Epcam immunoconjugates and therapeutic uses thereof

Abstract

The disclosure generally relates to methods of treatment using conditionally activatable masked immunoconjugates comprising antibodies and antibody fragments that specifically bind human EpCAM, as well as methods of using human EpCAM antibodies, human EpCAM antibody fragments, human EpCAM activatable antibodies, and human EpCAM conditionally activatable masked immunoconjugates, for the treatment of diseases such as cancer.

Description

[0001] Atty. Dkt. No.: 45325-0051WO1 EPCAM IMMUNOCONJUGATES AND THERAPEUTIC USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003]

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63 / 750,040, filed on January 27, 2025, and 63 / 802,846, filed on May 9, 2025. The contents of these applications are hereby incorporated by reference in their entireties.

[0004] SEQUENCE LISTING

[0005]

[0002] This application contains a Sequence Listing that has been submitted electronically as an XML file named “45325-0051 ST26 SL. XML.'’ The XML file, created on January 23, 2026, is 336.346 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety TECHNICAL FIELD

[0006]

[0003] The present disclosure is in the field of cancer therapeutics.

[0007] BACKGROUND

[0008]

[0004] Epithelial cell adhesion molecule (EpCAM), is a type I trans-membrane glycoprotein comprising an extracellular domain, a transmembrane domain, and a single intracellular domain.

[0009] EpCAM expression in human is epithelia-specific. The majority of epithelial cells express EpCAM, except squamous epithelium and some specific epithelium cell types, such as epidermal keratinocytes, hepatocytes, gastric parietal cells, and myoepithelial cells (Balzar etal., J. Mol. Med. 77:699-712 (1999); Momburg etal., Cancer Res 47:2883-2891 (1987)).

[0010]

[0005] EpCAM is abundantly and homogeneously expressed on human carcinomas of different origins (Went et al., Br. J. Cancer 94:128-35 (2006): Herlyn et al., Proc Natl. Acad. Set. USA 76:1438-1442 (1979); Went etal.. Hum. Pathol. 35:122-128 (2004)). EpCAM is overexpressed in the vast majority of epithelial cancers, including for example, ovarian cancers, colon cancers, stomach cancers, prostate cancers, and lung cancers. In addition, EpCAM has been shown to be expressed on the majority of primary, metastatic, and disseminated NSCLC (non-small cell lung cancer cells) (Passlick, Jnt. J. Cancer 87:548-552 (2000)), on gastric and gastro-esophageal junction adenocarcinomas (Martin, J. Clin. Pathol. 52:701-704 (1999)) and in cell lines derived from colorectal, pancreatic carcinomas and breast carcinomas (Szala. Proc. Natl. Acad. Sci. USA 87:3542-3546 (1990), Packeisen, Hybridoma 18:37-40 (1999)). In another study, immunohistochemical analysis of 108 samples of secondary' tumors has found that only 4% lacked EpCAM expression. EpCAM is overexpressed also in cancer-initiating or cancer stem cells isolated from colon, breast, pancreas and prostate carcinomas (O'Brien et al., Nature 445: 106-110 (2007); Marhaba et al.. Curt: Mol. Med. 8:784-804 (2008)).Atty. Dkt. No.: 45325-0051WO1

[0006] Normal cells express EpCAM on the basolateral side of the epithelial membrane, whereas cancer cells heavily express EpCAM on the apical surface. Antibody-based therapeutics have been designed to exploit this characteristic of EpCAM expression, as normal cellular EpCAM is less prominent and less exposed, meaning healthy cells cannot be as susceptible to binding by therapeutic anti-EpCAM antibodies.

[0011]

[0007] Camptothecin (CPT) is a pentacyclic alkaloid isolated from the bark and stem of Camptotheca acuminata (Camptotheca, Happy tree), a tree native to China. Camptothecin inhibits topoisomerase I. which leads to cell death. Because of its cytotoxic mechanism and broad-spectrum antitumor activity, there have been substantial efforts towards developing clinical analogues of camptothecin. Poor solubility and inactivity at physiological conditions, however, have limited the clinical development of suitable camptothecin analogues. Camptothecin and most of its derivatives are not soluble in aqueous buffers. Further, camptothecin is in equilibrium in an active lactone form and inactive hydrolyzed carboxylate form, thereby limiting its therapeutic efficacy.

[0012]

[0008] A number of antibodies to EpCAM have been used in the clinic but failed for various reasons.

[0013] The EpCAM antibodies tested take a number of formats, including naked antibodies, immunotoxins and bi- or tri-specific antibodies (Baeuerle, Br. J. Cancer, 96:417-423 (2007)). For example, adecatumumab (MT201), a naked anti-EpCAM antibody has been tested in clinical studies of treatment in colorectal, prostate and breast cancers. Safety issues facing the current anti-EpCAM antibody-based approaches include systemic intolerability and acute pancreatitis. Thus, although there have been several attempts to develop therapeutic antibodies to EpCAM, there is a significant need for the development of novel therapeutic EpCAM immunoconjugates that overcome the shortcomings and limitations of the previously developed antibodies or antibody derivatives.

[0014] SUMMARY

[0015]

[0009] In one aspect, the present disclosure provides a method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease in a subject in which diseased cells express epithelial cell adhesion molecule (EpCAM), the method comprising administering to the subject a dose of about 1.2 mg / kg to about 20 mg / kg of a conditionally activatable masked immunoconjugate comprising:

[0016] (a) an activatable antibody comprising:

[0017] (i) an EpCAM antibody or EpCAM -binding fragment thereof (AB), comprising:

[0018] (1) a heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence NYYIH (SEQ ID NO: 13):Atty. Dkt. No.: 45325-0051WO1

[0019] (2) a heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14);

[0020] (3) a heavy chain complementarity determining region 3 (VH-CDR3) comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15);

[0021] (4) a light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO:42):

[0022] (5) a light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence QTSNLAS (SEQ ID NO:40); and (6) a light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence AQNLELPNT (SEQ ID NO:41);

[0023] (ii) a cleavable moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169); and (iii) a masking moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein the masking moiety inhibits the binding of the antibody or antibody fragment to EpCAM when the activatable antibody is in an uncleaved state,

[0024] wherein the activatable antibody in an uncleaved state has a structural arrangement from N-terminus to C- terminus of: (masking moiety )-(cleavable moiety)-(antibody or antibody fragment) or (antibody or antibody fragment)-(cleavable moiety) -(masking moiety); and

[0025] (b) at least one linker-payload depicted by tire structure of Formula (X):

[0026] Formula (X)

[0027]

[0028] Atty. Dkt. No.: 45325-0051WO1 or a pharmaceutically acceptable salt thereof.

[0029]

[0010] In some instances, the dose is determined by actual body weight (ABW).

[0030]

[0011] In some instances, the dose is determined by adjusted ideal body weight (AIBW).

[0031]

[0012] In some instances, the at least one linker-payload is covalently bound to the AB.

[0032]

[0013] In some instances, the conditionally activatable masked immunoconjugate has a dnig-to- antibody ratio (DAR) of 8. In some instances, the conditionally activatable masked immunoconjugate has a drag -to-anti body ratio (DAR) of 4.

[0033]

[0014] In some instances, the activatable antibody is a full length human IgGl EpCAM antibody¬ having eight cysteines which, when in a native conformation form four interchain disulfide bonds.

[0034]

[0015] In some instances, each linker-payload is individually covalently bound to the activatable antibody via one of the eight cysteines (each referred to herein as an “interchain cysteine”), e.g., via a thioether bond. Each of the eight interchain cysteines are shown in FIG. 3. depicted ’ith an “S” and a thioether bond links the linker-payload to the AB.

[0035]

[0016] In some instances, the EpCAM antibody or EpCAM-binding fragment thereof comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO:54 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0036]

[0017] In some instances, the activatable antibody component of the conditionally activatable masked immunoconjugate comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179. In certain of these specific instances, the activatable masked immunoconjugate comprises having a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179, wherein the one or more linker-pay loads of Formula (X) are each covalently bound to an interchain cysteine of the AB (e.g., via a thioether bond) at a DAR of 8. In certain other specific instances, the activatable masked immunoconjugate comprises an activatable antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179, wherein the one or more linker-payloads of Formula (X) are each covalently bound to an interchain cysteine of the AB (e.g., via a thioether bond) at a DAR of 4.

[0037]

[0018] In some instances, the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, and a dAb.

[0038]

[0019] In some instances, the MM is linked to the CM such that the activatable antibody in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: MM- CM-AB or AB-CM-MM. In some instances, the activatable antibody further comprises a first linking peptide (LP1 ) and a second linking peptide (LP2), wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1 -CM-LP2-ABAtty. Dkt. No.: 45325-0051WO1 or AB-LP2-CM-LP1-MM, and wherein each ofLPl and LP2 is a peptide of about 1 to 20 amino acids in length. In some instances, the two linking peptides are not identical to each other.

[0039]

[0020] In some instances, the disease is cancer. In some instances, the cancer is a solid tumor cancer. In some instances, the cancer is advanced or metastatic colorectal cancer. In some instances, the cancer is advanced or metastatic gastric cancer. In some instances, the cancer is advanced or metastatic ovarian cancer. In some instances, the cancer is advanced or metastatic endometrial cancer. In some instances, the subject is human.

[0040]

[0021] In some instances, the cancer is gastroesophageal junction carcinoma. In some instances, the cancer is triple-negative breast cancer (TNBC). In some instances, the cancer is non-small cell lung cancer (NSCLC), In some instances, the cancer is pancreatic ductal adenocarcinoma (PDAC)

[0041]

[0022] In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 2.4 mg / kg to about 11 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 1.2 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 2.4 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 4.8 mg / kg. In certain instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 6.0 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 7.2 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 8.6 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 10.0 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 11.0 mg / kg. In some instances, the conditionally activatable masked immunocon jugate is administered at a dose of about 11 mg / kg to about 20 mg / kg.

[0042]

[0023] In some instances, the conditionally activatablc masked immunoconjugate is administered at a dose of about 12.0 mg / kg.

[0043]

[0024] In some instances, the dose is determined by actual body weight (ABW).

[0044]

[0025] In some instances, the dose is determined by adjusted ideal body weight (AIBW).

[0045]

[0026] In some instances, the administering occurs once every week, once every’ two weeks, once every three weeks or once every four weeks. In some instances, the administering occurs once every three weeks.

