-1 inhibitor is nivolumab, and optionally where the PD-L1 inhibitor 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 or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22 and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23, and the subject has locally advanced or metastatic head and neck cancer.

[0046] In some aspects of the methods provided herein, for example, but not limited to, the methods described in the paragraph immediately preceding this one (paragraph

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

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

[0048] In some aspects of the methods provided herein, including, but not limited to, the methods described in the first to third paragraphs prior to 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 where the PD-1 inhibitor is nivolumab, and optionally where the PD-L1 inhibitor 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 monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23, and the subject has gastric cancer or esophageal cancer.

[0050] In some aspects of the methods provided herein, including but not limited to the methods described in the paragraph immediately preceding this one (paragraph

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

[0051] In some aspects of the methods provided herein, the subject has progressed or relapsed following platinum-based therapy or chemotherapy including a fluoropyrimidine.

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

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

[0054] In some embodiments of the methods provided herein, the antibody or antigen-binding fragment thereof comprises a CDR H1 comprising the amino acid sequence of SEQ ID NO:9, a CDR H2 comprising the amino acid sequence of SEQ ID NO:10, a CDR H3 comprising the amino acid sequence of SEQ ID NO:11; a CDR L1 comprising the amino acid sequence of SEQ ID NO:12, a CDR L2 comprising the amino acid sequence of SEQ ID NO:13, and a CDR L3 comprising the amino acid sequence of SEQ ID NO:14.

[0055] In some embodiments of the methods provided herein, the antibody or antigen-binding fragment thereof comprises a CDR H1 comprising the amino acid sequence of SEQ ID NO:16, a CDR H2 comprising the amino acid sequence of SEQ ID NO:17, a CDR H3 comprising the amino acid sequence of SEQ ID NO:18; a CDR L1 comprising the amino acid sequence of SEQ ID NO:19, a CDR L2 comprising the amino acid sequence of SEQ ID NO:20, and a CDR L3 comprising the amino acid sequence of SEQ ID NO:21.

[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 an amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7, and a light chain comprising an amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8.

[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 aspects of the methods provided herein, the antibody is a fully human antibody.

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

[0061] In some embodiments of the methods provided herein, the antibody drug conjugate has the following structure: TIFF0007657774000001.tif36160, where L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10.

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

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

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

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

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

[0067] In some embodiments of the methods provided herein, the extender unit has the structure of formula (1) below, the amino acid unit is valine citrulline, and the spacer unit is a PAB group having the structure of formula (2) below. TIFF0007657774000002.tif69128

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

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

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

[0071] In some embodiments of the methods provided herein, the antibody drug conjugate comprises 3-5 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 pharma- ceutically acceptable excipients including 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 an antibody drug conjugate at a concentration of 1-20 mg / mL, 5-15 mg / mL, or 8-12 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 5-50 mM, 10-40 mM, 15-35 mM, 15-30 mM, or 15-25 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 ranges from 0.001-0.1% (v / v), 0.0025-0.075% (v / v), 0.005-0.05% (v / v), or 0.01-0.03% (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 1-20% (w / v), 2-15% (w / v), 3-10% (w / v), or 4-6% (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 aspects 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 to 300 mM, 75 mM to 250 mM, 100 mM to 200 mM, or 130 mM to 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 to 6.5 or 5.7 to 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, between 15°C and 27°C, at about 4°C, or at about 25°C.

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

[0093] In some embodiments 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 HCl, having a pH of about 6.0 at 25° C.

[0094] In some embodiments 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 has a pH of about 6.0 at 25° C.

[0095] In some embodiments 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, having a pH of about 6.0 at 25° C.

[0096] In some embodiments 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 has a pH of about 6.0 at 25° C.

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

[0098] In some embodiments 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 embodiments 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 embodiments of the methods provided herein, the antibody drug conjugate is administered by intravenous (IV) injection or infusion.

[0101] 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 two cycles every three 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 every three-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 three cycles every four 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 every four-week cycle. [The present invention 1001] 1. A method of 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 or antigen-binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23; the subject has hormone receptor positive and human epidermal growth factor receptor 2 negative (HR+ / HER2-) breast cancer; method. [The present invention 1002] The method of the present invention 1001, wherein the HR+ / HER2- breast cancer is estrogen receptor (ER) positive and / or progesterone receptor (PR) positive and HER2 negative. [The present invention 1003] The method of claim 1001 or 1002, wherein the subject has locally advanced or metastatic cancer. [The present invention 1004] The method of any of claims 1001 to 1003, wherein the subject has previously received at least one endocrine therapy and a cyclin-dependent kinase (CDK) 4 / 6 inhibitor in the metastatic or locally advanced setting. [The present invention 1005] The method of any of claims 1001 to 1004, wherein the subject has previously received treatment with a taxane or anthracycline. [The present invention 1006] Any of the methods of claims 1001-1005, wherein the subject has a deleterious germline mutation in a breast cancer susceptibility gene (BRCA) 1 or BRCA2 and has been previously treated with a poly ADP ribose polymerase (PARP) inhibitor. [The present invention 1007] 1. A method of 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 or antigen-binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23; 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 of claim 1007, wherein the subject has locally advanced or metastatic cancer. [The present invention 1009] The method of claim 1007 or 1008, wherein the subject has previously received at least two systemic therapies. [The present invention 1010] The method of claim 1009, wherein the subject has previously undergone treatment with a taxane. [The present invention 1011] Any of the methods of claims 1007-1010, wherein the subject has a deleterious germline mutation in a breast cancer susceptibility gene (BRCA) 1 or BRCA2 and has been previously treated with a poly ADP ribose polymerase (PARP) inhibitor. [The present invention 1012] 1. A method of 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 or antigen-binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23; the subject has squamous non-small cell lung cancer (NSCLC), method. [The present invention 1013] The method of claim 1012, wherein the subject has locally advanced or metastatic cancer. [The present invention 1014] The method of claim 1012 or 1013, wherein the subject has progressed or relapsed after platinum-based therapy. [The present invention 1015] The method of claim 1014, wherein the subject has progressed or relapsed within 12 months after platinum-based therapy. [The present invention 1016] Any of the methods of claims 1012 to 1015, wherein 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 wherein the PD-1 inhibitor is nivolumab, and optionally wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab. [The present invention 1017] 1. A method of 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 or antigen-binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23; the subject has non-squamous NSCLC, method. [The present invention 1018] The method of claim 1017, wherein the subject has wild-type epidermal growth factor receptor (EGFR) and wild-type anaplastic lymphoma kinase (ALK). [The present invention 1019] The method of claim 1017 or 1018, wherein the subject has locally advanced or metastatic cancer. [The present invention 1020] Any of the methods of claims 1017 to 1019, wherein the subject has progressed or relapsed after platinum-based therapy. [The present invention 1021] The method of claim 1020, wherein the subject has progressed or relapsed within 12 months after platinum-based therapy. [The present invention 1022] Any of the methods of claims 1017 to 1021, wherein 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 wherein the PD-1 inhibitor is nivolumab, and optionally wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab. [The present invention 1023] 1. A method of 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 or antigen-binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23; the subject has locally advanced or metastatic head and neck cancer; method. [The present invention 1024] The method of claim 1023, wherein the subject has progressed or relapsed after platinum-based therapy. [The present invention 1025] The method of claim 1024, wherein the subject has progressed or relapsed within 6 months after platinum-based therapy. [The present invention 1026] Any of the methods of claims 1023 to 1025, wherein 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 wherein the PD-1 inhibitor is nivolumab, and optionally wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab. [The present invention 1027] 1. A method of 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 or antigen-binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23; the subject has gastric cancer or esophageal cancer; method. [The present invention 1028] The method of claim 1027, wherein the subject has locally advanced or metastatic cancer. [The present invention 1029] The method of claim 1027 or 1028, wherein the subject has progressed or relapsed after platinum-based therapy and / or chemotherapy comprising a fluoropyrimidine. [The present invention 1030] The method of claim 1029, wherein the subject has progressed or relapsed within 6 months after platinum-based therapy or chemotherapy including a fluoropyrimidine. [The present invention 1031] The method of any of claims 1027 to 1030, wherein the gastric or esophageal cancer is a HER2 positive cancer and the subject has previously undergone a HER2 directed therapy. [The present invention 1032] the antibody or antigen-binding fragment thereof comprises a CDR H1 comprising the amino acid sequence of SEQ ID NO:9, a CDR H2 comprising the amino acid sequence of SEQ ID NO:10, a CDR H3 comprising the amino acid sequence of SEQ ID NO:11; a CDR L1 comprising the amino acid sequence of SEQ ID NO:12, a CDR L2 comprising the amino acid sequence of SEQ ID NO:13, and a CDR L3 comprising the amino acid sequence of SEQ ID NO:14; or the antibody or antigen-binding fragment thereof comprises CDR H1 comprising the amino acid sequence of SEQ ID NO:16, CDR H2 comprising the amino acid sequence of SEQ ID NO:17, 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; Any of the methods of 1001 to 1031 of the present invention. [The present invention 1033] The method of any of claims 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. [The present invention 1034] Any of the methods of the present inventions 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] Antigen-binding fragments are Fab and F(ab') 2 , Fv or scFv fragment. [The present invention 1036] The method according to any one of claims 1001 to 1035, wherein the antibody is a fully human antibody. [The present invention 1037] The method of any of claims 1001 to 1036, wherein the antibody or antigen-binding fragment thereof is recombinantly produced. [The present invention 1038] The antibody drug conjugate has the structure: TIFF0007657774000003.tif36160 wherein L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10. Any of the methods of 1001 to 1037 of the present invention. [The present invention 1039] The method of claim 1038, wherein p is 2 to 8. [The present invention 1040] The method of claim 1038, wherein p is 3 to 5. [The present invention 1041] The method of any of claims 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] The method of claim 1041, wherein the linker is an enzyme-cleavable linker and forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof. [The present invention 1043] Linker is -A a -W w -Y y -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 present invention 1044] The method of the present invention 1043, wherein the extender unit has the structure of the following formula (1), the amino acid unit is valine citrulline; and the spacer unit is a PAB group having the structure of the following formula (2): TIFF0007657774000004.tif78128 。 [The present invention 1045] The method of claim 1043, wherein the extender unit forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof and the spacer unit is linked to MMAE via a carbamate group. [The present invention 1046] The method of any of claims 1001 to 1037, wherein the antibody drug conjugate comprises 1 to 10 units of MMAE per antibody or antigen-binding fragment thereof. [The present invention 1047] The method of any of claims 1001 to 1037, wherein the antibody drug conjugate comprises 2 to 8 units of MMAE per antibody or antigen-binding fragment thereof. [The present invention 1048] The method of any of claims 1001 to 1037, wherein the antibody drug conjugate comprises 3 to 5 units of MMAE per antibody or antigen-binding fragment thereof. [The present invention 1049] The method of any of claims 1001 to 1048, 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. [The present invention 1050] The method of claim 1049, wherein the antibody drug conjugate is administered at a dosage of about 1 mg / kg of subject body weight. [The present invention 1051] The method of claim 1049, wherein the antibody drug conjugate is administered at a dose of about 1.25 mg / kg of subject body weight. [The present invention 1052] The method of any of claims 1001 to 1051, wherein the antibody drug conjugate is administered by intravenous (IV) injection or infusion. [The present invention 1053] The method of any of claims 1001 to 1051, wherein the antibody drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes in a cycle of two times every three weeks. [The present invention 1054] The method of any of claims 1001 to 1051, wherein the antibody drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes on days 1 and 8 of every three week cycle. [The present invention 1055] The method of any of claims 1001 to 1051, wherein the antibody drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes for three cycles every four weeks. [The present invention 1056] The method of any of claims 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 days 1, 8, and 15 of every four week cycle. [Brief description of the drawings]

[0105] 4. Brief description of the drawings [Figure 1A-1] The nucleotide and amino acid sequences of the 191P4D12 protein are shown. [Figure 1A-2] The nucleotide and amino acid sequences of the 191P4D12 protein are shown. [Figure 1B] 1 shows the nucleotide and amino acid sequences of the heavy chain of Ha22-2(2.4)6.1. [Figure 1C] 1 shows the nucleotide and amino acid sequences of the light chain of Ha22-2(2.4)6.1. [Figure 1D] 1 shows the amino acid sequence of the heavy chain of Ha22-2(2.4)6.1. [Figure 1E] The amino acid sequence of the light chain of Ha22-2(2.4)6.1 is shown. [Diagram 2] Shows the efficacy of Ha22-2(2,4)6.1-vcMMAE in human lung cancer xenografts AG-L4 established subcutaneously in SCID mice. The results show that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of AG-L4 lung cancer xenografts implanted subcutaneously in nude mice compared to both treated and untreated controls. [Diagram 3] Shows the efficacy of Ha22-2(2,4)6.1-vcMMAE in human breast cancer xenografts BT-483 established subcutaneously in SCID mice. Results show that treatment with Ha22-2(2,4)6.1-vcMMAE significantly inhibited the growth of BT-483 breast tumor xenografts implanted subcutaneously in SCID mice compared to treated and untreated control ADCs. [Figure 4-1] Detection of 191P4D12 protein in cancer patient specimens by IHC. Figures 4A and 4B show breast cancer specimens. Figures 4C and 4D show lung cancer specimens. [Figure 4-2]Detection of 191P4D12 protein in cancer patient specimens by IHC. Figures 4E and 4F show esophageal cancer specimens. Figures 4G and 4H show head and neck cancer specimens. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0107] 5.1 Definition The techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed by those of skill in the art using conventional methodology, such as the widely utilized 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 defined herein, technical and scientific terms used in this description have the meanings that are commonly understood by those skilled in the art.For the purpose of interpreting this specification, the following explanations of terms shall apply, and where necessary, terms used in the singular form shall also include the plural form and vice versa.If the explanations of terms described herein contradict the documents incorporated herein by reference, the explanations of terms described below shall prevail.