[0046]

[0027] In some instances, the administering is via intravenous infusion. In some instances, the administering is via intravenous infusion for about 30 minutes to about 2 hours. In some instances, the administering is via intravenous infusion for about 60 minutes.Atty. Dkt. No.: 45325-0051WO1

[0028] In some instances, the conditionally activatable masked immunoconjugate is formulated as a pharmaceutical composition. In some instances, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0047]

[0029] Also disclosed herein is a method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease m a subject in which diseased cells express epithelial cell adhesion molecule (EpCAM), the method comprising administering to the subject a dose of about 1.0 mg / kg to about 20.0 mg / kg of an conditionally activatable masked immunoconjugate comprising:

[0048] (a) an activatable antibody comprising:

[0049] (iv) an EpCAM antibody or EpCAM-binding fragment thereof (AB), comprising:

[0050] (1) a heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence NYYIH (SEQ ID NO: 13);

[0051] (2) a heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14);

[0052] (3) a heavy chain complementarity determining region 3 (VH-CDR3) comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15);

[0053] (4 ) a light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO:42);

[0054] (5) a light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence QTSNLAS (SEQ ID NO:40); and (6) a light chain complementarity determining region 3 ( VL-CDR3) comprising the amino acid sequence AQNLELPNT (SEQ ID NO:41 );

[0055] (v) a cleavable moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 168); and

[0056] ( vi) a masking moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein the masking moiety inhibits the binding of the antibody or antibod}' fragment to EpCAM when the activatable antibody is in an uncleaved state,

[0057] wherein the activatable antibody in an uncleaved state has a structural arrangement from N-terminus to C-terminus of: (masking moiety)-(cleavable moiety)-(antibody or antibody fragment) or (antibody or antibody fragment)-(cleavable moiety)-(masking moiety); and

[0058] (b) at least one linker-payload depicted by the structure of Formula (X):Atty. Dkt. No.: 45325-0051WO1

[0059]

[0060] Formula (X)

[0061] or a pharmaceutically acceptable salt thereof Formula (X), wherein the at least one linker-payload is covalently bound to the AB

[0062]

[0030] In some instances, the dose is determined by actual body weight (ABW).

[0063]

[0031] In some instances, the dose is determined by adjusted ideal body weight (AIBW).

[0064]

[0032] In some instances, the conditionally activatable masked immunoconjugate has a drug-to- antibody ratio (DAR) of 8. In some instances, the conditionally activatable masked immunoconjugate has a drug -to-anti body ratio (DAR) of 4.

[0065]

[0033] In some instances, the activatable antibody is a full length human IgGl EpCAM antibody having eight cysteines which, when in a native conformation form four interchain disulfide bonds.

[0066]

[0034] In some instances, each linker-payload is individually covalently bound to the activatable antibody via one of the eight cysteines.

[0067]

[0035] In some instances, the EpCAM antibody or EpCAM-binding fragment thereof comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO:54 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0068]

[0036] In some instances, the antigen binding fragment thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, and a dAb.

[0069]

[0037] In some instances, the MM is linked to the CM such that the activatable antibody in an uncleaved state comprises the structural arrangement from N-terminus to C -terminus as follows: MM- CM-AB or AB-CM-MM. In some instances, the activatable antibody further comprises a first linking peptide (LP1) and a second linking peptide (LP2). wherein the activatable antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-LP1 -CM-LP2-AB or AB-LP2-CM-LP 1 -MM. and wherein each of LP 1 and LP2 is a peptide of about 1 to 20 amino acids in length. In some instances, the two linking peptides are not identical to each other.Atty. Dkt. No.: 45325-0051WO1

[0038] In some instances, the activatable antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179. In some instances, the activatable antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179. wherein the linker-payload of Formula (X) is conjugated to an interchain Cys of the AB via a thioether bond, and wherein the conditionally activatable masked immunoconjugate has a drug-to-antibody ratio (DAR) of 8. In some instances, the activatable antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179. wherein the linker-payload of Formula (X) is conjugated to an interchain Cys of the AB via a thioether bond, and wherein the conditionally activatable masked immunoconjugate has a drug-to- antibody ratio (DAR) of 4.

[0070]

[0039] In some instances, the disease is cancer. In some instances, the cancer is a solid tumor cancer. In some instances, the cancer is advanced or metastatic colorectal cancer. In some instances, the cancer is advanced or metastatic gastric cancer. In some instances, the cancer is advanced or metastatic ovarian cancer. In some instances, the cancer is advanced or metastatic endometrial cancer. In some instances, the subject is human.

[0071]

[0040] In some instances, the cancer is gastroesophageal junction carcinoma. In some instances, the cancer is triple-negative breast cancer (TNBC). In some instances, the cancer is non-small cell lung cancer (NSCLC). Tn some instances, the cancer is pancreatic ductal adenocarcinoma (PDAC).

[0072]

[0041] In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 2.4 mg / kg to about 11 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 1.2 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 2.4 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 4.8 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 7.2 mg / kg. In some instances, the conditionally activatable masked immunoconj ugate is administered at a dose of about 8.6 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 10 0 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 11.0 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 11 mg / kg to about 20 mg / kg.

[0073]

[0042] In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 12.0 mg / kg.Atty. Dkt. No.: 45325-0051WO1

[0043] In some instances, the dose is determined by actual body weight (ABW). In some instances, the EpCAM immunoconjugate can be administered with an anti-VEGF agent. In some instances, the anti- vascular endothelial growth factor (anti-VEGF) agent includes bevacizumab. In some instances, bevacizumab is administered at a dose of about 5 mg / kg ABW once every 2 weeks. In some instances, bevacizumab is administered at a dose of about 7.5 mg / kg ABW once every 3 weeks. In some instances, prophylactic agents can be administered in combination with (i) the EpCAM immunoconjugate or (ii) the EpCAM immunoconjugate and a second agent. One possible side effect of treatment can be diarrhea. In some instances, the prophylactic agent is a prophylactic agent against diarrhea (e.g., an agent that mitigates, reduces the incidence of and / or delays tire onset of diarrhea). In some instances, loperamide is provided to the subject as a prophylactic agent against diarrhea. In some instances, budesonide is provided to the subject as a prophylactic agent against diarrhea. Budesonide can be administered orally, rectally, nasally, or via inhalation.

[0074]

[0044] In some instances, the dose is determined by adjusted ideal body weight (AIBW).

[0075]

[0045] In some instances, if ABW of the subject is less than AIBW. then the dose administered is based on the ABW of the subject. In some instances, if the height of the subject is less than 60 inches, then the AIBW is used and is calculated as follows:

[0076] AIBW’:::{IBW + [0.4 x (ABW-IBW)]} - (5 pounds / inch)(60 inches - the subject’s height in inches).

[0077]

[0046] In some instances, the weight of the subject is greater than 100 kg, and the dose for the subject is based on an ABW that is fixed at 100 kg.

[0078]

[0047] In some instances, the EpCAM immunoconjugate can be administered with an anti-VEGF agent. In some instances, the anti-vascular endothelial growth factor (anti-VEGF) agent includes bevacizumab. In some instances, bevacizumab is administered at a dose of about 5 mg / kg ABW once every 2 weeks. In some instances, bevacizumab is administered at a dose of about 7.5 mg / kg ABW once every 3 weeks In some instances, prophylactic agents can be administered in combination with (i) the EpCAM immunoconjugate or (ii) the EpCAM immunoconjugate and a second agent. One possible side effect of treatment can be diarrhea. In some instances, the prophylactic agent is a prophylactic agent against diarrhea (e g., an agent that mitigates, reduces the incidence of and / or delays the onset of diarrhea). In some instances, loperamide is provided to the subject as a prophylactic agent against diarrhea. In some instances, budesonide is provided to the subject as a prophylactic agent against diarrhea. Budesonide can be administered orally, rectally, nasally, or via inhalation.

[0079]

[0048] In some instances, the administering occurs once every’ week, once every' two weeks, once every three weeks or once every' four weeks. In some instances, the administering occurs once every' three weeks.Atty. Dkt. No.: 45325-0051WO1

[0049] In some instances, the administering occurs once every four weeks.

[0080]

[0050] In some instances, the administering is via intravenous infusion. In some instances, the administering is via intravenous infusion for about 30 minutes to about 2 hours. In some instances, the administering is via intravenous infusion for about 60 minutes.

[0081]

[0051] In some instances, the methods also include administering one or more additional agents. In some instances, the one or more additional agents comprise an anti-VEGF agent, an anti-epidermal growth factor receptor agent, an immune checkpoint inhibitor, a v-Raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor, a mitogen-activated protein kinase / ERK Kinase (MEK) inhibitor, a human epidermal growth factor receptor 2 (HER2) inhibitor, or a Kirsten rat sarcoma viral oncogene homolog (KRAS) inhibitor. In some instances, the one or more additional agents comprise an anti- VEGF agent, an anti-epidermal growth factor receptor agent, an immune checkpoint inhibitor, a v-Raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor, a mitogen-activated protein kinase / ERK Kinase (MEK) inhibitor, a human epidermal growth factor receptor 2 (HER2) inhibitor, or a Kirsten rat sarcoma viral oncogene homolog (KRAS) inhibitor. In some instances, the anti-VEGF agent is bevacizumab, wherein the bevacizumab is administered at a dose of about 5 mg / kg ABW once every two weeks or wherein the bevacizumab is administered at a dose of about 7.5 mg / kg ABW once every three weeks

[0082]

[0052] In some instances, the conditionally activatable masked immunoconjugate is formulated as a pharmaceutical composition In some instances, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0083]

[0053] Also disclosed herein is a method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease in a subject in which diseased cells express EpCAM, the method comprising administering to the subject:

[0084] (I) a dose of about 1.0 mg / kg to about 20 mg / kg ABW or AIB W of a conditionally activatable masked immunoconjugate comprising:

[0085] (a) an activatable antibody comprising:

[0086] (i) an EpCAM antibody or EpCAM -binding fragment thereof (AB), comprising:

[0087] ( 1 ) a VH-CDR 1 comprising the amino acid sequence NYYTH (SEQ ID NO: 13):

[0088] (2) a VH-CDR2 comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14),

[0089] (3) a VH-CDR3 comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15):Atty. Dkt. No.: 45325-0051WO1

[0090] (4) a VL-CDR 1 comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO:42);

[0091] (5) a VL-CDR2 comprising the amino acid sequence QTSNLAS (SEQ ID NO:40); and