[0109] The terms "antibody," "immunoglobulin," or "Ig" are used interchangeably herein and are used in the broadest sense, specifically including monoclonal antibodies (including agonist, antagonist, neutralizing, full-length or intact monoclonal antibodies), antibody compositions having polyepitopic or monoepitopic specificity, polyclonal or univalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, and fragments thereof, e.g., as described below. Antibodies can be human, humanized, chimeric and / or affinity matured antibodies, as well as antibodies from other species, e.g., mouse and rabbit. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides that are capable of binding 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 each amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids, and each 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 specific embodiments, specific molecular antigens can be bound by the antibodies provided herein, including polypeptides or epitopes. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above, where a functional fragment refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.In particular, the antibody provided herein includes immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, such as molecules that contain antigen-binding domains or antigen-binding sites that bind to antigen (e.g., one or more CDRs of an antibody).Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995); Huston et al., 1993, Cell Biophysics 22: 189-224; Pluckthun and Skerra, 1989, Meth. Enzymol. 178: 497-515; and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecule. The antibodies can be agonist or antagonist antibodies.

[0110] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. In contrast to polyclonal antibody preparations, which may include different antibodies 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. A 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, e.g., present on or within a cell, e.g., on or within a cancer cell.

[0112] An "intact" antibody is one that comprises an antigen-binding site and a CL and at least a heavy chain constant region, CH1, CH2 and CH3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0113] The terms "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," and similar terms refer to the portion of an antibody (e.g., CDRs) that contains amino acid residues that interact with an antigen and confer specificity and affinity to the binding agent for the antigen. As used herein, "antigen-binding fragment" includes a portion of an intact antibody, e.g., an "antibody fragment" that contains the antigen-binding region or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies and di-diabodies (e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. 90:6444-48; Lu et al., 2005, J. Biol. Chem. 280:19665-72; Hudson et al., 2005, J. Biol. Chem. 280:19665-72; al., 2003, Nat. Med. 9:129-34; WO 93 / 11161; and U.S. Pat. Nos. 5,837,242 and 6,492,123); single chain antibody molecules (see, e.g., U.S. Pat. Nos. 4,946,778; 5,260,203; 5,482,858; and 5,476,786); dual variable domain antibodies (see, e.g., U.S. Pat. No. 7,612,181); single variable domain antibodies (sdAbs) (see, e.g., Woolven et al., 1999, Immunogenetics 50:98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA. 101:12444-49); and multispecific antibodies formed from antibody fragments.

[0114] The term "bind" or "binding" refers to interactions between molecules, including, for example, forming a complex. The interactions can be non-covalent interactions, including, for example, hydrogen bonding interactions, ionic bonding interactions, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of all non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The dissociation rate (k off ) and association rate (k on ) ratio (k off / k on ) is the dissociation constant K D which is inversely proportional to the affinity. D The lower the value, the higher the affinity of the antibody. D The value of k varies for different antibody-antigen complexes. on and k off The dissociation constant K of the antibodies provided herein depends on both D can be determined using any method provided herein or any other method known to those skilled in the art. The affinity at one binding site does not necessarily reflect the true strength of the interaction between antibody and antigen. When a complex antigen that contains multiple repeated antigenic determinants, such as a multivalent antigen, contacts an antibody that contains 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 the context of an antibody or antigen-binding fragment thereof described herein, terms such as "binds to," "specifically binds to," and similar terms are also used interchangeably herein to 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 an antigen or specifically binds to an antigen may be cross-reactive with related antigens. In certain embodiments, an antibody or antigen-binding fragment that binds to an antigen or specifically binds to an antigen does not cross-react with other antigens. An antibody or antigen-binding fragment that binds to an antigen or specifically binds to an antigen can be identified, for example, by immunoassay, Octet®, Biacore®, or other techniques known to those skilled in the art. In some embodiments, an antibody or antigen-binding fragment binds to an antigen or specifically binds to an antigen if it binds to the antigen with a higher affinity than any cross-reactive antigens as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response is at least two times the background signal or noise, and may be more than 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989). In certain embodiments, the extent of binding of an antibody or antigen-binding fragment to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its particular target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. Terms such as "specific binding," "specifically binds to," or "specific for" refer to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target.Antibodies or antigen-binding fragments that bind to an antigen include those that can bind to the antigen with sufficient affinity such that the binding molecule is useful, for example, as a diagnostic agent in targeting the antigen. In certain embodiments, antibodies or antigen-binding fragments that bind to an antigen have an affinity of less than 1000 nM, less than 800 nM, less than 500 nM, less than 250 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, less than 0.4 nM. A dissociation constant (K) of less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM, or of 1000 nM, 800 nM, 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. D In certain embodiments, the antibody or antigen-binding fragment binds to an epitope of an antigen that is conserved among antigens from different species (e.g., between the human and cynomolgus monkey species).

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

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

[0118] In certain embodiments, the antibody or antigen-binding fragment may comprise a portion of a "humanized" form of a non-human (e.g., murine) antibody, which is a chimeric antibody comprising a human immunoglobulin (e.g., recipient antibody) in which native CDR residues are replaced with residues from a corresponding CDR (e.g., donor antibody) of a non-human species, such as mouse, rat, rabbit, or non-human primate, that contains the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. The heavy or light chain of the humanized antibody may comprise substantially all of at least one or more variable regions, with all or substantially all of the CDRs corresponding to the CDRs of a non-human immunoglobulin and all or substantially all of the FRs being FRs of a human immunoglobulin sequence. In certain embodiments, the humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region (Fc). 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 "fully human antibody" or a portion of a "human antibody," which terms are used interchangeably herein and refer to an antibody that includes a human variable region and, for example, a human constant region. In specific embodiments, these terms refer to an antibody that includes a variable region and a constant region of human origin. A "fully human" antibody can also encompass, in certain embodiments, an antibody that binds a polypeptide and is encoded by a nucleic acid sequence that is a naturally occurring somatic variant of a human germline immunoglobulin nucleic acid sequence. The term "fully human antibody" includes antibodies that include variable and constant regions that correspond to human germline immunoglobulin sequences as described by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or that has been made using any of the techniques for making human antibodies. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581) and yeast display libraries (Chao et al., 2006, Nature Protocols 1:755-68). Methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77(1985); Boerner et al., 1991, J. Immunol. 147(1):86-95; and van Dijk and van de Winkel, 2001, Curr. Opin. Pharmacol. 5:368-74 can also be used to prepare human monoclonal antibodies.Human antibodies can be prepared by administering antigen to transgenic animals, e.g., mice, that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled (see, e.g., Jakobovits, 1995, Curr. Opin. Biotechnol. 6(5):561-66; Bruggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4):455-58; and U.S. 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 aspects, an antibody or antigen-binding fragment can comprise a portion of a "recombinant human antibody," which phrase includes human antibodies that are prepared, expressed, created or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse or a cow) that is transgenic and / or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, LD et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

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

[0122] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. For IgG, the four-chain unit is generally 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 and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at the other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. It is believed that certain amino acid residues form the interface between the light chain variable domain and the heavy chain variable domain. The pairing of VH and VL forms a single antigen-binding site. The structure and properties of various classes of antibodies are described in, for example, Basic and Clinical Immunology 71 (Stites et al. eds., 8th ed. 1994); and Immunobiology (Janeway et al. eds., 5th ed. 1995). th ed. 2001).

[0123] The term "Fab" or "Fab region" refers to the antibody region that binds to an antigen. A conventional IgG usually 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. The VH, CH1, VL, and CL within the Fab region can be arranged in various ways to confer antigen-binding capability according to the present disclosure. For example, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on separate polypeptides, as in the Fab region of a conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in different orders, as described in more detail in the following sections.

[0124] The terms "variable region," "variable domain," "V region," or "V domain" refer to the portion of an antibody's light or heavy chain that is generally located at the amino terminus of the light or heavy chain, has a length of about 120 amino acids to about 130 amino acids in the heavy chain and about 100 amino acids to about 110 amino acids in the light chain, and is used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as "VH." The variable region of the light chain may be referred to as "VL." The term "variable" refers to the fact that certain segments of the variable region vary widely in sequence between antibodies. The V region mediates antigen binding and defines the specificity of a particular antibody for a particular antigen. However, the variability is not evenly distributed across the 110 amino acid span of the variable region. Instead, the V region consists of less variable (e.g., relatively invariant) stretches called framework regions (FR) of about 15 amino acids 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. Each of the variable regions of the heavy and light chains comprises four FRs that adopt a mainly β-sheet shape, connected by three hypervariable regions that form loops that connect and sometimes form part of the β-sheet structure. The hypervariable regions of each chain are held together in close 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, for example, 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 shows various effector functions, such as the antibody's involvement in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable region varies widely in sequence between different antibodies. In a specific embodiment, the variable region is a human variable region.

[0125] The term "variable region residue numbering according to Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refers to the numbering system used for the heavy or light chain variable regions of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to truncations of or insertions into the FRs or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b and 82c, etc., according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat numbering sequence at the regions of homology. The Kabat numbering system is commonly used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al. supra). The "EU numbering system" or "EU index" is commonly used when referring to residues in 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 have been described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0126] The term "heavy chain", when used in reference to an antibody, 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 five well-known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG, and IgM, respectively, including the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4.

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

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

[0129] CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (see, for example, Kabat et al. supra). Instead, Chothia refers to the position of structural loops (see, for example, Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). The end of the Chothia CDR-H1 loop when numbered using Kabat's numbering rules varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, for example, 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 dedicated to immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. Herein, CDRs are referred to both in terms of amino acid sequence and position within the light or heavy chain. The "locations" of the CDRs within the structure of immunoglobulin variable domains are conserved across species and reside in structures called loops, so that the CDR and framework residues are readily identified by using a numbering system that aligns the variable domain sequences according to structural features.This information can be used to graft and replace CDR residues from one species of immunoglobulin into an acceptor framework, typically from a human antibody. A further numbering system (AHon) has been developed by Honegger and Pluckthun, 2001, J. Mol. Biol. 309:657-70. For example, the correspondence between numbering systems, including the Kabat numbering and the IMGT specific numbering system, is well known to those skilled in the art (see, for example, 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) TIFF0007657774000005.tif77137

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

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

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

[0134] The term "framework" or "FR" refers to 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" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is often defined as extending from the amino acid residue at position Cys226 or the amino acid residue at position 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 production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody. 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 residue has not been removed, and an antibody population that includes a mixture of antibodies with and without 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), and the like. Such effector functions generally require that the Fc region be combined with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays known to those of skill in the art. A "variant Fc region" comprises 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 compared to the native sequence Fc region or the Fc region of a parent polypeptide, e.g., 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 parent polypeptide. The variant Fc region herein may have at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, for example at least about 95% homology thereto.

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

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

[0138] As used herein, the term "pharmaceutical acceptable" means approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, and more particularly in humans.

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

[0140] In one embodiment, each component is "pharmaceutical 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 reaction, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutical acceptable excipient is non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. In some embodiments, the pharma- ceutically acceptable excipient is an aqueous pH buffered solution.

[0141] The abbreviation "MMAE" refers to monomethylauristatin E.