[0092] (6) a VL-CDR3 comprising the amino acid sequence AQNLELPNT (SEQ ID NO:41 ):

[0093] (ii) a cleavable moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 168); and

[0094] (iii) a masking moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises tire amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein the masking moiety inhibits the binding of the antibody or antibody fragment to EpCAM when the activatable antibody is in an uncleaved state,

[0095] wherein the activatable antibody in an uncleaved state has a structural arrangement from N- terminus to C -terminus of: (masking moiety)-(cleavable moiety)-(antibody or antibody fragment) or (antibody or antibody fragment)-(cleavable moiety) -(masking moiety): and (b) at least one linker-payload depicted by the structure of Formula (X):

[0096]

[0097] Formula (X)

[0098] or a pharmaceutically acceptable salt thereof (Formula (X)), wherein the at least one linker-payload is covalently bound to the AB; and

[0099] (II) bevacizumab.Atty. Dkt. No.: 45325-0051WO1

[0100]

[0054] Also disclosed herein is a method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease in a subject in which diseased cells express epithelial cell adhesion molecule (EpCAM), the method comprising administering to the subject

[0101] (1) a dose of about 1.0 mg / kg to about 20 mg / kg ABW or AIB W of a conditionally activatable masked immunoconjugate comprising:

[0102] (a) an activatable antibody comprising:

[0103] (i) an EpCAM antibody or EpCAM -binding fragment thereof (AB), comprising:

[0104] (1) a VH-CDR1 comprising the amino acid sequence NYYIH (SEQ ID NO: 13);

[0105] (2) a VH-CDR2 comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14);

[0106] (3) a VH-CDR3 comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15);

[0107] (4) a VL-CDR1 comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO:42);

[0108] (5) a VL-CDR2 comprising the amino acid sequence QTSNLAS (SEQ ID NO:40); and

[0109] (6) a VL-CDR3 comprising the amino acid sequence AQNLELPNT (SEQ ID NO:41);

[0110] ( ii) a cleavable moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169); and

[0111] (iii) a masking moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155). wherein the masking moiety inhibits the binding of the antibody or an tibody fragment to EpCAM when the activatable antibody is in an uncleaved state,

[0112] wherein the activatable antibody in an uncleaved state has a structural arrangement from N- terminus to C -terminus of: (masking moiety)-(cleavable moiety)-(antibody or antibody fragment) or (antibody' or antibody fragment)-(cleavable moiety)-(masking moiety ); and (b) at least one linker-payload depicted by the structure of Formula (X):Atty. Dkt. No.: 45325-0051WO1

[0113]

[0114] Formula (X)

[0115] or a pharmaceutically acceptable salt thereof (Formula (X)), wherein the at least one linkerpayload is covalently bound to the AB; and

[0116] (II) bevacizumab.

[0117]

[0055] In some instances, the conditionally activatable masked immunoconjugate has a drug-to- antibody ratio (DAR) of 8.

[0118]

[0056] In some instances, the conditionally activatable masked immunoconjugate has a drug-to- antibody ratio (DAR) of 4.

[0119]

[0057] In some instances, the EpCAM antibody or EpCAM-binding fragment thereof comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO:54 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0120]

[0058] In some instances, the activatable antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179.

[0121]

[0059] In some instances, the activatable antibody comprises a heavy chain comprising the ammo acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179, wherein the linker-payload of Formula (X) is conjugated to an interchain Cys of the AB via a thioether bond, and wherein the conditionally activatable masked immunoconjugate has a drug-to-antibody ratio (DAR) of 8.

[0122]

[0060] In some instances, the activatable antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179, wherein the linker-payload of Formula (X) is conjugated to an interchain Cys of the AB via a thiocthcrAtty. Dkt. No.: 45325-0051WO1 bond, and wherein the conditionally activatable masked immunoconjugate has a drug-to-antibody ratio (D AR) of 4.

[0123]

[0061] In some instances, the disease is cancer. In some instances, the cancer is a solid tumor cancer. In some instances, the cancer is advanced or metastatic colorectal cancer. In some instances, the cancer is advanced or metastatic gastric cancer. In some instances, the cancer is gastroesophageal junction carcinoma, triple-negative breast cancer, non-small cell lung cancer, or pancreatic ductal adenocarcinoma. In some instances, the cancer is advanced or metastatic ovarian cancer. In some instances, the cancer is advanced or metastatic endometrial cancer. In some instances, the subject is human.

[0124]

[0062] In some instances, the dose is based on actual body weight (ABW) of the subject.

[0125]

[0063] In some instances, the dose is based on adjusted ideal body weight (AIBW) of the subject In some instances, if ABW of the subject is less than AIBW, then the dose administered is based on the ABW of the subject. In some instances, if the height of the subject is less than 60 inches, then the AIBW is used and is calculated as follows: AIBW" = {IBW + [0.4 x (ABW-IBW)]} - (5 pounds / inch)(60 inches - the subject’s height m inches).

[0126]

[0064] In some instances, the weight of the subject is greater than 100 kg, and wherein the dose for the subject is based on an ABW that is fixed at 100 kg.

[0127]

[0065] In some instances, the subject is administered the conditionally activatable masked immunoconjugate once every week. In some instances, the subject is administered the conditionally activatable masked immunoconjugate once every 2 weeks. In some instances, the subject is administered the conditionally activatable masked immunoconjugate once every 3 weeks. In some instances, the subject is administered the conditionally activatable masked immunoconjugate once every 4 weeks.

[0128]

[0066] In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 1.2 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 2.4 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 4.8 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is admini stered at a dose of about 6.0 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 7.2 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 8.6 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 10.0 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 11.0 mg / kg. In some instances, the conditionally activatable masked immunoconjugate is administered at a dose of about 12 mg / kg.Atty. Dkt. No.: 45325-0051WO1

[0067] In some instances, the conditionally activatable masked immunoconjugate is administered via intravenous infusion. In some instances, the conditionally activatable masked immunoconjugate is administered via intravenous infusion for about 30 minutes to about 2 hours. In some instances, the conditionally activatable masked immunoconjugate is administered via intravenous infusion for about 60 minutes.

[0129]

[0068] In some instances, the conditionally activatable masked immunoconjugate is formulated as a pharmaceutical composition. In some instances, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0130]

[0069] In some instances, the subject is administered the bevacizumab once every 2 weeks. In some instances, the subject is administered the bevacizumab once every 3 weeks. In some instances, the subject is administered the bevacizumab once every 4 weeks.

[0131]

[0070] In some instances, the bevacizumab is administered at a dose of about 5 mg / kg. In some instances, the bevacizumab is administered at a dose of about 7.5 mg / kg.

[0132]

[0071] In some instances, the bevacizumab is administered via intravenous infusion.

[0133]

[0072] In some instances, any of the methods also include administering a prophylactic agent to the subject. In some instances, the prophylactic agent is an anti-diarrheal agent. In some instances, the anti- diarrheal agent is loperamide. In some instances, the loperamide is administered orally. In some instances, the loperamide is administered at a dose of about 1 mg to about 15 mg. In some instances, the loperamide is administered at a dose of about 4 mg to about 12 mg. In some instances, the loperamide is administered at a dose of about 4 mg. In some instances, the loperamide is administered one time daily. In some instances, the loperamide is administered two times daily. In some instances, the loperamide is administered three times daily. In some instances, the loperamide is administered for seven days.

[0134]

[0073] In some instances, the anti-diarrheal agent is budesonide. In some instances, tire budesonide is administered orally, rectally, nasally, or via inhalation. In some instances, the budesonide is administered at a dose of about 1 mg to about 15 mg. In some instances, the budesonide is administered at a dose of about 4 mg to about 12 mg. In some instances, the budesonide is administered at a dose of about 9 mg. In some instances, the budesonide is administered one time daily. In some instances, the budesonide is administered two times daily. In some instances, the budesonide is administered three times daily. In some instances, the budesonide is administered for seven days.

[0135] BRIEF DESCRIPTION OF THE FIGURES

[0136]

[0074] FIG. 1 shows the camptothecin derivative linker-payload of use in accordance with certain embodiments of the disclosure.Atty. Dkt. No.: 45325-0051WO1

[0075] FIG. 2 shows the conjugation of a camptothecin-derived (CPT66) linker-payload to a conditionally activatable EpCAM antibody in accordance with certain embodiments of the disclosure

[0076] FIG. 3 shows a cartoon depicting conditionally activatable masked EpCAM antibody immunoconjugate in accordance with certain embodiments of the disclosure. The depicted embodiment comprises an EpCAM antibody, a cleavable substrate linker, and a mask. In an uncleaved (inactive) state, the mask inhibits the binding of the EpCAM antibody to EpCAM. The cleavable substrate linker is cleavable by a protease. Upon cleavage, the mask is released and the antibody is free to bind to EpCAM. The depicted embodiment includes 8 conjugated camptothecin-derived (CPT66) linker¬ payloads (shown as ovals in cartoon). The conjugation of the linker-payloads to the activatable EpCAM antibody is stochastic, with conjugation occurring at the antibody's inter-chain cysteines.

[0137]

[0077] FIG. 4 shows a treatment overview of the disclosure.

[0138]

[0078] FIG. 5 shows H& E staining (left image) and an IHC image (right images) of EpCAM expression. Scale of left and middle image: 250 pm.

[0139]

[0079] FIG. 6 shows baseline characteristics for subjects in phase 1 cohort.1Patients treated with at least one EpCAM-CPT66 dose between 2.4 mg / kg and 10 mg / kg; MSS = microsatellite stable.

[0140]

[0080] FIG. 7 shows anti-tumor activity at doses selected for expansion. There was a confirmed overall response rate (ORR): 28% (5 / 18) overall, with 43% (3 / 7) at 10 mg / kg.1Maximum immunohistochemistry (IHC) H-Score is 300; H-score captures the proportion of EpCAM+ cells in the biopsy and intensity of EpCAM expression.

[0141]

[0081] FIG. 8 shows anti-tumor activity in subjects treated with 7.2 mg / kg to 10 mg / kg. 94% (17 / 18) disease control with continuing tumor reduction overtime. PD = progressive disease; PR = partial response; SD stable disease.

[0142]

[0082] FIG. 9 shows months of treatment since initial therapy in subjects treated with 7.2 mg / kg, 8.6 mg / kg, and 10 mg / kg. The preliminary median progression free survival 5.8 months. ID = investigator decision; SW = subject withdrawal.