[0142] Unless otherwise specified, the term "alkyl" refers to a saturated straight or branched chain hydrocarbon containing from about 1 to about 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), preferably from about 1 to about 8 carbon atoms. 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.Alkyl groups, whether alone or as part of another group, include: -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', -S3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -N and optionally substituted with one or more groups, preferably 1 to 3 groups (and optional further substituents selected from halogen), including but not limited to -H(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-(C The -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl groups are defined as follows: -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 It may be further substituted with one or more groups including, but not limited to, (O)N(R'')2, -NHC(O)R'', -SR'', -SOR'', -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 and branched carbon chains containing from about 2 to about 20 carbon atoms (and all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), with from about 2 to about 8 carbon atoms being preferred. Alkenyl chains have at least one double bond in the chain, and alkynyl chains have 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, -isobutylenyl, -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.Alkenyl and alkynyl groups, either alone or as part of another group, include: -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', -S3R', -S(O)2R', -S(O)R', -OH, =O, -N3, and -R' is optionally substituted with one or more groups, preferably one to three groups (and optional further substituents selected from halogen), including but not limited to -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), The -O-(C2-C8 alkynyl), -aryl, -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl groups are defined as follows: -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'', - It may be further substituted with one or more substituents including, but not limited to, C(O)N(R'')2, -NHC(O)R'', -SR'', -SOR'', -S(O)R'', -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 (and all combinations and subcombinations 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 the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylenes include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decalene, 1,4-cyclohexylene, and the like.Alkylene groups, whether alone or as part of another group, include -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', -S3R', -S(O)2R', -S(O)R', -OH, =O, -N3, -NH2, -N and optionally substituted with one or more groups, preferably 1 to 3 groups (and optional further substituents selected from halogen), including but not limited to -H(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-(C The -C1-C8 alkyl, -C2-C8 alkenyl, and -C2-C8 alkynyl groups are defined as -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( and -N(R'')2, -NHC(O)R'', -SR'', -SOR'', -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.

[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 noted, 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 (-CHC≡C-), and 4-pentynyl (-CHCHCHC≡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 the removal of one hydrogen atom from a carbon atom of a parent aromatic ring system. Some aryl groups are represented as "Ar" in the 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] Aryl groups, either alone or as part of another group, include -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', -SOR', -S(O) and -R', -S(O)R', -OH, -NO2, -N3, -NH2, -NH(R'), -N(R')2, and -CN, each R' being independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, each of which is independently substituted with one or more, preferably 1 to 5, or even 1 to 2 groups, including but not limited to ... The C8 alkynyl, O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), and -aryl groups are defined as: -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'', -SOR'', -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.

[0149] Unless otherwise specified, the term "arylene" refers to an optionally substituted aryl group that is divalent (i.e., derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent aromatic ring system) and can be in the ortho, meta, or para configuration as shown in the following structure with phenyl as an exemplary aryl group. TIFF0007657774000006.tif22128 Typical "-(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-naphthylethene-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and the like.

[0150] Unless otherwise specified, the term "heterocycle" refers to a monocyclic, bicyclic, or polycyclic ring system having 3 to 14 ring atoms (also referred to as ring members), in which 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 subcombinations of ranges and specific numbers of carbon atoms and heteroatoms therein). A heterocycle can have 1 to 4 ring heteroatoms independently selected from N, O, P, or S. One or more N, C, or S atoms in a heterocycle can be oxidized. A monocyclic heterocycle 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 a bicyclic heterocycle 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 a heteroatom can be aromatic or non-aromatic.Unless otherwise specified, a heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure.Heterocycles are described in Paquette, "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950-present), especially volumes 13, 14, 16, 19, and 28; and J.Am.Chem.Soc.82:5566 (1960).Examples of "heterocycle" groups 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-dithiazinyl, thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthyl Thenyl, 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 Preferred "heterocyclic" 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, and tetrazolyl.Heterocyclic groups, either alone or as part of another group, include -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( and -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl ... (C2-C8 alkynyl), -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, and -aryl groups are -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 It may be further substituted with one or more substituents including, but not limited to, (O)N(R'')2, -NHC(O)R'', -SR'', -SOR'', -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: 2-, 3-, 4-, 5-, or 6-position of pyridine; 3-, 4-, 5-, or 6-position of pyridazine; 2-, 4-, 5-, or 6-position of pyrimidine; 2-, 3-, 5-, or 6-position of pyrazine; 2-, 3-, 4-, or 5-position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole; 2-, 4-, or 5-position of oxazole, imidazole, or thiazole; 3-, 4-, or 5-position of isoxazole, pyrazole, or isothiazole; 2- or 3-position of aziridine; 2-, 3-, or 4-position of azetidine; 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of quinoline; or 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of isoquinoline. Even more typically, the carbon-linked 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, nitrogen-linked heterocycles can be attached 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; at the 2-position of isoindole or isoindoline; at the 4-position of morpholine; and at the 9-position of carbazole or β-carboline. Even more typically, nitrogen-linked heterocycles include 1-aziridyl, 1-azetyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0153] Unless otherwise specified, the term "carbocycle" refers to a saturated or unsaturated non-aromatic monocyclic, bicyclic, or polycyclic ring system having 3 to 14 ring atoms (as well as all combinations and subcombinations of ranges and specific numbers of carbon atoms therein), all of which are carbon atoms. Monocyclic carbocycles preferably have 3 to 6 ring atoms, and even more preferably 5 or 6 ring atoms. Bicyclic carbocycles preferably have 7 to 12 ring atoms, for example, 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 "carbocycle" includes, for example, a monocyclic carbocycle fused to an aryl ring (e.g., a monocyclic carbocycle fused to a benzene ring). Carbocycles preferably have 3 to 8 carbon ring atoms.Carbocyclic groups, either alone or as part of another group, include, 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', -SOR', -S(O)2R' and -C(O)R', -OH, =O, -N3, -NH2, -NH(R'), -N(R')2, and -CN, preferably one or two groups (and optional further substituents selected from halogen), where each R' is independently selected from -H, -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl, and wherein said -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl are each independently selected from -C1-C8 alkyl, -C2-C8 alkenyl, -C2-C8 alkynyl, or -aryl. The alkenyl, -C2-C8 alkynyl, -O-(C1-C8 alkyl), -O-(C2-C8 alkenyl), -O-(C2-C8 alkynyl), and -aryl groups are -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) It may be further substituted with one or more substituents including, but not limited to, NHR'', -C(O)N(R'')2, -NHC(O)R'', -SR'', -SOR'', -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-cyclopent-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 an optionally substituted carbocyclic group, as defined above, that is divalent (i.e., derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent carbocyclic system).

[0156] Unless the context dictates otherwise, a hyphen (-) designates the point of attachment to the pendant molecule. Thus, the term "-(C1-C8 alkylene)aryl" or "-C1-C8 alkylene(aryl)" refers to a C1-C8 alkylene radical, as defined herein, where the alkylene radical is attached to a pendant molecule at any of the alkylene radical's carbon atoms, and one of the hydrogen atoms attached to the alkylene radical's carbon atom is replaced with an aryl radical, as defined herein.

[0157] When a particular group is "substituted," the group may have one or more substituents independently selected from a list of substituents, preferably 1 to 5 substituents, more preferably 1 to 3 substituents, and most preferably 1 to 2 substituents. However, the group may have any number of substituents, generally selected from halogen. Groups that are substituted are also designated as such. It is intended that the definition of any substituent or variable at a particular location in a molecule be independent of its definition elsewhere in that molecule. It is understood that the substituents and substitution patterns on the compounds of the invention may 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 methods described herein.

[0158] A protecting group, as used herein, refers to a group that selectively blocks, temporarily or permanently, one reactive site in a multifunctional compound. Suitable hydroxy protecting groups for use in the present invention are pharma- ceutically 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 described in Protective Groups in Organic Synthesis by TW Greene and PG M Huts (John Wiley & sons, 31, 2001), incorporated herein by reference in its entirety for all purposes. rdsee, for example, the ether (e.g., alkyl and silyl ethers, including dialkylsilyl ethers, trialkylsilyl ethers, dialkylalkoxysilyl ethers), ester, carbonate, carbamate, sulfonate, 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, guaiacol methyl ether, t-butoxymethyl ether, 4-pentenyloxymethyl 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;Substituted 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-butyl ether, Preferred protecting groups include, but are not limited to, alkyl dimethylsilyl ether, diphenylmethylsilyl ether, benzoyl formate 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, benzylsulfonate, tosylate, methylene acetal, ethylidene acetal, and t-butyl methylidene ketal. 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 (heterocycle), -C2~C 20 Alkenylene (heterocycle), or -C2~C 20 Alkynylene (heterocycle), wherein said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, and heterocycle radicals, either alone or as part of another group, may be substituted.

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

[0160] The term "compound" refers to and includes the chemical compound per se, as well as the following, whether or not expressly stated, and unless it is clear from the context that the following are to be excluded: amorphous and crystalline forms of the compound, including polymorphs, which may be part of a mixture or in isolation; the 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 isomers and non-chiral isomers, which optical isomers include isolated optical isomers as well as mixtures of optical isomers, including racemic and non-racemic mixtures; where the isomers may be in isolated form or in mixtures with one or more other isomers. isotopes of the compounds, including compounds containing radioisotopes, including deuterium-containing and tritium-containing compounds, and including therapeutically and diagnostically effective radioisotopes; multimeric forms of the compounds, including dimeric, trimeric, etc. forms; salts of the compounds, preferably pharma- ceutically acceptable salts, including acid addition salts and base addition salts, including salts with organic and inorganic counterions, and including zwitterionic forms, where the compound is associated with two or more counterions, the two or more counterions may be the same or different; and solvates of the compounds, including hemisorbates, monosorbates, disor- vates, etc., including inorganic solvates, including organic solvates and hydrates, where 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 references to one or more of the above forms, e.g., salts and / or solvates; however, this reference is merely for emphasis and should not be construed to exclude other of the above forms identified above.

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

[0162] Table 2: Amino acid abbreviations TIFF0007657774000007.tif112157

[0163] Table 3: Amino acid substitution or similarity matrix Adapted from GCG software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely the substitution is found in related native proteins. TIFF0007657774000008.tif135140

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

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

[0166] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be achieved using mathematical algorithms.A preferred non-limiting example of the mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc.Natl.Acad.Sci.USA 87:2264 2268, modified as in Karlin and Altschul, 1993, Proc.Natl.Acad.Sci.USA 90:5873 5877.Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J.Mol.Biol.215:403.BLAST nucleotide searches can be performed using, for example, the NBLAST nucleotide program parameters set to score=100, word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using, for example, XBLAST program parameters set to score 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively, PSI BLAST can be used to perform an iterative search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11 17.Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

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

[0168] The term "cytotoxic agent" refers to a substance that inhibits or prevents the expression activity, function, and / or causes destruction of a cell. The term is intended to include toxins, such as radioisotopes, chemotherapeutic agents, and small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants. Examples of cytotoxic agents include auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), aureomycin, maytansinoids, ricin, ricin A chain, combrestatins, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α-sarcin, gelonin, mitogenin, restrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitors, and glucocorticoids and other chemotherapeutic agents, as well as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 Or Bi213 , P 32 and Lu 177 Examples of radioisotopes of Lu include, but are not limited to, Lu radioisotopes, including Lu 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 29, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,

[0169] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a binding molecule (e.g., an antibody) or pharmaceutical composition provided herein sufficient to effect a desired result.

[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., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, diagnosed with a condition or disorder. In another embodiment, a subject is a mammal, e.g., 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 present outside the body to a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art.

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

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

[0174] The term "cancer" or "cancer cell" is used herein to refer to tissue or cells found in a neoplasm that have characteristics that distinguish it from normal tissue or tissue cells. Such characteristics include, but are not limited to, the degree of anaplasia, irregularity in shape, unclear cell outlines, nuclear size, changes in nuclear or cytoplasmic structure, other phenotypic changes, the presence of cellular proteins indicative of a cancerous or precancerous condition, an increased number of mitoses, and the ability to metastasize. Words related to "cancer" include carcinoma, sarcoma, tumor, epithelioma, leukemia, lymphoma, polyp, and scirrhous carcinoma, transformation, 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 of a given value or range.

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

[0177] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided. Also, whenever an embodiment is described herein with the phrase "consisting essentially of," it is understood that other similar embodiments described in terms of "consisting of" are also provided.

[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 in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0179] The term "variant" refers to a molecule that exhibits a variation from a described type or reference, such as a protein that has 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). Analogs are 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 FIG. 1), as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated / produced 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 parts or fragments of different 191P4D12 proteins, and fusion proteins of 191P4D12 proteins with heterologous polypeptides. Such 191P4D12 proteins are collectively referred to as 191P4D12-related proteins, proteins of the present invention, or 191P4D12. The term "191P4D12-related protein" refers to a 191P4D12-related protein that is ... Also refers to a polypeptide fragment or 191P4D12 protein sequence of 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 Methods of Treating Cancer Provided herein are methods of treating various cancers, including breast cancer (e.g., HER+ / HER2- breast cancer and triple-negative breast cancer that is ER-, PR-, and HER2- negative (ER- / PR- / HER2-)), 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 antibody drug conjugates (ADCs) that bind to 191P4D12. In some embodiments, the ADC is enfortumab vedotin (anti-191P4D12-ADC, also known as 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 monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23, 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 monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23, 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 monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23, 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 monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23, 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 monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22 and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23, 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 or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23, and the subject has gastric cancer or esophageal cancer.

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

[0188] to

[0194] ), therapeutic agents that may be used are described in Section 5.3, selection of patients for treatment is described herein and exemplified in Sections 5.2 and 6, dosing regimens and pharmaceutical compositions for administering therapeutic agents are described in Sections 5.4 and 6 below, biomarkers that can be used to identify therapeutic agents, select patients, determine outcomes of these methods, and / or benchmark in any manner for these methods are described herein and exemplified in Sections 5.2 and 6, and the therapeutic outcomes of the methods provided herein may be improvements in the biomarkers described herein, such as the biomarkers described and exemplified in Sections 5.2 and 6. Thus, one of 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 embodiments, the methods provided herein are used for treating breast cancer in a subject. In some embodiments, the breast cancer is hormone receptor positive and human epidermal growth factor receptor 2 negative (HR+ / HER2-) breast cancer. In some embodiments, the breast cancer is estrogen receptor (ER) positive and / or progesterone receptor (PR) positive and HER2 negative. In some embodiments, the breast cancer is ER positive, PR positive and HER2 negative. In some embodiments, the breast cancer is ER positive and HER2 negative. In some embodiments, the breast cancer is PR positive and HER2 negative. In some embodiments, the breast cancer is histologically, cytologically, or both histologically and cytologically confirmed, including, for example, 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. In some embodiments, histological, cytological, or both histological and cytological confirmation is performed according to American Society of Clinical Oncology / College of American Pathologists (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, 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 / College of American Pathologists (ASCO / CAP) guidelines based on the most recently analyzed tissue.

[0191] In some embodiments, subjects with breast cancer and treated with the methods provided herein have received one or more endocrine therapies and cyclin-dependent kinase (CDK) 4 / 6 inhibitors in metastatic or locally advanced settings. In some embodiments, subjects with breast cancer and treated with the methods provided herein have received prior treatment with taxanes or anthracyclines in any setting. In some embodiments, subjects with breast cancer and treated with the methods provided herein have deleterious germline mutations in breast cancer susceptibility genes (BRCA) 1 or 2 and must have been treated with a poly ADP-ribose polymerase (PARP) inhibitor.

[0192] In some specific embodiments, subjects treated with the methods provided herein must have histologically or cytologically confirmed HR+ / HER2- breast cancer, defined as ER positive and / or progesterone receptor (PR) positive and HER2 negative according to American Society of Clinical Oncology / College of American Pathologists (ASCO / CAP) guidelines based on most recently analyzed tissue, have locally advanced or metastatic disease, have received one or more endocrine therapies and a cyclin-dependent kinase (CDK) 4 / 6 inhibitor in the metastatic or locally advanced setting, have received prior treatment with a taxane or anthracycline in any setting, and / or have a deleterious germline mutation in the breast cancer susceptibility genes (BRCA) 1 or 2 and 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 ASCO / CAP guidelines for TNBC histology (ER negative / PR negative / HER2 negative) based on the most recently analyzed tissue. In some embodiments, the TNBC is locally advanced or metastatic. In some embodiments, the subject with TNBC and treated with the methods provided herein has undergone two or more systemic therapies. In some embodiments, the subject with TNBC and treated with the methods provided herein has undergone two or more systemic therapies, including, in any situation, a taxane. In some embodiments, the subject with TNBC and treated with the methods provided herein has a deleterious germline mutation in BRCA1, BRCA2, or both BRCA1 and BRCA2. In some embodiments, the subject with TNBC and treated with the methods provided herein has been treated with a PARP inhibitor. In some embodiments, the subjects treated by the methods provided herein for TNBC have any permutation or combination of the characteristics described in this paragraph.

[0194] In some specific embodiments, subjects treated with the methods provided herein have histologically or cytologically confirmed TNBC, defined as definite TNBC histology (ER negative / PR negative / HER2 negative) according to ASCO / CAP guidelines based on most recently analyzed tissue, have locally advanced or metastatic disease, have received two or more systemic therapies, including a taxane in any setting, have a deleterious germline mutation in BRCA1 or BRCA2 or both, and / or have 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 with squamous NSCLC and treated with the methods provided herein has progressed or relapsed after platinum-based therapy, for example, if the relapse occurs within 12 months after completion of platinum-based therapy, including platinum therapy administered as adjuvant therapy. In some embodiments, the subject with squamous NSCLC and treated with the methods provided herein has undergone 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 with the methods provided herein has histologically or cytologically confirmed squamous NSCLC, has locally advanced or metastatic disease, has progressed or relapsed after platinum-based therapy, counted as a regimen if relapse occurred within 12 months after completion of platinum-based therapy, including platinum therapy administered as adjuvant therapy, 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.

[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 by local laboratory standards. In some embodiments, the non-squamous NSCLC is locally advanced or metastatic. In some embodiments, the subject with squamous NSCLC and treated with the methods provided herein has progressed or relapsed after platinum-based therapy, for example, when relapse occurs within 12 months after completion of platinum-based therapy in metastatic or locally advanced setting, including platinum therapy administered as adjuvant therapy. In some embodiments, a subject having squamous NSCLC and treated with 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.

[0198] In some specific embodiments, the subject treated with the methods provided herein has histologically or cytologically confirmed non-squamous NSCLC that is EGFR wild-type and ALK wild-type by local laboratory standards, has locally advanced or metastatic disease, has progressed or relapsed after platinum-based therapy if recurrence occurred within 12 months following completion of platinum-based therapy in the metastatic or locally advanced setting, including, for example, platinum therapy administered as adjuvant therapy, and 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.

[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 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 with head and neck cancer and treated with the methods provided herein has progressed or relapsed after a platinum-containing regimen in a metastatic or locally advanced setting, which does not include a platinum-containing regimen administered as part of a multimodality treatment in a curative setting, unless the subject relapses or progresses within 6 months after completion. In some embodiments, the subject with head and neck cancer and treated with 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.

[0200] In some specific aspects, the subject treated with the methods provided herein has histologically or cytologically confirmed head and neck cancer, has locally advanced or metastatic disease, has progressed or relapsed after a platinum-containing regimen in the metastatic or locally advanced setting, which does not include a platinum regimen administered as part of multimodality therapy in the curative setting, unless the subject relapses or progresses within six months of completion, and 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.

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

[0202] In some specific embodiments, the subject treated with the methods provided herein has histologically or cytologically confirmed gastric or esophageal cancer, has locally advanced or metastatic disease, has progressed or relapsed after a chemotherapy regimen comprising fluoropyrimidines and platinums for metastatic or locally advanced disease, which does not include a neoadjuvant or adjuvant regimen unless the subject relapses or progresses within 6 months after completion, has HER2-positive cancer, and is receiving a HER2-directed therapy. In another specific embodiment, the subject treated with the methods provided herein has histologically or cytologically confirmed gastric or esophageal cancer, has locally advanced or metastatic disease, has progressed or relapsed after a chemotherapy regimen comprising fluoropyrimidines and platinums for metastatic or locally advanced disease, which does not include a neoadjuvant or adjuvant regimen unless the subject relapses or progresses within 6 months after completion.

[0203] In certain embodiments, the methods provided herein are used to treat subjects with cancers that express 191P4D12 RNA, express 191P4D12 protein, or express both 191P4D12 RNA and 191P4D12 protein.In certain embodiments, the methods provided herein are used to treat subjects with cancers that express 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 embodiments, the 191P4D12 RNA expression in cancer is determined by polynucleotide hybridization, sequencing (to evaluate the relative abundance of sequences), and / or PCR (including RT-PCR). In some embodiments, 191P4D12 protein expression in cancer is determined by IHC, fluorescence activated cell sorting (FACS) analysis, and / or Western blotting. In some embodiments, 191P4D12 protein expression in cancer is determined by two methods: IHC.

[0204] In certain embodiments, the methods provided herein are used to treat a subject with cancer, the cancer expressing 191P4D12 RNA, expressing 191P4D12 protein, or expressing both 191P4D12 RNA and 191P4D12 protein, and the cancer being sensitive to cytotoxic agents that block microtubule polymerization (such as Vinca and MMAE). In certain embodiments, the methods provided herein are used to treat a subject with cancer expressing both 191P4D12 RNA and 191P4D12 protein, and being sensitive to cytotoxic agents that block microtubule polymerization (such as Vinca and MMAE), 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, subjects that can be treated with the methods provided herein are subjects with solid tumors, including, for example, subjects with hormone receptor positive and human epidermal growth factor receptor 2 negative (HR+ / HER2-) breast cancer, subjects with ER negative, PR negative and HER2 negative (ER- / PR- / HER2-) breast cancer, subjects with NSCLC, subjects with non-squamous NSCLC, subjects with head cancer, subjects with neck cancer, subjects with head and neck cancer, subjects with gastric cancer, subjects with esophageal cancer, and / or subjects with gastric or esophageal cancer.

[0206] In certain embodiments, subjects that can be treated with the methods provided herein further include those with locally advanced solid tumors, metastatic solid tumors (including metastatic malignant tumors), and locally advanced and metastatic solid tumors. In some embodiments, the solid tumors that can be treated with 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 aspects, the solid tumor that can be treated with the methods provided herein is 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 malignancies), and locally advanced and metastatic solid tumors are confirmed histologically, cytologically, or both histologically and cytologically.

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

[0209] In certain embodiments, the subject that can be treated by the methods provided herein has previously received at least 2, 3, 4, 5, or 6 types of systemic therapy. Such systemic therapy can be any therapy that uses a substance that travels through the bloodstream and reaches and affects cells throughout the body. Such systemic therapy can be one described in the previous paragraph (paragraph

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

[0210] In certain embodiments, subjects that can be treated with the methods provided herein have progressed or relapsed 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 other treatments, including, but not limited to, any or any combination of treatments described in the paragraph two paragraphs prior to this paragraph (paragraph

[0215] ). In some specific embodiments, the subject has progressed or relapsed within 6 months after platinum-based therapy or chemotherapy including fluoropyrimidine. In other specific embodiments, the subject has progressed or relapsed within 6 months after platinum-based therapy. In further embodiments, the subject has progressed or relapsed within 12 months after platinum-based therapy.

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

[0212] In some embodiments, subjects who can be treated with the methods provided herein have any combination or permutation of having undergone one or more other cancer treatments such as those described in the previous paragraph (paragraph

[0218] ) and having progressed or relapsed after one or more other cancer treatments such as those described in the paragraph four paragraphs prior to this one (paragraph

[0215] ).

[0213] In some embodiments, subjects that can be treated with the methods provided herein have 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 deleterious germline mutations in breast cancer susceptibility genes (BRCA)1, BRCA2, or both BRCA1 and BRCA2. In one embodiment, the subject has ER-negative, PR-negative and HER2-negative (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 or 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, deleterious germline mutations in breast cancer susceptibility genes (BRCA)1, BRCA2, or both BRCA1 and BRCA2 are 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 based on the most recently analyzed tissue as described in the American Society of Clinical Oncology / College of American Pathologists (ASCO / CAP) guidelines, which are incorporated herein by reference in their entirety.

[0216] In some embodiments, the phenotypic or genotypic characteristics are determined by sequencing, including next-generation sequencing (e.g., Illumina, Inc.'s NGS), 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., Illumina, Inc.'s NGS), 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., Illumina, Inc.'s NGS), 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 from Illumina, Inc.), DNA hybridization, and / or RNA hybridization. In one embodiment, deleterious germline mutations in breast cancer susceptibility genes (BRCA) 1, BRCA2, or both BRCA1 and BRCA2 are determined by sequencing including next generation sequencing (e.g., NGS from 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 from 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 (eg, NGS from Illumina, Inc.), DNA hybridization, and / or RNA hybridization.In one embodiment, wild-type anaplastic lymphoma kinase (ALK) is determined by sequencing, including next-generation sequencing (e.g., NGS from Illumina, Inc.), DNA hybridization, and / or RNA hybridization. In one embodiment, 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 from Illumina, Inc.), DNA hybridization, and / or RNA hybridization.

[0217] In some embodiments, the one or more other therapies 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 embodiments, the PD-1 inhibitor is pembrolizumab or nivolumab. In other embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab. Other examples of PD-l / PD-L1 inhibitors include, but are not limited to, those described in U.S. Pat. Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; and 8,217,149, as well as PCT Publication Nos. WO 2003042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO 2011159877, WO 2011082400, and WO 2011161699, all of which are incorporated herein 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 the fusion protein AMP-224. In another embodiment, the PD-1 antibody is BGB-A317. BGB-A317 is a monoclonal antibody that has been specifically engineered for its ability to bind to Fcγ receptor I and has a unique binding signature with high affinity and excellent target specificity for PD-1.