[0143]

[0083] FIG. 10 shows scans of a 46-year old subject (further described in Example 4) with metastatic colorectal cancer. Left images: baseline; right images: 6 weeks after therapy.

[0144]

[0084] FIG. 11 shows the most frequent treatment-related adverse events (TRAE).

[0145]

[0085] FIG. 12 shows pharmacokinetic data plotted as concentration as a function of time since last dose of EpCAM-CPT66 (the EpCAM conditionally activatable antibody immunoconjugate of Example 1). Intact: reflects only masked ADC; Total: reflects masked + unmasked forms of ADC; Free CAMP59 refers to the free camptothecin-derived pay load.Atty. Dkt. No.: 45325-0051WO1

[0146] DETAI LED DESCRIPTION

[0147]

[0086] Hie disclosure generally relates to antibodies and antibody fragments that specifically bind human EpCAM, EpCAM activatable antibodies, and immunoconjugates thereof, as well as methods of using the antibodies, antibody fragments, activatable antibodies, and immunoconjugates, for the treatment of diseases such as cancer.

[0148]

[0087] Antibody drug conjugates (ADCs) are monoclonal antibodies (mAbs) that have been conjugated covalently to cytotoxic small molecules through a linker, allowing for tissue-specific delivery of the conjugate payload driven by the target antigen expressed on the surface of tumor cells. ADCs have transformed the field of clinical oncology in a very short span of time because they offer the potential of targeted drug delivery with minimal off-tumor toxicity. To date. ADCs have generated a robust commercial pipeline of 140 agents currently in clinical trials, including 11 that are in a late stage of clinical development, 3 of which are likely to submit a marketing application in 2023-2024. This adds to the 13 ADCs that are presently approved for marketing worldw ide, including 6 targeting hematological malignancies and 7 for solid tumors (Dumontet et al. 2023). The 3 components of an ADC (targeted antibody, linker, and cytotoxic payload) all play vital roles towards determining the stability, efficacy, and toxicity profile of the drug (Samantasinghar et al., 2023). and they need to be selected carefully with the right combination for the appropriate tumor indications.

[0149]

[0088] EpCAM antibodies and immunoconjugates are described in WO 2024 / 015830, which is incorporated by reference in its entirety,

[0150]

[0089] Judicious target antigen selection for the monoclonal antibody (mAb) component is particularly important to maximize tumor selectivity and potency while minimizing on-target toxicities. Ideal targets are those that are preferentially expressed with sufficient density and homogeneity on the extracellular membrane of tumor cells, with minimal expression on normal cells to optimize the therapeutic index, and with efficient antibody -dependent internalization and lysosomal trafficking (Esapa et al., 2023). However, most targets are expressed to some extent on normal cells and therefore require careful balance of safety and efficacy. Epithelial cell adhesion molecule (EpCAM) represents an example of such a target, being a cell surface glycoprotein that is overexpressed in most epithelial cancers, but also expressed at the basolateral cell membrane of proliferating epithelia in most adult tissue with the highest expression in normal colon (Schnell et al., 2013; Eyvazi et al., 2018). EpCAM is a multifunctional transmembrane protein involved in the regulation of cell adhesion, proliferation, migration, sternness, and epithelial-to- mesenchymal transition of carcinoma cells (Schnell et al., 2013). EpCAM has been shown to have homogeneous high expression by immunohistochemistry in a high percentage of tumor samples across a majority of epithelial -derived carcinoma cancer types w i th a high degree of correlation between primary tumors and both local and distant metastases (Spizzo et al., 2011). High EpCAMAtty. Dkt. No.: 45325-0051WO1 expression is associated with poor prognosis in multiple indications, including breast, colorectal, and ovarian cancer (Gires et al., 2020).

[0151]

[0090] Targeting of EpCAM through multiple therapeutic modalities (including mAbs, bispecific T-cell engaging antibodies, and ADCs) has been shown to provide clinical benefit in early studies (Liu and Wang 2022). However, expression of EpCAM across multiple normal epithelial tissues, to date, has limited the effective systemic use of these EpCAM-targeted therapeutics.

[0152]

[0091] Conditionally activatable masked immunoconjugate therapeutics are fully recombinant prodrugs conditionally activated by protease activity upregulated in the tumor microenvironment (TME). Conditionally activatable masked immunoconjugate take advantage of a fundamental difference in protease biology between tumors and normal tissues. Upregulated extracellular protease activity is abundant in the TME and is a hallmark of most human cancers, maintaining key elements of the transformed phenotype, including growth, invasion, and metastasis (Kessenbrock et al., 2010; Sevenich and Joyce, 2014). Conversely, protease activity in normal tissues is under tight control by redundant mechanisms to avoid unregulated protease activity harmful to normal cellular homeostasis. As a result of the differential between protease activity in normal versus tumor cellular environments, conditionally activatable masked immunoconjugate therapeutics have the potential to minimize off- tumor toxicities while maintaining efficacy against tumor cells.

[0153]

[0092] There is a high unmet need for safe and effective treatments of tumors having high EpCAM expression. For example, in 2023, it is estimated that there were approximately 153,000 new cases of CRC in the United States and approximately 52,500 deaths. Overall, CRC has a 5-year relative survival of 65%, but patients with metastatic disease have a 5-year relative overall survival of only 15% (SEER, Cancer Stat Facts - Colorectal). Other EpCAM expressing tumors, including gastric, ovarian and endometrial cancers, similarly have relatively poor 5-year survival outcomes (6-32%) for patients with metastatic disease (SEER, Cancer Stat Facts - General).

[0154]

[0093] Current therapies for many solid tumors are systemically administered chemotherapy in combination or sequentially with monoclonal antibodies, antibody drug conjugates, small molecule pathway inhibitors and / or immune checkpoint inhibitors. For example, for the treatment of CRC, current guidelines from the American Society of Clinical Oncology (Moms et al., 2023) recommend doublet chemotherapy (folinic acid, fluorouracil [FIJ], and oxaliplatin [FOLFOX]: or folinic acid, FU, and irinotecan [FOLF1RI]), triplet chemotherapy (folinic acid, FU, oxaliplatin, and irinotecan [FOLFOX1RI]), or pembrolizumab (if tumor is microsatellite instability -high or deficient mismatch repair) for patients with previously untreated, initially unresectable metastatic disease. Addition of anti- epidermal growth factor receptor or anti-vascular endothelial growth factor antibody is suggested for some patients. For certain patients who have progressed after previous therapy, encorafenib plusAtty. Dkt. No.: 45325-0051WO1 cetuximab is recommended. Similar recommendations are provided for other EpCAM- expressing indications.

[0155]

[0094] Since current standard therapies do not confer prolonged survival, the treatment of metastatic disease continues to be an area of unmet medical need. Thus, investigation of new therapeutic agents is warranted.

[0156]

[0095] To address these unmet needs, the disclosure provides immunoconjugates having antibodies and antigen-binding antibody fragments that specifically bind human EpCAM, including conditionally activatable masked antibodies (activatable forms of the EpCAM antibodies or EpCAM -binding antibody fragments thereof). In some instances, the conditionally activatable masked immunoconjugates comprise a camptothecin derivative linker-payload with the following structure (where the following structure represents the linker-payload prior to conjugation to the antibody, i.e. the linker-payload reactant), shown as Formula (I) below:

[0157]

[0158] Thus, in some instances, the immunoconjugates comprise a camptothecin derivative linker-payload with the following structure (wherein tire ** denotes the covalent attachment position to the EpCAM antibody or conditionally activatable masked antibody), shown as Formula (X) below:Atty. Dkt. No.: 45325-0051WO1

[0159] OH

[0160]

[0161] Formula (X).

[0162]

[0096] EpCAM is known to be associated with cell-cell adhesion in epithelia and to be involved in cell signaling, differentiation, proliferation, and migration. The overexpression of EpCAM has been implicated in the pathogenesis of diseases and disorders, such as cancer. For example, EpCAM is highly expressed in a variety of cancer types such as, for example, breast cancer, lung cancer, liver cancer, stomach cancer, head & neck cancer, prostate cancer, pancreatic cancer, ovarian cancer, and colon cancer, and most cancers (and metastases) of epithelial origin. EpCAM is also highly expressed in tumor initiating / cancer stem cells, The provided EpCAM immunoconjugates have uses that include treating such diseases and cancers

[0163]

[0097] In some instances, the cancer is gastroesophageal junction carcinoma. In some instances, the cancer is triple-negative breast cancer (TNBC). In some instances, the cancer is non-small cell lung cancer (NSCLC). In some instances, the cancer is pancreatic ductal adenocarcinoma (PDAC).

[0164] DEFINITIONS

[0165]

[0098] To facilitate an understanding, a number of terms and phrases are defined below.

[0166]

[0099] Tire terms “epithelial cell adhesion molecule” or " EpCAM", as used herein, refers to any native human EpCAM unless otherwise indicated. The term also encompasses naturally occurring variants of EpCAM, e.g., splice variants, allelic variants and isoforms. EpCAM polypeptides can be isolated from a variety of sources, such as from human or cynomolgus tissue or other biological samples, or prepared by known recombinant or synthetic methods. EpCAM is also known as CD326. 17-1A antigen, HEA125, MK-1, EGP-2. EGP314. EGP40, GA733-2, KSA, TACSTD1, TROP1, KS1 / 4, M4S1, DIAR5, MIC 18, HNPCC8, and ESA. Examples of EpCAM sequences include, but are not limited to NCBI reference number NP_002345.2 (amino acid residues 24-314 correspond to mature EpCAM, amino acids 24-265 correspond to the extracellular region of mature EpCAM (SEQ ID NO: 1)). TheAtty. Dkt. No.: 45325-0051WO1 extracellular region of mature EpCAM can further be divided into three domains: DI (amino acids 1-36 of SEQ ID NO: 1 (SEQ ID NO:2)), D2 (amino acids 43-112 of SEQ ID NO: 1 (SEQ ID NO:3)), and D3 (amino acids 113-243 of SEQ ID NO:1 (SEQ ID NO:4)).

[0167]

[0100] The terms “antibody" and “antigen-binding antibody fragment” and the like, as used herein, include any protein- or peptide -containing molecule that comprises at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one complementarity determining region (CDR) of a heavy or light chain or an antigen binding portion thereof. Such antibody optionally further affects at least one EpCAM activity, such as, but not limited to, where such antibody modulates, decreases, increases, antagonizes, agonizes, partially agonizes, partially antagonizes, mitigates, alleviates, blocks, inhibits, abrogates and / or interferes with at least one EpCAM activity or binding in vitro, in situ, in vivo and / or ex vivo. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof affects at least one EpCAM-mediated activity or function selected from: ligand binding, receptor signaling, membrane association, cell migration, cell proliferation, receptor binding activity,, RNA, DNA or protein production and / or synthesis.

[0168]

[0101] Antibodies are heterotetrameric glycoproteins, composed of two identical light chains (LC) and two identical heavy chains (HC). Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has spaced intrachain disulfide bridges. Each heavy chain has at one end a vanable region (VH) followed by a number of constant domains. Each light chain has a variable region at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable region is aligned with the variable region of the heavy chain. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4.

[0169]

[0102] The term "antibody" also includes fragments, specified portions and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and / or function of an antibody or specified fragment or portion thereof, including single chain antibodies and antigen (e.g., EpCAM)-binding antibody fragments. Functional fragments include antigen-binding fragments that bind to a mammalian antigen, such as EpCAM, alone or in combination with other antigens. For example, antibody fragments capable of binding to antigen or portions thereof, including, but not limited to. Fab (e.g., by papain digestion), Fab' (e.g.. by pepsin digestion and partial reduction) and F(ab')2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasminAtty. Dkt. No.: 45325-0051WO1 digestion). Fd (e.g.. by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the disclosure (see, e.g., Colligan, Immunology).

[0170]

[0103] Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and / or as disclosed herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combination gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CHI domain and / or hinge region of the heavy chain. The various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.

[0171]

[0104] The term "antibody fragment" refers to a portion of an intact antibody, generally the antigen binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')2, single chain (scFv) and Fv fragments, diabodies; linear antibodies; singlechain antibody molecules; single Fab arm “one arm" antibodies and multispecific antibodies formed from antibody fragments (each having a VH and VL), among others.

[0172]

[0105] Antibody fragments include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to, at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, or at least one portion of an antigen or antigen receptor or binding protein, which can be incorporated into an EpCAM antibody provided herein.

[0173]

[0106] Term “full length antibody" refers to an antibody in its substantially intact form, and not antibody fragments as defined above, Tire term particularly refers to an antibody with heavy chains that contain a hinge region and an Fc region, including a modified Fc region (e.g., a mutated and / or clipped hinge region).

[0174]

[0107] The term "variable" refers to the fact that certain portions of the variable regions of antibodies differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen However, the vanability is not evenly distributed throughout tire variable regions of antibodies. The variability is concentrated in three segments called complementarity -determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable regions. The more highly conserved portions of variable regions are called the framework (FR). The vanable regions of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. Hie CDRs in each chain are held togetherAtty. Dkt. No.: 45325-0051WO1 in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. Tire constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability ( / .<?.. Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed.. 1991, National Institutes of Health. Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., J Make. Biol. 273:927-948 (1997)). In addition, combinations of these two approaches are sometimes used in tire art to determine CDRs.

[0175]

[0108] The Kabat numbering system is generally used when referring to a residue in the variable region (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g.. Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service. National Institutes of Health, Bethesda, Md. (1991)).

[0176]

[0109] The amino acid position numbering as in Kabat, refers to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al.. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Insti tutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable region. For example, a heavy’ chain variable region can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody’ with a "standard" Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia et al., J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because tire Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35 A is present, the loop ends at 33; if both 35 A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.

[0177]

[0110] Hie terms " EpCAM antibody", " EpCAM antibody”, "antibody that specifically binds to EpCAM", "‘EpCAM -binding antibody fragments thereof’, and “antibody fragment that specifically binds EpCAM" refer to an antibody that is capable of binding EpCAM with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting EpCAM. The extent of binding of an EpCAMAtty. Dkt. No.: 45325-0051WO1 antibody to an unrelated, non-EpCAM protein is less than about 10% of the binding of the antibody to EpCAM as measured, e.g., by a radioimmunoassay (RIA).

[0178]

[0111] The amino acid position numbering as in Rabat, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al.. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g.. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a ''standard" Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), wherein the loops are also identified as light and heavy chain CDRs (e.g., LI = LC CDR1, etc.). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35 A 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 3.5B are present, the loop ends at 34). The AbM hypcrvariablc regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.

[0179] Table 1. Amino Acid Position Numbering Systems

[0180] Loop Kabat AbM Chothia

[0181] L24-I.34 1,24-134 1,24-134

[0182] L50-L56 L50-L56 L50-L56

[0183] L89-L97 L89-L97 L89-L97

[0184] H31-H35B H26-H35B H26- -1132.34

[0185] (Kabat Numbering)

[0186] H26-H35 H25-H32

[0187] >: Chothia Numbering)

[0188] H2 H50-H65

[0189] H3 H95-H102

[0190]

[0191]

[0112] The term "epitope" refers to a protein determinant capable of specific binding to an antibody.

[0192] Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein.Atty. Dkt. No.: 45325-0051WO1 Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

[0193]

[0113] " Blocking" antibody is one which inhibits or reduces the biological activity of the antigen it binds such as EpCAM. Particular blocking antibodies substantially or completely inhibit the biological activity of the antigen. Desirably, the biological activity is reduced by 10%. 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%. In one embodiment, the blocking antibody reduces the EpCAM associated tyrosine kinase activity 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

[0194]

[0114] An "isolated” antibody is one separated and / or recovered from its natural environment.

[0195] Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In particular aspects, the antibody is purified (1) to greater than 95% by¬ weight of antibody as determined by, for example, the Lowry method, and in some instances more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N -terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) under reducing or non-reducing conditions using Coomassie blue or, in some instances, silver stain. Isolated antibody includes the EpCAM antibody in situ within recombinant cells since at least one component of the antibody's natural environment is not present. Ordinarily, however, an isolated antibody is prepared by at least one purification step.

[0196]

[0115] A "human antibody” refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes mtact or full-length antibodies, activatable antibodies, antigen (e.g., human and / or cynomolgus EpCAM)-binding antibody fragments, and / or antibodies comprising at least one human heavy and / or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.

[0197]

[0116] The term "chimeric antibodies," as used herein, refer to antibodies wherein the sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.

[0198]

[0117] The term "humanized antibody," as used herein, refers to forms of non-buman (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or antigen-binding antibody fragments that contain minimal non-human (e.g., murine) sequences. Typically, humanizedAtty. Dkt. No.: 45325-0051WO1 antibodies are human immunoglobulins in which residues from the complementarity determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (Jones et al.. Nature 321:522-525 (1986); Riechmann et al.. Nature 332:323-327 (1988): Verhoeyen et al., Science 239: 1534-1536 (1988)). In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and / or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and / or capability. In general, the antibody comprises substantially all of at least one, and typically two or three, variable regions containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U. S. Pat. No, 5,225,539.

[0199]

[0118] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell -mediated cytotoxicity (ADCC); and antibody -dependent cell-mediated phagocytosis (ADCP).

[0200]

[0119] "Human effector cells" are leukocytes which express one or more FcRs and perform effector functions. In certain aspects, the cells express at least FcyRIII and perform ADCC or ADCP effector function(s). Examples of human leukocytes which mediate ADCC or ADCP include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils. Tire effector cells can be isolated from a native source, e.g., from blood.

[0201]

[0120] The term “Fc region” as used herein includes the polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG. and the last three constant region immunoglobulin domains of TgE and IgM, and the flexible hinge N -terminal to these domains. For IgA and IgM, Fc can include the J chain. For IgG, Fc comprises immunoglobulin domains CH2 and CH3 and the hinge between CHI and CH2. Although the boundaries of the Fc region can vary-, the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxylterminus, wherein the numbering is according to the EU index as in Kabat et al.. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). The " EU index as set forth in Kabat" refers to the residue numbering of the human IgGl EU antibody as described in Kabat etAtty. Dkt. No.: 45325-0051WO1 al., supra. Fc can refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein An Fc variant protein can be an antibody, Fc fusion, or any protein or protein domain that comprises an Fc region. Particular proteins comprise variant Fc regions, which are non-naturally occurring variants of an Fc. Polymorphisms have been observed at a number of Fc positions, including, but not limited to, Kabat 270. 272, 312, 315, 356, and 358, and thus slight differences between the presented sequence and sequences in the prior art can exist and would be understood by one skilled in the art based on the present teachings.

[0202]

[0121] Hie term “conjugate”, “immunoconjugate”, “ADC”, or “AADC” as used herein, refers to a compound or a derivative thereof that is linked to a cell binding agent (i.e.. an EpCAM antibody, EpCAM-binding antibody fragments thereof, or EpCAM conditionally activatable masked immunoconjugate) and is defined by a generic formula: C-L-A, wherein C= compound. L= linker, and A= EpCAM-binding agent (EpBA) (e.g.. an EpCAM antibody. EpCAM-binding antibody fragments thereof, or EpCAM conditionally activatable masked immunoconjugate, as disclosed herein). In some embodiments, the generic formula: D-L-A, wherein D=drug, L=linker and A=cell binding agent

[0203]

[0204] an EpCAM antibody, EpCAM-binding antibody fragments thereof, or EpCAM conditionally activatable masked immunoconjugate), can also be used in the same manner. In some instances, the immunoconjugate described herein comprises a camptothecin derivative linker-payload.

[0205]

[0122] A “linker” is any chemical moiety that is capable of linking a compound, usually a drug, such as a maytansinoid, a camptothecin derivative, or an indolinobenzodi azepine compound, to a cell-binding agent such as an anti EpCAM antibody or an EpCAM-binding antibody fragments thereof. Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Linkers also include peptide linkers and charged linkers, and hydrophilic forms thereof, as disclosed herein and know' in the art.

[0206]

[0123] An “interchain cysteine” refers to one of about 8 cysteine ammo acids in the AB, which when the AB is in a native (unconjugated) conformation, otherwise form four interchain disulfide bondsAn AB can have 1, 2, 3, 4, 5, 6, 7, 8, or more interchain cysteine. These interchain cysteines are located in the internal peptide sequence of the AB.. The linker-payload of tlie present disclosure may be conjugated to one or more interchain cysteines of the AB.