[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 subjects that can be treated with the methods provided herein are mammals. In some embodiments, the subjects that can be treated with the methods provided herein are humans.

[0221] 5.3 Anti-191P4D12 Antibody Drug Conjugates In general, the methods provided herein utilize anti-191P4D12 ADCs described herein and / or in U.S. Patent No. 8,637,642, the entire contents of which are incorporated herein by reference. The anti-191P4D12 antibody drug conjugates provided herein comprise an antibody or antigen-binding fragment thereof that binds to 191P4D12, conjugated to one or more units of a cytotoxic agent (or drug unit). The cytotoxic agent (or drug unit) 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 a pharma- ceutically acceptable salt or solvate thereof: L-(LU-D) p (I) During the ceremony, L is an antibody unit, e.g., an anti-191P4D12 antibody or antigen-binding fragment thereof, provided in Section 5.3.1 below; (LU-D) is the Linker Unit-Drug Unit moiety; Where: LU- is a linker unit, D is a drug entity having cytostatic or cytotoxic activity against a target cell; p is an integer from 1 to 20.

[0223] 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 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 a pharma- ceutically acceptable salt or solvate thereof: L-(A a -W w -Y y -D) p (II) During the ceremony, L is an antibody unit, e.g., an anti-191P4D12 antibody or an antigen-binding fragment thereof, provided in Section 5.3.1 below; and -A a -W w -Y y - is a linker unit (LU), where -A- is an extender unit, a is 0 or 1, each -W- is independently an amino acid unit; w is an integer ranging from 0 to 12; -Y- is a self-immolative spacer unit; y is 0, 1 or 2; D is a drug entity having cytostatic or cytotoxic activity against a target cell; 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] For compositions comprising multiple antibodies or antigen-binding fragments thereof, drug loading is represented by p, the average number of drug molecules per antibody unit. Drug loading can range from 1 to 20 drugs (D) per antibody. The average number of drugs per antibody in a preparation of a conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of antibody drug conjugates with respect to p can also be determined. In some cases, separation, purification, and characterization of homogeneous antibody drug conjugates with a particular value of p from antibody drug conjugates with other drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis. In an exemplary embodiment, p is 2 to 8.

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

[0228] Antibodies that specifically bind to a 191P4D12 protein comprising the amino acid sequence of SEQ ID NO:2 include antibodies that can bind to other 191P4D12-related proteins. For example, antibodies that bind to a 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 embodiments, 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 (cDNA sequence of SEQ ID NO:3) and / or a light chain comprising the amino acid sequence of SEQ ID NO:6 (cDNA sequence of SEQ ID NO:5), as shown in Figures 1B and 1C.

[0231] In some embodiments, an anti-191P4D12 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a CDR comprising the amino acid sequence of the complementarity determining region (CDR) of the heavy chain variable region set forth in SEQ ID NO:22 (which is the amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 136 (serine) of SEQ ID NO:7), and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23 (which is the amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 130 (arginine) of SEQ ID NO:8). SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:7 and SEQ ID NO:8 are shown in Figures 1D and 1E and are listed below. TIFF0007657774000009.tif172166

[0232] CDR sequences can be determined according to well-known numbering systems. As mentioned above, CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (see, for example, Kabat et al. supra). Instead, Chothia refers to the position of structural loops (see, for example, Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). The end of Chothia CDR-H1 loop when numbered using Kabat numbering rules varies between H32 and H34 depending on the length of the loop (this is because Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (see, for example, 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 dedicated to immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. Herein, CDRs are referred to both in terms of amino acid sequence and position within the light or heavy chain.Since the "location" of the CDRs in the structure of immunoglobulin variable domains is conserved among species and resides in structures called loops, the CDR and framework residues are easily identified by using a numbering system that aligns the variable domain sequences according to structural features. This information can be used when grafting and substituting CDR residues from one species of immunoglobulin into an acceptor framework, typically from a human antibody. A further numbering system (AHon) has been developed by Honegger and Pluckthun, 2001, J. Mol. Biol. 309:657-70. For example, the correspondence between numbering systems, including the Kabat numbering and the IMGT specific numbering system, is well known to those skilled in the art (see, for example, Kabat supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al. supra). Residues from each of these hypervariable regions or CDRs are 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 a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22 according to the Kabat numbering, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23 according to the Kabat numbering.

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

[0235] In another embodiment, the anti-191P4D12 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 as set forth in SEQ ID NO:22 according to the Chothia numbering, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region as set forth in SEQ ID NO:23 according to the Chothia numbering.

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

[0237] In yet another embodiment, the anti-191P4D12 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 as set forth in SEQ ID NO: 22 according to the IMGT numbering, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region as set forth in SEQ ID NO: 23 according to the IMGT numbering.

[0238] As mentioned above, CDR sequences according to different numbering systems can be easily determined using online tools such as those provided by Antigen Receptor Numbering and Receptor Classification (ANARCI). For example, the heavy chain CDR sequences in SEQ ID NO: 22 and the light chain CDR sequences in SEQ ID NO: 23 according to Kabat numbering as determined by ANARCI are listed in Table 4 below.

[0239] (Table 4) TIFF0007657774000010.tif31166

[0240] As another example, the heavy chain CDR sequences in SEQ ID NO:22 and the light chain CDR sequences in SEQ ID NO:23 according to the IMGT numbering as determined by ANARCI are listed in Table 5 below.

[0241] (Table 5) TIFF0007657774000011.tif31166

[0242] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR H1 comprising the amino acid sequence of SEQ ID NO:9, a CDR H2 comprising the amino acid sequence of SEQ ID NO:10, a CDR H3 comprising the amino acid sequence of SEQ ID NO:11, a CDR L1 comprising the amino acid sequence of SEQ ID NO:NO:12, a CDR L2 comprising the amino acid sequence of SEQ ID NO:NO:13, and a 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 a CDR H1 comprising the amino acid sequence of SEQ ID NO:16, a CDR H2 comprising the amino acid sequence of SEQ ID NO:17, a CDR H3 comprising the amino acid sequence of SEQ ID NO:18, a CDR L1 comprising the amino acid sequence of SEQ ID NO:NO:19, a CDR L2 comprising the amino acid sequence of SEQ ID NO:NO:20, and a 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 the amino acid sequence ranging from amino acid 20 (glutamic acid) to amino acid 466 (lysine) of SEQ ID NO:7 and a light chain comprising the amino acid sequence ranging from amino acid 23 (aspartic acid) to amino acid 236 (cysteine) of SEQ ID NO:8.

[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, reduced immunogenicity, or solubility. Thus, in addition to the antibodies described herein, it is contemplated that antibody variants may be prepared. 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 may alter post-translational processes of the antibody, for example, may alter the number or position of glycosylation sites, or may alter 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 antibodies that are chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cellular ligands or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, and the like. Additionally, the antibody can include one or more non-classical amino acids.

[0248] Mutations can be substitutions, deletions or insertions of one or more codons encoding the single domain antibody or polypeptide resulting in a change in the amino acid sequence compared to the original antibody or polypeptide. Amino acid substitutions can be, for example, conservative amino acid substitutions resulting from replacing one amino acid with another amino acid containing similar structural and / or chemical properties, such as replacement of a leucine with a serine. Standard techniques known to those of skill in the art can be used to introduce mutations into the nucleotide sequences encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Insertions or deletions can range from about 1 to 5 amino acids. In certain embodiments, substitutions, deletions or insertions include fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the original molecule. In specific embodiments, the substitutions are conservative amino acid substitutions made 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 parent antibody.

[0249] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing multiple residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue.

[0250] Antibodies generated by conservative amino acid substitution are included in the present disclosure. In conservative amino acid substitution, an amino acid residue is replaced with an amino acid residue that contains a side chain with a similar charge. As mentioned above, families of amino acid residues that contain side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), 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. Following mutagenesis, the encoded protein can be expressed, the activity of the protein can be determined, and conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) can be made to maintain or not significantly change the properties.

[0251] Amino acids may 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, naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0252] For example, any cysteine ​​residue not involved in maintaining the proper conformation of the antibody may be substituted with another amino acid, such as, for example, alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

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

[0254] Covalent modification of antibodies is included within the scope of this disclosure. Covalent modification includes reacting targeted amino acid residues of antibodies with organic derivatizing agents that can react with selected side chains or N- or C-terminal residues of antibodies. Other modifications include deamidation of glutaminyl and asparaginyl residues to corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of α-amino groups of lysine, arginine and histidine side chains (see, for example, Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0255] Other types of covalent modifications of antibodies that fall within the scope of the present disclosure include altering the native glycosylation pattern of the antibody or polypeptide (see, e.g., Beck et al., 2008, Curr. Pharm. Biotechnol. 9:482-501; and Walsh, 2010, Drug Discov. Today 15:773-80) and linking the antibody to one of a variety of nonproteinaceous polymers, such as polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, e.g., by methods described in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337.

[0256] In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 70% homology or identity to the heavy chain shown in SEQ ID NO:NO:7. In some embodiments, the antibodies or antigen-binding fragments provided herein comprise a heavy chain having greater than 75% homology or identity to the heavy chain 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to the light chain shown in SEQ ID NO:8.

[0258] In some embodiments, the anti-191P4D12 antibodies provided herein comprise the heavy and light chain CDR regions of the antibody designated Ha22-2(2,4)6.1 produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, or heavy and light chain CDR regions comprising amino acid sequences that are 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 the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0259] In some aspects, the antibodies or antigen-binding fragments thereof provided herein comprise a humanized heavy chain variable region and a humanized light chain variable region, wherein: (a) the heavy chain variable region comprises a CDR that comprises the amino acid sequence of the heavy chain variable region CDR shown in the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267; (b) the light chain variable region comprises a CDR comprising the amino acid sequence of the light chain variable region CDR shown in the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0260] In some embodiments, the anti-191P4D12 antibody provided herein comprises the heavy and light chain variable regions of the antibody designated Ha22-2(2,4)6.1 (see FIG. 3) produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, or the heavy and light chain variable regions comprising amino acid sequences that are 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 antibody provided herein. The constant region of the antibody of the present invention can be selected from any subclass. In one embodiment, a human IgG1 constant region can be used as the heavy chain constant region, and a human Igκ constant region can be used as the light chain constant region.

[0261] In some embodiments, the anti-191P4D12 antibodies provided herein comprise the heavy and light chains of the antibody designated Ha22-2(2,4)6.1 (see FIG. 3) produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267, or heavy and light chains comprising amino acid sequences homologous to the amino acid sequences of the heavy and light chains of Ha22-2(2,4)6.1, wherein the antibody retains desired functional properties of the anti-191P4D12 antibodies provided herein.

[0262] In some aspects, the antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous or identical to the heavy chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267; and (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous or identical to the light chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0263] In some embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 85% homologous or identical to the heavy chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 90% homologous or identical to the heavy chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In yet other embodiments, the heavy chain variable region comprises an amino acid sequence that is at least 95% homologous or identical to the heavy chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the heavy chain variable region can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical to the heavy chain variable region amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0264] In some embodiments, the light chain variable region comprises an amino acid sequence that is at least 85% homologous or identical to the light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the light chain variable region comprises an amino acid sequence that is at least 90% homologous or identical to the light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In yet other embodiments, the light chain variable region comprises an amino acid sequence that is at least 95% homologous or identical to the light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the light chain variable region can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical to the light chain variable region amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0265] In other aspects, the antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain and a light chain, wherein: (a) the heavy chain comprises an amino acid sequence that is at least 80% homologous or identical to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267; and (b) the light chain comprises an amino acid sequence that is at least 80% homologous or identical to the light chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0266] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 85% homologous or identical to the heavy chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the heavy chain comprises an amino acid sequence that is at least 90% homologous or identical to the heavy chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In yet other embodiments, the heavy chain comprises an amino acid sequence that is at least 95% homologous or identical to the heavy chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the heavy chain can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical to the heavy chain amino acid sequence of the antibody produced by the hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[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 No. PTA-11267. In other embodiments, the light chain comprises an amino acid sequence that is at least 90% homologous or identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In yet other embodiments, the light chain comprises an amino acid sequence that is at least 95% homologous or identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267. In other embodiments, the light chain can be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical to the light chain amino acid sequence of an antibody produced by a hybridoma deposited under American Type Culture Collection (ATCC) Accession No. PTA-11267.