[0207]

[0124] " Aberrant cell proliferation”, as used herein, unless otherwise indicated, refers to cell growth that is independent of normal regulatory' mechanisms (e.g., loss of contact inhibition). This includes, for example, the abnormal growth of: (1) tumor cells (tumors) that proliferate by expressing a mutatedAtty. Dkt. No.: 45325-0051WO1 tyrosine kinase or over expression of a receptor tyrosine kinase; (2) benign and malignant cells of other proliferative diseases in which aberrant tyrosine kinase activation occurs; (3) any tumors that proliferate by receptor tyrosine kinases; (4) any tumors that proliferate by aberrant serine / threonine kinase activation; (5) benign and malignant cells of other proliferative diseases in which aberrant serine / threonine kinase activation occurs, and (6) benign and malignant cells of other proliferative diseases.

[0208]

[0125] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated ceil growth. A "tumor" comprises one or more cancerous cells. The term “cancer” or “cancerous” as defined herein, includes “pre -cancerous” conditions that, if not treated, can evolve into a cancerous condition In some embodiments, the cancer is an epithelial cancer. In some embodiments, the cancer expresses EpCAM. Examples of cancers include, breast cancer, lung cancer, non-small cell lung cancer, stomach cancer, colorectal cancer, prostate cancer, bladder cancer, ovarian cancer, colon cancer, rectal cancer, a cancer comprising a cancer stem cell, uterine cancer, gastric cancer, head and neck cancer, endometrial cancer, and pancreatic cancer,

[0209]

[0126] Other exemplary cancers include gastroesophageal junction (GEJ) carcinoma, triple-negative breast cancer (TNBC), non-small cell lung cancer (NSCLC). or pancreatic ductal adenocarcinoma (PDAC).

[0210]

[0127] The terms "cancer cell," "tumor cell," and grammatical equivalents refer to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells).

[0211]

[0128] As used herein, the term “cytotoxic agent” refers to a substance that inhibits or prevents one or more cellular functions and / or causes cell death

[0212]

[0129] As used herein, “treatment” refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include pre venting occurrence or recurrence of disease, alleviation of sy mptoms, dimmishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, methods and compositions provided herein are useful in attempts to delay development of a disease or disorder.

[0213]

[0130] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both 1) therapeutic measures that cure, slow down, Jessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent and / or slow the development of a targeted pathologic condition or disorder. Thus, those in need ofAtty. Dkt. No.: 45325-0051WO1 treatment include those already with the disorder; those prone to have the disorder: and those in whom the disorder is to be prevented. In certain embodiments, a subject is successfully "treated" for cancer according to the methods provided herein if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction m the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity, tumorigenic frequency, or tumorigcnic capacity, of a tumor; reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non-tumorigenic state: or some combination of effects,

[0214]

[0131] As used herein, the term “ABW” refers to actual body weight. The term “mg / kg (ABW)” refers herein to a weight-based dosage of therapeutic agent in which the quantity of therapeutic agent (in milligrams) is calculated on the basis of a subject’s actual body w eight in kilograms.

[0215]

[0132] As used herein, the term “AIBW” refers to adjusted ideal body weight. The tenn "adjusted ideal body weight" (AIBW) refers to a size descriptor that accounts for sex. total body weight. and height. AIBW is calculated as follows:

[0216] Step 1: Calculate the Ideal Body weight (IBW)

[0217] Men: IBW (kg) = 50 + [2.3 kg x (height in inches - 60)]

[0218] Women: IBW (kg) = 45,5 kg + [2.3 x (height in inches - 60)] Step 2: Calculate the Adjusted Ideal Body Weight (AIBW) using the same formula for men and women:

[0219] AIBW = IBW + [0.4 x (ABW - IBW)], where ABW = Actual Body Weight (kg). The tenn “mg / kg (AIBW)” refers herein to a weight-based dosage of therapeutic agent in which the quantity of therapeutic agent (in milligrams) is calculated on the basis of a subject’s adjusted ideal body weight (AIBW) in kilograms.

[0220]

[0133] An "effective amount" of an antibody as disclosed herein is an amount sufficient to cany out a specifically stated purpose. A "therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A “therapeutically effective amount” of a therapeutic agent (e.g., a conjugate or immunoconjugate) can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.Atty. Dkt. No.: 45325-0051WO1

[0134] A '‘therapeutic agent"’ encompasses both a biological agent such as an antibody, a peptide, a protein, an enzyme, a chemotherapeutic agent, or a conjugate or immunoconjugate.

[0221]

[0135] The terms "subject," "individual," "animal," "patient," and "mammal," refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include but are not limited to humans, non-human primates, domestic animals, farm animals, rodents, and the like, which is to be the recipient of a particular treatment.

[0222]

[0136] Tire terms “polynucleotide” or “nucleic acid”, as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. Hie nucleotides can be deoxy ribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g.. methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g.. phosphorothioates, phosphorodithioates, etc ), those containing pendant moieties, such as, for example, proteins e.g.. nucleases, toxins, antibodies, signal peptides, ply-L -lysine, etc.), those with intercalators (e.g.. acridine, psoralen, etc., those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g.. alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or can be conjugated to solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls can also be denvatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example. 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2' azido-ribose, carbocyclic sugar analogs, alpha- anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. 'These alternative linking groups include, but are not limited to, embodiments, wherein phosphate is replaced by P(O)S ("thioate”). P(S)S ("dithioate"). "(0)NR2 ("amidate"), P(O)R, P(O)OR, CO or CH2 ("formacetal"), m which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionallyAtty. Dkt. No.: 45325-0051WO1 containing an ether (—0—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0223]

[0137] The term “vector" means a construct, which is capable of delivering, and optionally expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0224]

[0138] The terms “polypeptide", “peptide", and “protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified ammo acids, and it can be interrupted by non-amino acids. Tire terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc. ). as well as other modifications known in the art.

[0225]

[0139] The term ‘‘identical’’ or percent “identity", as known in the art, is a measure of the relationship between two polynucleotides or two polypeptides, as determined by comparing their sequences.Identity or similarity with respect to a sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical ( / .e., same residue) or similar (i.e., amino acid residue from the same group based on common side-chain properties, see below) to EpCAM antibody residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. None of N-terminal, C -terminal, or internal extensions, deletions, or insertions into tire antibody sequence outside of the variable region shall be construed as affecting sequence identity or similarity. In general, the two sequences to be compared are aligned to give a maximum correlation between the sequences. The alignment of the two sequences is examined and the number of positions giving an exact amino acid or nucleotide correspondence between the two sequences determined, divided by the total length of the alignment and multiplied by 100 to give a % identity figure. This % identity' figure can be determined over the whole length of the sequences to be compared, which is particularly suitable for sequences of the same or very similar length and which are highly homologous, or over shorter defined lengths, which is more suitable for sequences of unequal length or which have a lower level of homology Likewise percent similarity can be determined in an analogous manner based on the presence of both identical and similar residues.Atty. Dkt. No.: 45325-0051WO1

[0140] The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. 87:2264-2268 (1990), as modified in Karlin etal., Proc. Natl. Acad. Sci. 90:5873-5877 (1993), and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments. Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res.

[0226] 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul etal, Meth. Enzym. 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR®) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in GCG software (e.., using a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14. 12. 10. 8. 6, or 4, and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS 4:11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue Table, a gap length penalty- of 12 and a gap penalty of 4. Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art. In certain embodiments, the default parameters of the alignment software used. In certain embodiments, the percentage identity " X” of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y / 'Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence is longer than the percent identity of the second sequence to the first sequence.

[0227]

[0141] As a non-limiting example, whether any particular polynucleotide has a certain percentage sequence identity (e.g., is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequence can, in certain embodiments, be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive. Madison, WI 53711) Bestfit uses the local homology algorithm of Smith and Waterman, Advances in AppliedAtty. Dkt. No.: 45325-0051WO1 Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence as provided herein, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0228]

[0142] A ‘‘conservative amino acid substitution'’ is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including, for example, basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g.. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.. tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In some embodiments, conservative substitutions in the sequences of the polypeptides and antibodies provided herein do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s), to which the polypeptide or antibody binds. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well- known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993): Kobayashi etal., Protein Eng. 12(10):879-884 (1999); and Burks etal., Proc. Natl. Acad. Set. USA 94:412-417 (1997)).

[0229]

[0143] “Alkyl" as used herein refers to a saturated linear or branched-chain monovalent hydrocarbon radical of one to twenty carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, 1 -propyl, 2 -propyl, 1-butyl, 2-methyl-l -propyl, -CH2CH(CH3)2), 2 butyl, 2 -methy -2-propyl, 1 -pentyl, 2 -pentyl 3-pentyl, 2 -methyl -2 -butyl, 3 -methyl -2 -butyl, 3 methyl- 1-butyl, 2-methyl-l -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, 3,3 dimethyl-2 -butyl, 1 -heptyl, 1-octyl, and the like. In some embodiments, the alkyl has one to ten carbon atoms In some embodiments, the alkyl has one to four carbon atoms.

[0230]

[0144] The number of carbon atoms in a group can be specified herein by the prefix “Cx-xx”, wherein x and xx are integers. For example, “Cl-4alkyl” is an alkyl group having from 1 to 4 carbon atoms.

[0231]

[0145] The term “compound'’ or “cytotoxic compound,’’ or “cytotoxic agent" are used interchangeably.

[0232] They are intended to include compounds for which a structure or formula or any derivative thereof has been disclosed herein or a structure or formula or any derivative thereof that has been incorporated by¬ reference. The term also includes, stereoisomers, geometric isomers, tautomers, solvates, metabolites,Atty. Dkt. No.: 45325-0051WO1 and salts (e.g. pharmaceutically acceptable salts) of a compound of ail the formulae disclosed herein. Tire term also includes any solvates, hydrates, and polymorphs of any of the foregoing. The specific recitation of ‘‘stereoisomers,’’ “geometric isomers,” “tautomers,” “solvates,” “metabolites,” “salt”, “conjugates,” “conjugates salt,” “solvate,” “hydrate,” or “polymorph” in certain embodiments, provided herein shall not be interpreted as an intended omission of these forms in other disclosed embodiments, where the term “compound” is used without recitation of these other forms. In some instances, the compound comprises a camptothecin derivative linker-payload

[0233]

[0146] Hie term “imine reactive reagent” refers to a reagent that is capable of reacting with an imine group. Examples of imine reactive reagent includes, but is not limited to, sulfites (H2SO3, H2SO2 or a salt of HSO3, SO32-or HSO3-formed with a cation), metabisulfite (H2S2O5 or a salt of S2O52-formed with a cation), mono, di. tri. and tetra- thiophosphates (PO3SH3. PO2S2H3. POS3H3, PS4H3 or a salt of PO3S3', PO2S23-, POS33-or PS43-formed with a cation), thio phosphate esters ((RiO)2PS(ORi), RiSH, RiSOH, RiSOiH, RiSOsH), various amines (hydroxyl amine (e.g., NH2OH), hydrazine (e.g. NH2NH2), NH2O-R1, Ri’NH-Ri, NH2-R1), NH2-CO-NH2, H;-Ci== S)-NH;. thiosulfate (H2S2O3 or a salt of S2O- formed with a cation), dithionite (H2S2O4 or a salt of S2O42-formed with a cation), phosphorodithioate (P(=S)(ORk)(SH)(OH) or a salt thereof formed with a cation), hydroxamic acid (RkC(=O)NHOH or a salt formed with a cation), hydrazide (RkCONHNH2), formaldehyde sulfoxylate (HOCH2SO2H or a salt of HOCH2SO2 formed with a cation, such as HOCH2SO2-Na+glycated nucleotide (such as GDP- raannose), fludarabine or a mixture thereof, wherein Ri and Ri’ are each independently a linear or branched alkyl having 1 to 10 carbon atoms and are substituted with at least one substituent selected from N(Rj)2, -CO2H, SO3H, and POSH; Ri and Ri’ can be further optionally substituted with a substituent for an alkyl disclosed herein: Rj is a linear or branched alkyl having 1 to 6 carbon atoms; and Rk is a linear, branched or cyclic alky], alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heterocyclyl or heteroaryl (in some embodiments, Rk is a linear or branched alkyl having 1 to 4 carbon atoms; in some embodiments, Rk is methy l, ethyl or propyl). In some embodiments, the cation is a monovalent cation, such as Na+or K”. In some embodiments, the imine reactive reagent is selected from sulfites, hydroxyl amine, urea and hydrazine. In some embodiments, the imine reactive reagent is NaHSCh or KHSO3.

[0234]

[0147] The term “cation” refers to an ion with positive charge. The cation can be monovalent (e.g..

[0235] Na+, K+, NH4+etc.), bi-valent (e.g., Ca2+, Mg2+, etc.) or multi-valent (e.g., Al’+etc.). In some embodiments, the cation is monovalent.

[0236]

[0148] The phrase “pharmaceutically acceptable salt” as used herein, refers to pharmaceutically acceptable organic or inorganic salts of a compound provided herein. Exemplary / salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,Atty. Dkt. No.: 45325-0051WO1 bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate. saccharate, formate, benzoate, glutamate, methanesulfonate ‘"mesylate,” ethanesulfonate, benzenesulfonate, p- toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt can involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion. The counter ion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt can have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counter ion.

[0237]

[0149] If the compound provided herein is a base, the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, me thane sulfonic acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.

[0238]

[0150] If the compound provided herein is an acid, the desired pharmaceutically acceptable salt can be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like. Illustrative Examples of suitable salts include, but are not limited to. organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

[0239]

[0151] As used herein, the term ‘“solvate” means a compound that further includes a stoichiometric or non-stoichiometric amount of solvent such as water, isopropanol, acetone, ethanol, methanol, DMSO. ethyl acetate, acetic acid, and ethanolamine dichloromethane, 2 propanol, or the like, bound by non- covalent intermolecular forces. Solvates or hydrates of the compounds are readily prepared by addition of at least one molar equivalent of a hydroxy lie solvent such as methanol, ethanol, 1 -propanol, 2- propanol or water to the compound to result in solvation or hydration of tire imine moiety.

[0240]

[0152] A “metabolite” or “catabolite” is a product produced through metabolism or catabolism in the body of a specified compound, a derivative thereof, or a conjugate thereof, or salt thereof. Metabolites of a compound, a derivative thereof, or a conjugate thereof, can be identified using routine techniquesAtty. Dkt. No.: 45325-0051WO1 known in the art and their activities determined using tests such as those disclosed herein. Such products can result for example from the oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like, of the administered compound. Accordingly, the disclosure includes metabolites of compounds, a derivative thereof, or a conjugate thereof, of the disclosed EpCAM compositions disclosed herein, including compounds, derivatives thereof, or conjugates thereof, produced by a process comprising contacting a disclosed EpCAM compound, a derivative thereof, or a conjugate thereof, with a mammal for a period of time sufficient to yield a metabolic product thereof.

[0241]

[0153] I’he phrase “'pharmaceutically acceptable'" indicates that the substance or composition must be compatible chemically and / or toxicologically, with the other ingredients comprising a formulation, and / or the mammal being treated therewith.

[0242]

[0154] The term ‘‘protecting group’’ or “protecting moiety” refers to a substituent that is commonly employed to block or protect a particular functionality while reacting other functional groups on tire compound, a derivative thereof, or a conjugate thereof. For example, an “amine -protecting group"’ or an “ammo-protecting moiety” is a substituent attached to an amino group that blocks or protects the amino functionality in the compound Such groups are well known in the art (see, for example, P. Wuts and T. Greene, 2007, Protective Groups in Organic Synthesis, Chapter 7. J. Wiley & Sons, NJ) and exemplified by carbamates such as methyl and ethyl carbamate, FMOC, substituted ethyl carbamates, carbamates cleaved by 1,6-p-elimination (also termed “self immolative”), ureas, amides, peptides, alkyl and aryl derivatives. Suitable amino-protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9-fluorenylmethylenoxycarbonyl (Fmoc). For a general description of protecting groups and their use. see P. G. M. Wuts & T. W. Greene, Protective Groups in Organic Synthesis. John Wiley & Sons, New York, 2007.

[0243]

[0155] Tire term “amino acid” refers to naturally occurring amino acids or non-naturally occurring amino acid. In one embodiment, the amino acid is represented by NH2-C(Raa’Raa)-C(:;:O)OH, wherein Raa and Raa’ are each independently H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyi having 1 to 10 carbon atoms, aryl, heteroaryl or heterocyclyl or Raa and the N- terminal nitrogen atom can together form a heteroycyclic ring (e.g.. as in proline). Tire term “amino acid residue” refers to the corresponding residue when one hydrogen atom is removed from the amine and / or carboxy end of the amino acid, such as -NH-C(Raa’Raa)-C( ())()-.

[0244]

[0156] The term “peptide” refers to short chains of amino acid monomers linked by peptide (amide) bonds. In some embodiments, the peptides contain 2 to 20 amino acid residues. In other embodiments, the peptides contain 2 to 10 amino acid residues. In yet other embodiments, the peptides contain 2 to 5 amino acid residues. As used herein, when a peptide is a portion of a cytotoxic agent or a linkerAtty. Dkt. No.: 45325-0051WO1 disclosed herein represented by a specific sequence of amino acids, the peptide can be connected to the rest of the cytotoxic agent or the linker in both directions. For example, a dipeptide XI -X2 includes XI -X2 and X2-X1. Similarly, a tripeptide X1-X2-X3 includes X1-X2-X3 and X3-X2-X1 and a tetrapeptide X1-X2-X3-X4 includes X1-X2-X3-X4 and X4-X3-X2-X1. XI, X2, X3 and X4 represents an amino acid residue,

[0245]

[0157] The term ‘"reactive ester group” refers to a group an ester group that can readily react with an amine group to form amide bond. Exemplary reactive ester groups include, but are not limited to, N- hydroxy succinimide esters, N-hydroxyphthalimide esters, N-hydroxy sulfo-succinimide esters, paranitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters and their derivatives, wherein the derivatives facilitate amide bond formation. In certain embodiments, the reactive ester group is an N- hydroxysuccinimide ester or an N hydroxy sulfo-succinimide ester.

[0246]

[0158] The term ‘"amine reactive group” refers to a group that can react with an amine group to form a covalent bond. Exemplary' amine reactive groups include, but are not limited to, reactive ester groups, acyl halides, sulfonyl halide, imidoester, or a reactive thioester groups. In certain embodiments, tire amine reactive group is a reactive ester group. In one embodiment, the amine reactive group is an N- hydroxysuccinimide ester or an N-hydroxy sulfo-succinimide ester.

[0247]

[0159] The term ‘"thiol-reactive group” refers to a group that can react with a thiol (-SH) group to form a covalent bond. Exemplary thiol-reactive groups include, but are not limited to, maleimide, haloacetyl. haloacetamide, vinyl sulfone, vinyl sulfonamide or vinyl pyridine. In one embodiment, the thiolreactive group is maleimide.

[0248]

[0160] As used in the present disclosure and claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly? dictates otherwise.

[0249]

[0161] The term "and / or" where used herein is to be taken as specific disclosure of each of the two or more specified features or components with or without the other, Tirus, the term "and / or" as used in a phrase such as " A and / or B" herein is intended to include " A and B," " A or B," " A" (alone), and " B" (alone). Likewise, the term "and / or" as used in a phrase such as " A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and (. A and B; B and C; A (alone); B (alone); and C (alone).

[0250]

[0162] Wherever embodiments, are disclosed herein with the language “comprising,” otherwise analogous embodiments, described in terms of “consisting of’ and / or “consisting essentially of' are also provided.Atty. Dkt. No.: 45325-0051WO1 IMMUNOCON JU GATES COMPRISING EPCAM ANTIBODIES, EPCAM-BINDING ANTIBODY FRAGMENTS THEREOF, AND EPCAM CONDITIONALLY ACTIVATABLE MASKED IMMUNOCONJUGATES

[0251]

[0163] Disclosed herein are immunoconjugates comprising EpCAM antibodies and EpCAM-binding antibody fragments thereof. These proteins specifically bind human EpCAM and are provided herein. In some embodiments, the proteins are referred to herein as " EpCAM-binding agents" or EpBAs.” EpCAM expression, particularly high EpCAM expression, can be detected in many different tumors.

[0252]

[0164] In additional embodiments, immunoconjugates disclosed herein comprise the EpCAM-binding agent, which is an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM conditionally activatable masked immunoconjugate. In some embodiments, the EpBA is a full-length EpCAM antibody (re., a full-length antibody that specifically binds EpCAM). In some embodiments, the EpCAM antibody is a monoclonal antibody. In some embodiments, the EpCAM antibody is a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody; a multi -specific antibody e.g., a bi-specific antibody), or an EpCAM-binding antibody fragment thereof. In some embodiments, the EpCAM antibody specifically binds human EpCAM. In further embodiments, the EpCAM antibody specifically binds human EpCAM and cyno EpCAM. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof is a mouse, other rodent, chimeric, humanized or fully human monoclonal antibody.