[0268] The engineered antibodies provided herein include those in which modifications have been made to framework residues in VH and / or VL (e.g., to improve the properties of the antibody). Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis (e.g., "backmutating" leucine to methionine). Such "backmutated" 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 regions, or even within one or more CDR regions, to remove T cell epitopes and thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further 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 or CDR regions, the antibodies of the present invention may 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 antigen-dependent cellular cytotoxicity. Additionally, the anti-191P4D12 antibodies provided herein may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to modify its glycosylation, also to modify one or more functional properties of the antibody. Each of these aspects is described in further detail below.

[0271] In one embodiment, the hinge region of CH1 is modified to change, for example, increase or decrease, the number of cysteine ​​residues in the hinge region.This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al.The number of cysteine ​​residues in the hinge region of CH1 is changed, for example, to facilitate assembly of light and heavy chains, or to increase or decrease the stability of anti-191P4D12 antibody.

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

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

[0274] In yet another embodiment, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to modify the effector function(s) 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 the effector ligand but retains the antigen binding ability of the parent antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0275] The reactivity of anti-191P4D12 antibody with 191P4D12-related protein can be established by many well-known means, including Western blot, immunoprecipitation, ELISA, and FACS analysis, using 191P4D12-related protein, 191P4D12-expressing cells or their extracts as necessary. 191P4D12 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators or enzymes. Furthermore, bispecific antibodies specific for two or more 191P4D12 epitopes can be generated using methods 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, wherein the heavy chain of Ha22-2(2,4)6.1 consists of the amino acid sequence ranging from residue E at position 20 to residue K at position 466 of SEQ ID NO:7, and the light chain of Ha22-2(2,4)6.1 consists of the amino acid sequence ranging from residue D at position 23 to residue C at position 236 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), PO Box 1549, Manassas, VA 20108 on August 18, 2010, and assigned accession number PTA-11267.

[0278] 5.3.2 Cytotoxic Agents (Drug Units) In some embodiments, the ADC comprises an antibody or antigen-binding fragment thereof conjugated to a dolastatin or auristatin, a peptide analog and derivative of dolostatin (U.S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anticancer activity (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug unit may be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug unit (WO 02 / 088172).

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

[0280] In some embodiments, the auristatin is MMAE (where the wavy line indicates the covalent attachment to the linker of the antibody drug conjugate). TIFF0007657774000012.tif24140

[0281] In some embodiments, an exemplary embodiment comprising MMAE and a linker moiety (further described herein) has the following structure (where L represents an antibody and p ranges from 1 to 12): TIFF0007657774000013.tif36160

[0282] Typically, peptide-based drug units can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to 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 may be prepared according to the methods of U.S. Pat. No. 5,635,483; U.S. Pat. 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, GR, et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol 21(7):778-784.

[0283] 5.3.3 Linker Typically, an antibody drug conjugate comprises a linker unit between the drug unit (e.g., MMAE) and the antibody unit (e.g., an anti-191P4D12 antibody or antigen-binding fragment thereof). In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In 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 cleavage agent present in the intracellular environment (e.g., in lysosomes or endosomes or caveolae). The linker can be, for example, a peptidyl linker that is cleaved by intracellular peptidase or protease enzymes, including but not limited to lysosomal or endosomal proteases. In some embodiments, the peptidyl linker is at least 2 amino acids long or at least 3 amino acids long. Cleavage agents can include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release active drugs in target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typical is a peptidyl linker that is cleavable by enzymes present in 191P4D12-expressing cells. For example, a peptidyl linker that can be cleaved by cathepsin B, a thiol-dependent protease that is highly expressed in cancerous tissues, 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. Pat. No. 6,214,345, which is incorporated by reference in its entirety for all purposes. In a specific embodiment, the peptidyl linker that can be cleaved by intracellular proteases is a Val-Cit linker or a Phe-Lys linker (see, for example, U.S. Pat. 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 certain pH value. Typically, pH sensitive linker is hydrolyzable under acidic conditions. For example, acid labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis aconitic amide, orthoester, acetal, ketal, etc.) that is hydrolyzable in lysosomes can be used (see, for example, U.S. Pat. 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 below pH 5.5 or below pH 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker, such as a thioether attached to the therapeutic agent via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929).

[0286] In yet other embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), which can be formed using 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. Pat. 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, incorporated herein by reference in its entirety for all purposes).

[0289] Typically, the linker is not substantially sensitive to the extracellular environment. As used herein, "not substantially sensitive to the extracellular environment" in the context of the linker means that when the antibody drug conjugate is present in an extracellular environment (e.g., in plasma), about 20% or less of the linker in the sample of the antibody drug conjugate is cleaved, typically about 15% or less, more typically about 10% or less, even more typically about 5% or less, about 3% or less, or about 1% or less. Whether the linker is not substantially sensitive to the extracellular environment can be determined, for example, by incubating the 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-mutually exclusive, embodiments, the linker promotes cellular internalization. In certain embodiments, the linker promotes cellular internalization when conjugated to a therapeutic agent (i.e., in the context of the linker-therapeutic agent portion of an antibody-drug conjugate compound described herein). In yet other embodiments, 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 present invention are described in WO 2004-010957, U.S. 2006 / 0074008, U.S. 20050238649, and U.S. 2006 / 0024317, each of which is 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 embodiments, the Linker unit has the formula: -A a -W w -Y y - having where -A- is an extender unit, a is 0 or 1, each -W- is independently an amino acid unit; w is an integer ranging from 0 to 12; -Y- is a self-immolative spacer unit; y is 0, 1, or 2.

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

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

[0295] In one embodiment, the extender unit forms a bond with a sulfur atom of an antibody unit. The sulfur atom may be derived from a sulfhydryl group of an antibody. Exemplary extender units of this embodiment are shown in brackets in formulas IIIa and IIIb below, where L-, -W-, -Y-, -D, w and y are as defined above, and R 17 -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 and r is an integer ranging from 1 to 10, wherein the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, carbocyclo, heterocyclo, and arylene radicals, either alone or as part of another group, can be substituted. In some embodiments, the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocycle, carbocyclo, heterocyclo, and arylene radicals, either alone or as part of another group, are unsubstituted.

[0296] In some embodiments, R 17 -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-, and r is an integer ranging from 1 to 10, wherein the alkylene group is unsubstituted and the remaining groups are optionally substituted.

[0297] It should be understood that from all exemplary embodiments, even if not explicitly stated, 1-20 drug units (p=1-20) can be linked to an antibody unit. TIFF0007657774000014.tif57128

[0298] Exemplary extender units include R 17 is an extender unit of formula IIIa where is -(CH2)5-. TIFF0007657774000015.tif24128

[0299] Another exemplary extender unit is R 17 -(CH2CH2O) r and r is 2. TIFF0007657774000016.tif24128

[0300] Exemplary extender units include R 17 Arylene- or Arylene-C1-C 10 The extender unit of formula IIIa is alkylene-. In some embodiments, the aryl group is an unsubstituted phenyl group.

[0301] Yet another exemplary extender unit is R 17 is an extender unit of formula IIIb where is -(CH2)5-. TIFF0007657774000017.tif20128

[0302] In certain embodiments, the extender 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 extender unit. A representative extender unit of this embodiment is depicted within the brackets of Formula IV, where R 17 , -L-, -W-, -Y-, -D, w and y are as defined above. TIFF0007657774000018.tif18128

[0303] It should be noted that throughout this application, the S moiety in the following formulae refers to the sulfur atom of the antibody unit, unless the context dictates otherwise. TIFF0007657774000019.tif12128

[0304] In certain structural descriptions of sulfur-linked ADCs herein, the antibody is represented as "L". It can also be represented as "Ab-S". The inclusion of "S" merely indicates the sulfur linkage feature and does not indicate that a particular sulfur atom has multiple linker-drug moieties. The left bracket in structures using the "Ab-S" designation can also be placed to the left of the sulfur atom between Ab and S, which is an equivalent designation for the ADCs of the invention described throughout this specification.

[0305] In yet other embodiments, the extender comprises a reactive site capable of forming a bond with a primary or secondary amino group of an antibody unit. Examples of these reactive sites include, but are not limited to, activated esters, such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. Representative extender units of this embodiment are depicted within the brackets of Formulas Va and Vb, where -R 17 -, L-, -W-, -Y-, -D, w and y are as defined above. TIFF0007657774000020.tif53128

[0306] In some embodiments, the extender comprises a reactive site that is reactive towards a modified carbohydrate (-CHO) group that may be present on an antibody unit. For example, the carbohydrate can be mildly oxidized using a reagent such as sodium periodate, and the resulting (-CHO) unit of the oxidized carbohydrate can be condensed with an extender that contains a functional group such as a hydrazide, oxime, primary or secondary amine, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide as described in Kaneko et al., 1991, Bioconjugate Chem. 2:133-41. Exemplary extender units of this embodiment are depicted within square brackets in formulas VIa, VIb, and VIc, where -R 17 -, L-, -W-, -Y-, -D, w and y are as defined above. TIFF0007657774000021.tif77128

[0307] 5.3.3.2 Amino acid units The amino acid unit (-W-), when present, links the extender unit to the spacer unit if the spacer unit is present, the extender unit to the drug unit if the spacer unit is not present, and the antibody unit to the drug unit if the extender unit and spacer unit are not 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 in brackets, where w is an integer ranging from 0 to 12: TIFF0007657774000022.tif29128In formula, R 19is 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, The file is TIFF0007657774000023.tif107148.

[0309] In some embodiments, the Amino Acid Unit can be enzymatically cleaved by one or more enzymes, including cancer or tumor associated proteases, to liberate the Drug Unit (-D), which in one embodiment, upon release, is protonated in vivo to provide the Drug (D).

[0310] In certain embodiments, the amino acid units include natural amino acids. In other embodiments, the amino acid units include unnatural amino acids. Exemplary Ww units are represented by Formulas VII-IX below: TIFF0007657774000024.tif21128In formula, R 20 and R 21 is as follows: TIFF0007657774000025.tif120147TIFF0007657774000026.tif21128In formula, R 20 , R 21 and R 22 is as follows: TIFF0007657774000027.tif37134TIFF0007657774000028.tif21128In formula, R 20 , R 21 , R 22 and R23 is as follows: TIFF0007657774000029.tif28137.

[0311] Exemplary amino acid units include R 20 is benzyl and R 21 is -(CH2)4NH2; R 20 is isopropyl, R 21 is -(CH2)4NH2; or R 20 is isopropyl, R 21 is -(CH2)3NHCONH2.

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

[0313] -W is useful because of enzymatic cleavage by certain enzymes, e.g., tumor-associated proteases. w - units can be designed and their selectivity optimized. w -units, the cleavage of which is catalyzed by cathepsins B, C and D, or plasmin proteases.

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

[0315] R 19 , R 20 , R21 , R 22 or R 23 Each carbon atom to which 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, tetraisoquinoline carboxylate lysine, cyclohexylalanine lysine, isonipecotic acid lysine, β-alanine lysine, glycine serine valine glutamine, and isonepecotic acid.

[0317] 5.3.3.3 Spacer Units The Spacer unit (-Y-), when present, links the Amino Acid unit to the Drug unit, if the Amino Acid unit is present. Alternatively, the Spacer unit links the Extender unit to the Drug unit, if the Amino Acid unit is not present. The Spacer unit also links the Drug unit to the Antibody unit, if both the Amino Acid unit and the Extender unit are not present.

[0318] Spacer units are of two general types: non-self-immolative or self-immolative. A non-self-immolative spacer unit is one in which some or all of the spacer unit remains attached to the drug unit after cleavage, particularly enzymatic cleavage, of the amino acid unit from the antibody drug conjugate. Examples of non-self-immolative 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 the glycine-drug unit is cleaved from L-Aa-Ww-. In one embodiment, an independent hydrolysis reaction occurs within the target cell, cleaving the glycine-drug unit bond and liberating the drug. TIFF0007657774000030.tif54131

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

[0320] In one embodiment, the spacer unit is absent (-Y y -).

[0321] Alternatively, an antibody drug conjugate comprising a self-immolative spacer unit can release -D. As used herein, the term "self-immolative spacer" refers to a bifunctional chemical moiety that can covalently link two spaced apart chemical moieties together into a stable tripartite molecule, which spontaneously separates from the second chemical moiety upon cleavage of the bond to the first moiety.

[0322] In some embodiments, -Y y - is a phenylene moiety Q mand p-aminobenzyl alcohol (PAB) units (see Schemes 2 and 3) substituted with Q, 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 ranging from 0 to 4. The alkyl, alkenyl and alkynyl groups, either alone or as part of another group, may be substituted.

[0323] In some embodiments, -Y- is linked to -W- through the amino nitrogen atom of the PAB group. w - is a PAB group linked to - and directly bonded to -D through a carbonate, carbamate or ether group. Without being bound to a particular theory or mechanism, Scheme 2 displays a possible drug release mechanism of a PAB group directly bonded to -D through a carbamate or carbonate group, as described by Toki et al., 2002, J. Org. Chem. 67:1866-1872. TIFF0007657774000031.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 ranging from 0 to 4, and p ranges from 1 to about 20. The alkyl, alkenyl, and alkynyl groups, either alone or as part of another group, can be optionally substituted.

[0325] Without being bound to any particular theory or mechanism, Scheme 3 depicts a possible drug release mechanism for a PAB group that is directly attached to -D via an ether or amine bond, where D contains an oxygen or nitrogen group that is part of the Drug unit. TIFF0007657774000032.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 ranging from 0 to 4, and p ranges from 1 to about 20. The alkyl, alkenyl, and alkynyl groups, either alone or as part of another group, can be optionally substituted.

[0327] Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are 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. Spacers that undergo cyclization upon hydrolysis of the amide bond can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (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 2-aminophenylpropionic acid amides (Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination of amine-containing drugs substituted at the alpha position of glycine (Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of a self-immolative spacer.

[0328] In one embodiment, the spacer unit is a branched bis(hydroxymethyl)-styrene (BHMS) unit as depicted in Scheme 4, which can be used to incorporate and release multiple drugs. TIFF0007657774000033.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 ranging from 0 to 4, n is 0 or 1, and p ranges from 1 to about 20. The alkyl, alkenyl, and alkynyl groups, either alone or as part of another group, can be optionally substituted.

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

[0331] In one aspect, the spacer unit (-Y y -) is represented by formulas X-XII: TIFF0007657774000034.tif28128 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 ranging from 0 to 4. The alkyl, alkenyl and alkynyl groups, either alone or as part of another group, may be substituted. TIFF0007657774000035.tif36128.

[0332] Embodiments of Formula I and II that include antibody-drug conjugate compounds can include: TIFF0007657774000036.tif19128, where w and y are 0, 1 or 2, TIFF0007657774000037.tif19128 (wherein w and y are each 0); TIFF0007657774000038.tif166128.

[0333] 5.3.3.4 Drug burden Drug loading is represented by p, which is the average number of drug units per antibody in the molecule. Drug loading can range from 1 to 20 drug units (D) per antibody. The ADCs provided herein include a collection of antibodies or antigen-binding fragments conjugated with drug units ranging from 1 to 20, for example. The average number of drug units per antibody in a preparation of ADCs 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, separation, purification and characterization of a homogenous ADC with a particular value of p from ADCs with other drug loadings can be achieved by means such as electrophoresis.

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

[0335] In certain embodiments, the drug loading of the ADCs provided herein is in the range of 1 to about 8, about 2 to about 6, about 3 to about 5, about 3 to about 4, about 3.1 to about 3.9, about 3.2 to about 3.8, about 3.2 to about 3.7, about 3.2 to about 3.6, about 3.3 to about 3.8, or about 3.3 to about 3.7.

[0336] In certain embodiments, the drug loading of the ADCs provided herein is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or more. In certain embodiments, the drug loading of the ADCs provided herein is about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, or about 3.9.

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

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

[0339] The loading (drug / antibody ratio) of the ADC can be controlled in a variety of ways, for example, by (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limited reduction conditions for cysteine ​​thiol modification, (iv) recombinantly engineering the amino acid sequence of the antibody such that the number and position of cysteine ​​residues are altered to control the number and / or position of linker-drug linkages (such as ThioMab or ThioFab prepared as disclosed herein and in WO 2006 / 034488, which is incorporated herein by reference in its entirety).

[0340] It should be understood that when multiple nucleophilic groups react with a drug-linker intermediate or linker reagent and subsequently with a drug unit reagent, the resulting product is a mixture of ADC compounds with a distribution of one or more drug units attached to an antibody unit. The average number of drugs per antibody can be calculated from the mixture by a dual antibody-specific and drug-specific ELISA antibody assay. Individual ADC molecules can be identified in a mixture by mass spectrometry and separated by HPLC, e.g., hydrophobic interaction chromatography (e.g., Hamblett, KJ, et al., "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate," Abstract No. 624, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004; Alley, SC, et al., "Controlling the location of drug attachment in antibody-drug conjugates," Abstract No. 627, American Association for Cancer Research, 2004 Annual Meeting, March 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). In certain embodiments, homogenous 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. Pat. No. 8,637,642, incorporated herein by reference in its entirety.

[0342] In some embodiments, the antibody drug conjugate for the methods provided herein is AGS-22M6E, prepared according to the methods described in U.S. Pat. No. 8,637,642 and having the following formula: TIFF0007657774000039.tif36160In the formula, L is Ha22-2(2,4)6.1 and p is 1 to 20.

[0343] In some embodiments, p is in the range of 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is in the range of 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is about 1. In other embodiments, p is about 2. In other embodiments, p is about 3. In other embodiments, p is about 4. In other embodiments, p is about 5. In other embodiments, p is about 6. In other embodiments, p is about 7. In other embodiments, p is about 8. In other embodiments, p is about 9. In other embodiments, p is about 10. In some embodiments, p is about 3.1. In some embodiments, p is about 3.2. In some embodiments, p is about 3.3. In some embodiments, p is about 3.4. In some embodiments, p is about 3.5. In other embodiments, p is about 3.6. In some embodiments, p is about 3.7. In some embodiments, p is about 3.8. In some embodiments, p is about 3.9. In some embodiments, p is about 4.0. In some embodiments, p is about 4.1. In some embodiments, p is about 4.2. In some embodiments, p is about 4.3. In some embodiments, p is about 4.4. In some embodiments, p is about 4.5. In other embodiments, p is about 4.6. In some embodiments, p is about 4.7. In some embodiments, p is about 4.8. In some embodiments, p is about 4.9. In some embodiments, p is about 5.0.

[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 (Challita-Eid PM et al,Cancer Res.2016;76(10):3003-13). Enfortumab vedotin induces antitumor activity by binding to the cell surface 191P4D12 protein, leading to internalization of the ADC-191P4D12 complex, which is then transported to the lysosomal compartment, where MMAE is released via proteolytic cleavage of the linker. The intracellular release of MMAE subsequently disrupts tubulin polymerization, leading to G2 / M cell cycle arrest and apoptotic cell death (Francisco JA et al,Blood.2003 Aug 15;102(4):1458-65).

[0345] As described above and in US Patent No. 8,637,642, AGS-22M6E is an ADC derived from a mouse hybridoma cell line. Enfortumab vedotin is the Chinese hamster ovary (CHO) cell line-derived equivalent of 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 has been confirmed through extensive analytical and biological characterization studies, such as binding affinity to 191P4D12, in vitro cytotoxicity, and in vivo antitumor activity.

[0346] 5.4 Pharmaceutical Compositions In certain embodiments of the methods provided herein, the ADC used in the methods is provided as a "pharmaceutical composition". Such pharmaceutical compositions include the antibody drug conjugates provided herein and one or more pharma- ceutically acceptable or physiologically acceptable excipients. In certain embodiments, the antibody drug conjugates are provided in combination with or separately from one or more additional agents. Also provided are compositions including such one or more additional agents and one or more pharma-ceutically acceptable or physiologically acceptable excipients. In certain embodiments, the antibody drug conjugates and the 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 carry out 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 cancer or tumor modulating antibody drug conjugates are provided.

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

[0349] A pharmaceutical composition typically comprises a therapeutically effective amount of at least one of the antibody drug conjugates provided herein and one or more pharma- ceutically acceptable formulation agents. In certain embodiments, the pharmaceutical composition further comprises one or more additional agents described herein.

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

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

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

[0353] In some embodiments, the pharmaceutical compositions provided herein comprise L-histidine, TWEEN-20, and at least one of trehalose dihydrate or sucrose. In some embodiments, the pharmaceutical compositions provided herein further comprise hydrochloric acid (HCl) or succinic acid.

[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 one particular embodiment, the concentration of TWEEN-20 is about 0.01% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.015% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.016% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.017% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.018% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.019% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.02% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.021% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.022% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.023% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.024% (v / v). In one particular embodiment, the concentration of TWEEN-20 is about 0.025% (v / v).

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

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

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

[0359] In certain embodiments, the molar concentration of sucrose is 50 mM to 300 mM. In other embodiments, the molar concentration of sucrose is 75 mM to 250 mM. In some embodiments, the molar concentration of sucrose is 100 mM to 200 mM. In other embodiments, the molar concentration of sucrose is 130 mM to 150 mM. In some embodiments, the molar concentration of sucrose is 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 comprise HCl. In other embodiments, the pharmaceutical compositions provided herein comprise 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 between 15° C. and 27° C. In yet other embodiments, the pH is measured at 4° C. In yet other embodiments, the pH is measured at 25° C.

[0363] In some embodiments, the pH is adjusted with HCl. In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH in the range of 5.5-6.5 at room temperature. In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH in the range of 5.7-6.3 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.7 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.8 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.9 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.0 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.1 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.2 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.3 at room temperature.

[0364] In some embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH ranging from 5.5 to 6.5 at 15°C to 27°C. In some embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH ranging from 5.7 to 6.3 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 5.7 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 5.8 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 5.9 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 6.0 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 6.1 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 6.2 at 15° C. to 27° C. In some more specific embodiments, the pharmaceutical composition comprises HCl and the pharmaceutical composition has a pH of about 6.3 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 5.5-6.5 at room temperature. In some embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH in the range of 5.7-6.3 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 ranging from 5.5 to 6.5 at 15°C to 27°C. In some embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH ranging from 5.7 to 6.3 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 5.7 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 5.8 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 5.9 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 6.0 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 6.1 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 6.2 at 15° C. to 27° C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid and the pharmaceutical composition has a pH of about 6.3 at 15° C. to 27° C.

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

[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 aspect, provided herein is a method for treating a pulmonary circulation comprising: (a) a structure: TIFF0007657774000040.tif36160, where L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharma- ceutically 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 aspect, the pharmaceutical compositions provided herein comprise: (a) a structure: TIFF0007657774000041.tif36160, where L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharma- ceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.5% (w / v) trehalose dihydrate, and succinic acid. wherein the 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 particular aspect, the pharmaceutical compositions provided herein comprise: (a) a structure: TIFF0007657774000042.tif36160, where L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; and (b) a pharma- ceutically acceptable excipient comprising about 20 mM L-histidine, about 0.02% (w / v) TWEEN-20, about 5.0% (w / v) sucrose, and HCl. wherein the antibody drug conjugate is at a concentration of about 10 mg / mL and the pH is about 6.0 at 25° C.

[0375] It is understood that although specific numbers (and numerical ranges thereof) are provided, in certain embodiments, numbers within, for example, 2%, 5%, 10%, 15% or 20% of said numbers (or numerical 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 aqueous or non-aqueous in nature.In addition, the vehicle can contain other pharma-ceutically acceptable excipients to modify or maintain the pH, osmolality, viscosity, sterility or stability of the pharmaceutical composition.In certain embodiments, the pharma-ceutically acceptable vehicle is an aqueous buffer.In other embodiments, the vehicle contains, for example, sodium chloride and / or sodium citrate.

[0377] The pharmaceutical compositions provided herein may further comprise other pharma- ceutically acceptable formulating agents for modifying or maintaining the release rate of the antibody-drug conjugate and / or additional agent as described herein. Such formulating agents include substances known to those skilled in the art in the preparation of sustained release formulations. For further references regarding pharma- ceutical and physiologically acceptable formulating 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 are applicable to the methods and compositions provided herein.

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

[0379] A pharmaceutical composition can be formulated to be compatible with its intended route of administration. Thus, the pharmaceutical composition includes excipients suitable for administration by routes including parenteral (e.g., subcutaneous (sc), intravenous, intramuscular, or intraperitoneal), intradermal, oral (e.g., ingestion), inhalation, intracavity, intracranial, and transdermal (topical). Other exemplary routes of administration are described herein.

[0380] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. The suspension may be formulated using suitable dispersing or wetting agents and suspending agents disclosed herein or known to those skilled in the art. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Acceptable diluents, solvents and dispersion media that may be used include water, Ringer's solution, isotonic sodium chloride solution, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS), ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any non-irritating fixed oil may be used, including synthetic mono- or diglycerides. 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 the inclusion of agents which delay absorption, for example, 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, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration.

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

[0383] In one aspect, a pharmaceutical composition intended for parenteral administration may contain one or more pharma- ceutically acceptable excipients, including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against microbial growth, stabilizers, solubility enhancers, isotonicity agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.

[0384] In one embodiment, suitable aqueous vehicles include, but are not limited to, water, saline, saline or phosphate buffered saline (PBS), sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection.Non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, and medium chain triglycerides of coconut oil, and palm seed oil.Water-miscible vehicles include, but are not limited to, ethanol, 1,3-butanediol, liquid polyethylene glycols (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N-methyl-2-pyrrolidone, N,N-dimethylacetamide, and dimethylsulfoxide.

[0385] In one embodiment, suitable antimicrobial agents or preservatives include, but are not limited to, phenol, cresol, mercurial, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate, thimerosal, benzalkonium chloride (e.g., benzethonium chloride), methyl- and propyl-paraben, and sorbic acid. Suitable isotonicity agents include, but are not limited to, sodium chloride, glycerin, and dextrose. Suitable buffers include, but are not limited to, phosphates and citrates. Suitable antioxidants 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 sequestering or chelating agents include, but are not limited to, EDTA. Suitable pH adjusting agents include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Suitable complexing agents include, but are not limited to, cyclodextrins, such as α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin, and sulfobutylether 7-β-cyclodextrin (CAPTISOL®, CyDex, Lenexa, KS).

[0386] In one aspect, the pharmaceutical compositions provided herein can be formulated for single or multiple doses. Single dose formulations are packaged in ampoules, vials, or syringes. Multi-dose parenteral formulations can contain bacteriostatic or fungistatic concentrations of antibacterial agents. 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 lyophilized powder and hypodermic tablets, 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-, sustained-, pulsed-, controlled-, targeted-, and programmed-release forms.

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

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

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

[0392] Lyophilized compositions can be produced by freeze-drying the liquid pharmaceutical compositions provided herein.In a specific embodiment, the pharmaceutical compositions provided herein are freeze-dried pharmaceutical compositions.In some embodiments, the pharmaceutical preparations are freeze-dried powders and can be reconstituted for administration as solutions, emulsions and other mixtures.They can also be reconstituted and formulated as solids or gels.

[0393] In some aspects, preparation of the lyophilized formulations provided herein involves batch processing of the formulated bulk solution for lyophilization, sterile filtration, filling into vials, freezing the vials in a lyophilization chamber, followed by lyophilization, stoppering and capping.

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

[0395] Lyophilized powder can be prepared by dissolving the antibody drug conjugate provided herein or its pharma- ceutically acceptable derivative 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 distributed 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, at about 4°C to room temperature.

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

[0397] An exemplary reconstitution procedure is as follows: (1) attach an 18 or 20 gauge needle to a 5 mL or 3 mL syringe and fill the syringe with water for injection (WFI) grade water; (2) measure the appropriate amount of WFI using the graduations on the syringe while ensuring that there are no air bubbles in the syringe; (3) insert the needle into the rubber stopper; (4) dispense the entire contents of the syringe through the vial wall into a container, remove the syringe and needle, and place in a sharps container; (4) swirl the vial continuously to carefully solubilize the entire contents of the vial until completely reconstituted (e.g., about 20 s to about 40 s), minimizing excessive agitation of the protein solution that can result in foaming.

[0398] 5.5 Methods of Using the Pharmaceutical Compositions in Combination Therapy Methods of inhibiting tumor cell proliferation using the pharmaceutical compositions provided herein in combination with chemotherapy or radiation or both include administering the pharmaceutical composition before, during, or after the initiation of chemotherapy or radiation therapy, as well as any combination thereof (i.e., before and during, before and after, during and after, or before, during and after chemotherapy and / or radiation therapy). Depending on the treatment protocol and the needs of the particular patient, the methods are performed to provide the most effective treatment and ultimately extend the patient's lifespan.

[0399] Administration of the chemotherapeutic agent can be accomplished in a variety of ways, including systemic administration by parenteral and enteral routes. In one embodiment, the chemotherapeutic agent is administered separately. Specific examples of chemotherapeutic agents or chemotherapies 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, cyclophosphamide ... , asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, 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 radiation source is outside the patient's body, the treatment is known as external beam radiation therapy (EBRT).When the radiation source is inside the patient's body, the treatment is called brachytherapy (BT).

[0401] The above therapeutic regimens may 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 WO 2005 / 092380 (Pfizer)) or other ligands, and cytokines.

[0402] When mammals undergo further chemotherapy, the above-mentioned chemotherapy agents can be used.In addition, growth factor inhibitors, biological response modifiers, antihormonal therapy, selective estrogen receptor modulators (SERM), angiogenesis inhibitors and antiandrogens can be used.For example, antihormonal agents, such as antiestrogens such as Nolvadex (tamoxifen), or antiandrogens such as Casodex (4'-cyano-3-(4-fluorophenylsulfonyl)-2-hydroxy-2-methyl-3'-(trifluoromethyl)propionanilide) can be used.

[0403] In some embodiments, the pharmaceutical compositions provided herein are used in combination with a second therapeutic agent, e.g., 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 reduces, inhibits, prevents or regulates one or more checkpoint proteins in whole or in part. 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-Ll and PD-L2 (Pardoll, Nature Reviews Cancer, 2012, 12, 252-264). These proteins appear to be involved in costimulatory 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 incorporated herein in their entirety. In one embodiment, the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP-675,206). In another embodiment, the anti-CTLA-4 antibody is ipilimumab (also known as MDX-010 or MDX-101). Ipilimumab is a fully human monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is commercially available under the trade name Yervoy™.

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

[0407] In one embodiment, the checkpoint inhibitor is a PD-1 inhibitor. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody. In one embodiment, the anti-PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558, or MDX1106) or pembrolizumab (also known as MK-3475, 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. CT-011 administered alone failed to show a response in the treatment of acute myeloid leukemia (AML) at relapse. 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 that is specifically engineered for its ability to bind to Fcγ receptor I and has a unique binding signature with high affinity and excellent target specificity for PD-1.

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

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

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

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

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

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

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

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

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

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

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

[0419] In certain embodiments, the combination therapies provided herein include two or more of the checkpoint inhibitors described herein (including checkpoint inhibitors of the same or different classes). Additionally, the combination therapies described herein can be used in combination with one or more second active agents described herein, where appropriate to treat a disease as described herein and understood in the art.

[0420] In some embodiments, the checkpoint inhibitor is administered prior to administration of the pharmaceutical composition. In other embodiments, the checkpoint inhibitor is administered simultaneously (e.g., during the same administration period) with the pharmaceutical composition provided herein. In yet other embodiments, the checkpoint inhibitor is administered after administration of the pharmaceutical composition provided herein.

[0421] In some embodiments, the amount of the checkpoint inhibitor can be determined by standard clinical techniques.

[0422] The dosage of the checkpoint inhibitor results in a serum titer of about 0.1 μg / ml to about 450 μg / ml, and in some embodiments is at least 0.1 μg / ml, at least 0.2 μg / ml, at least 0.4 μg / ml, at least 0.5 μg / ml, at least 0.6 μg / ml, at least 0.8 μg / ml, at least 1 μg / ml, at least 1.5 μg / ml, such as at least 2 μg / ml, at least 5 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least At least 25 μg / ml, at least 30 μg / ml, at least 35 μg / ml, at least 40 μg / ml, at least 50 μg / ml, at least 75 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 200 μg / ml, at least 250 μg / ml, at least 300 μg / ml, at least 350 μg / ml, at least 400 μg / ml, or at least 450 μg / ml can be administered to a human for the prevention and / or treatment of cancer. It should be understood that the precise dose of checkpoint inhibitor used will also depend on the route of administration, and the severity of the cancer in the subject, and should be determined according to the judgment of the practitioner and each patient's circumstances.

[0423] In some embodiments, the dosage of the checkpoint inhibitor (e.g., PD-1 inhibitor or PD-L1 inhibitor) administered to the patient is typically between 0.1 mg / kg and 100 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is between about 1 mg / kg and about 75 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is between 1 mg / kg and 20 mg / kg of the subject's body weight, e.g., between 1 mg / kg and 5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 1 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 1.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 2 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 2.5 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the patient is about 3 mg / kg of the subject's body weight. In some embodiments, the dosage administered to the ...

Claims

1. A medicament for preventing or treating locally advanced or metastatic head and neck cancer in a subject, comprising an antibody drug conjugate (ADC), the antibody drug conjugate comprises an antibody or antigen-binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethylauristatin E (MMAE), the antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising a complementarity determining region (CDR) comprising the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:22, and a light chain variable region comprising a CDR comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:23; (i) the subject has progressed or relapsed after platinum-based therapy, (ii) 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); or (iii) the subject has progressed or relapsed after platinum-based therapy and 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); Medicine.

2. The pharmaceutical agent of claim 1, wherein the subject has progressed or relapsed within 6 months after platinum-based therapy.

3. The pharmaceutical agent according to claim 1, wherein the PD-1 inhibitor is nivolumab.

4. The pharmaceutical composition of claim 1, wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

5. the antibody or antigen-binding fragment thereof comprises a CDR H1 comprising the amino acid sequence of SEQ ID NO:9, a CDR H2 comprising the amino acid sequence of SEQ ID NO:10, a CDR H3 comprising the amino acid sequence of SEQ ID NO:11; a CDR L1 comprising the amino acid sequence of SEQ ID NO:12, a CDR L2 comprising the amino acid sequence of SEQ ID NO:13, and a CDR L3 comprising the amino acid sequence of SEQ ID NO:14; or the antibody or antigen-binding fragment thereof comprises CDR H1 comprising the amino acid sequence of SEQ ID NO:16, CDR H2 comprising the amino acid sequence of SEQ ID NO:17, 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 pharmaceutical composition according to any one of claims 1 to 4.

6. The pharmaceutical according to any one of claims 1 to 4, 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.

7. The pharmaceutical of any one of claims 1 to 4, 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.

8. The antigen-binding fragment is selected from the group consisting of Fab, F(ab') 2 8. The pharmaceutical according to any one of claims 1 to 7, which is an Fv or scFv fragment.

9. The pharmaceutical according to any one of claims 1 to 8, wherein the antibody is a fully human antibody.

10. The method of any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof is recombinantly produced.

11. The antibody drug conjugate has the following structure: where L- represents an antibody or antigen-binding fragment thereof and p is 1 to 10; The pharmaceutical composition according to any one of claims 1 to 10.

12. The pharmaceutical composition of claim 11, wherein p is 2 to 8.

13. The pharmaceutical composition of claim 11, wherein p is 3 to 5.

14. The pharmaceutical agent according to any one of claims 1 to 13, wherein the antibody or antigen-binding fragment is linked to each unit of monomethylauristatin E (MMAE) via a linker.

15. The pharmaceutical according to claim 14, wherein the linker is an enzyme-cleavable linker that forms a bond with a sulfur atom of the antibody or its antigen-binding fragment.

16. Linker is -A a -W w -Y y 15. The pharmaceutical composition of claim 14, having the formula: -, wherein -A- is an extender unit, a is 0 or 1, -W- is an amino acid unit, w is an integer ranging from 0 to 12, -Y- is a spacer unit, and y is 0, 1, or 2.

17. 17. The pharmaceutical composition of claim 16, wherein the extender unit has the structure of the following formula (1), the amino acid unit is valine citrulline; and the spacer unit is a PAB group having the structure of the following formula (2): 。

18. The pharmaceutical of claim 16, wherein the extender unit forms a bond with a sulfur atom of the antibody or antigen-binding fragment thereof, and the spacer unit is linked to MMAE via a carbamate group.

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

20. The method of any one of claims 1 to 10, wherein the antibody-drug conjugate comprises 2 to 8 units of MMAE per antibody or antigen-binding fragment thereof.

21. The method of any one of claims 1 to 10, wherein the antibody-drug conjugate comprises 3 to 5 units of MMAE per antibody or antigen-binding fragment thereof.

22. The pharmaceutical agent according to any one of claims 1 to 21, wherein the antibody-drug conjugate is administered at a dose of 1 to 10 mg / kg of the subject's body weight, 1 to 5 mg / kg of the subject's body weight, 1 to 2.5 mg / kg of the subject's body weight, or 1 to 1.25 mg / kg of the subject's body weight.

23. The pharmaceutical composition of claim 22, wherein the antibody-drug conjugate is administered at a dose of about 1 mg / kg of subject body weight.

24. The pharmaceutical composition of claim 22, wherein the antibody-drug conjugate is administered at a dose of about 1.25 mg / kg of subject body weight.

25. The medicament of any one of claims 1 to 24, wherein the antibody-drug conjugate is used to be administered by intravenous (IV) injection or infusion.

26. The medicament of any one of claims 1 to 24, wherein the antibody drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes in a cycle of two times every three weeks.

27. The method of any one of claims 1 to 24, wherein the antibody drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes on days 1 and 8 of every 3-week cycle.

28. The medicament of any one of claims 1 to 24, 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.

29. The medicament of any one of claims 1 to 24, wherein the antibody drug conjugate is administered by intravenous (IV) injection or infusion over about 30 minutes on days 1, 8, and 15 of every 4-week cycle.

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