[0253]

[0165] In some embodiments, immunoconjugates disclosed herein comprise an EpCAM antibody’ that is an EpCAM-binding antibody fragment. In some embodiments, immunoconjugates disclosed herein comprise a non-human mammal antibody, a murine antibody, a chimeric antibody, a humanized antibody; or a human antibody. In some embodiments, immunoconjugates disclosed herein comprise a full-length antibody. In some embodiments, immunoconjugates disclosed herein comprise a human IgGl antibody.

[0254]

[0166] In some embodiments, immunoconjugates disclosed herein comprise a Fab, a Fab', a F(ab')2, an scFv, or a disulfide-linked Fv (dsFv). In additional aspects, EpCAM-binding antibody fragment is a single chain Fv (scFv), a disulfide linked Fv. an IgG CH2. minibody, an F(ab’)3^ a scAb. a tetrabody, a triabody, a diabody, DVD-Ig, an Fcab, a mAb2, a(scFv)2, a scFv-Fc or a bis-scFv.

[0255]

[0167] In some instances, the immunoconjugates disclosed herein comprise EpCAM antibodies and EpCAM-binding antibody fragments thereof provided herein that optionally bind EpCAM (e.g.. human EpCAM and / or murine EpCAM), with a wide range of affinities (KD). In a particular embodiment, the antibody binds human EpCAM with high affinity. For example, a human or human engineered or humanized or resurfaced mAb can bind human antigen with a KDequal to or less than about 10-7M, such as but not limited to, 0.1 -9.9 (or any range or value therein between) x 10’7, IO’8, 10’9, IO'1", 101’,Atty. Dkt. No.: 45325-0051WO1 or 10“12, or any range or value therein, as determined by flow cytometry base assays, enzyme-linked immunoabsorbent assay (ELISA), surface plasmon resonance (SPR) or the KinExA® method using standard operating procedures. In some embodiments, the EpCAM antibodies bind with a Kd of about 1O'SM or less, more specifically about ICC to 1O’]OM.

[0256]

[0168] The affinity or avidity for EpCAM of an antibody or antibody fragment thereof in an iromunoconjugate described herein can be determined experimentally using any suitable method known in the art, e.g., flow cytometry, enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., BIACORE™ analysis), using standard operating procedures. Direct binding assays as well as competitive binding assay formats can be routinely employed. See, e.g., Berzofsky, et al., " Antibody- ntigen Interactions." In Fundamental Immunology, Paul, W. E,, Ed., Raven Press: New- York, N Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, N. Y. (1992); and methods disclosed herein The measured affinity of a particular antibody-EpCAM interaction can vary if measured under different conditions (e.g., salt concentration, pH, temperature). Ulus, measurements of affinity and other EpCAM-binding parameters (e.g.. KD or Kd, Kon, Koff) are made with standardized solutions of antibody and EpCAM, and a standardized buffer, as know n in the art and such as the buffer disclosed herein.

[0257]

[0169] In one embodiment, binding assays are performed using flow- cytometry on cells expressing the EpCAM antigen on the surface. For example, such EpCAM-positive cells are incubated with varying concentrations of EpCAM antibodies using 1 xlO5cells per sample in 100 pL FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum). Then, the cells are pelleted, washed, and incubated for 1 h with 100 pL of FITC-conjugated goat anti -mouse IgG-antibody (such as obtainable from Jackson ImmunoResearch) m FACS buffer. The cells are pelleted again, washed with FACS buffer and resuspended in 200 pL of PBS containing 1% formaldehyde. Samples are acquired, for example, using a FACSCalibur™ flow cytometer with the HTS multiwell sampler and analyzed using CellQuest® Pro (all from BD Biosciences, San Diego, US). For each sample, the mean fluorescence intensity for FL I (MFI) is exported and plotted against the antibody concentration in a semi-log plot to generate a binding curve. A sigmoidal dose-response curve is fitted for binding curves and EC50 values are calculated using programs such as GraphPad Prism v4 'ith default parameters (GraphPad software, San Diego, CA). EC50 values can be used as a measure for the apparent dissociation constant “ d’’ or “KD” for each antibody.

[0258]

[0170] In certain embodiments, the EpCAM antibodies or fragments thereof in any of the immunoconjugates described herein are modified to alter their binding affinity for EpCAM and / or EpCAM antigenic fragments thereof. Binding properties can be determined by a variety of in vitro assay methods and employing standard operating procedures known in the art, including for example,Atty. Dkt. No.: 45325-0051WO1 enzyme-linked immunoabsorbent assay (ELISA), radioimmunoassay (RIA)), or kinetics (e g., BIACORE™ analysis),

[0259]

[0171] In one embodiment, the EpCAM antibody or EpCAM-binding antibody fragment thereof in any of the immunoconjugates described herein specifically binds human and / or cynomolgus EpCAM, and / or EpCAM antigenic fragments thereof, with a dissociation constant or KD or Kd (koffi'kon) of less than 10" M, or of less than 10bM, or ofless than 107M, or ofless than 1 O’8M, or of less than 10'9M, orofless than 1 O'10M, or of less than 10’l!M, or of less than 10'12' M, or of less than 10’13M. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof specifically binds human and / or cynomolgus EpCAM and / or EpCAM antigenic fragments thereof with a KD of 1.0 x 10'9M or less, 2,0 x 109M or less. 3,0 x 109M or less, 4.0 x 109M or less, 5.0 x 109M or less, 6,0 x 109M or less, 7.0 x 10'9M or less. 8.0 x 10’9M or less, or 9.0 x 109M or less. In certain embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof binds to both human and cynomolgus EpCAM and / or EpCAM antigenic fragments thereof with a KD of 3.0 x 10’9M or less. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof binds to human EpCAM with a KD of about 0.4 x 10’9. In some embodiments, the EpCAM antibody or EpCAM- binding antibody fragment thereof binds to human EpCAM with a KD of about 0.8 x 10’9. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof binds to cynomolgus EpCAM with a KD of about 0.8 x 10'9. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof binds to cynomolgus EpCAM with a KD of about 2.2 x 1 O’ ’. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof binds to cynomolgus EpCAM with a KD of about 2.8 x 10’9.

[0260]

[0172] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof in any of the immunoconjugates described herein specifically binds to an epitope within the extracellular region of human EpCAM (SEQ ID NO: 1). Tire extracellular region of human EpCAM can be further divided into three distinct domains: DI (SEQ ID NO:2), D2 (SEQ ID NO:3), and D3 (SEQ ID NO:4). In certain embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof specifically binds to an epitope within the first extracellular domain (DI) of human EpCAM.

[0261]

[0173] In one embodiment, the EpCAM antibody or EpCAM-binding antibody fragment thereof in any of the immunoconjugates described herein comprises a VH-CDR1 comprising X1YX3X4H, wherein Xi is selected from N and S. X3is selected from Y, N, F, S, H, D, L, I, and W, and X4 is selected from I and M; a VH-CDR2 comprising WX2X3PGX6VYIQYX12X13KFX17G, wherein X2 is selected from I and F, X3 is selected from Y and N. Xg is selected from N and D, X12 is selected from N and S, X13, is selected from E and Q. and X17 is selected from K and Q (SEQ ID NO:7); and a VH-CDR3 comprising X1GX3X4FAY, wherein Xi is selected from D and E, X3is selected from P, A, S, Y, F. G, T, and V, and X4 is selected from Y and W (SEQ ID NO:8).Atty. Dkt. No.: 45325-0051WO1

[0174] In additional embodiments, the disclosure provides an EpCAM antibody or EpCAM-binding antibody fragment thereof in any of the immunoconjugates described herein, comprising a light chain CDR1 (VL-CDR1) comprising RSSX₄SLLHSX₁₀GX₁₂TYLX₁₆!wherein 4 is selected from R and K, X10 is selected from N and D, X12 is selected from F and I, and Xig is selected from Y and S (SEQ ID NO: 10); a light chain VL-CDR2 comprising QTSNLAS (SEQ ID NO:40); and a VL-CDR3 comprising X₁QX₃LELPX₈T, wherein Xi is selected from A. L, and Q. \ is selected from S, G, Y, and N, and Xg is selected from N and W (SEQ ID NO: 11). In some embodiments, tire disclosure provides an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a heavy chain CDR1 (VH-CDR1) comprising the sequence of SEQ ID NO: 13; a heavy chain CDR2 (VH-CDR2) comprising the sequence of SEQ ID NO: 14: a heavy chain CDR3 (VH-CDR3) comprising the sequence of SEQ ID NO: 15; a light chain CDR1 (VL-CDR1) comprising the sequence of SEQ ID NO:42; a light chain CDR2 (VL- CDR2) comprising the sequence of SEQ ID NO:40; and a light chain CDR3 (VL-CDR3) comprising the sequence of SEQ ID NO:41.

[0262]

[0175] In some embodiments, the VH-CDR1 comprises the sequence NYX₃IH, wherein X3 is selected from Y, N. F, S, H. D, L. I. and W (SEQ ID NO:6). In some embodiments, the VH-CDR3 comprises the sequence DGPX₄FAY. wherein X4 is selected from Y and W (SEQ ID NO:9). In some embodiments, the VL-CDR3 comprises the sequence AQX₃LELPNT, wherein X₃ is selected from S, G, Y, and N (SEQ ID NO: 12). In some embodiments, the disclosure provides an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a heavy chain CDR1 (VH-CDR1) composing the sequence of SEQ ID NO: 13; a heavy chain CDR2 (VH-CDR2) comprising the sequence of SEQ ID NO: 14; a heavy chain CDR3 (VH-CDR3) comprising the sequence of SEQ ID NO: 15; a light chain CDR1 (VL-CDR1) comprising the sequence of SEQ ID NO:42; a light chain CDR2 (VL-CDR2) comprising the sequence of SEQ ID NO:40; and a light chain CDR3 (VL-CDR3) comprising the sequence of SEQ ID NO:41.

[0263]

[0176] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof in any of the immunoconjugates described herein comprises a set of complementarity determining regions (CDRs): heavy chain variable region (VH)-CDRl. VH-CDR2, VH-CDR3, light chain variable region ( VL) CDR1. VL-CDR2 and VL-CDR3, wherein the heavy chain CDRs are disclosed in Table 2.

[0264] Table 2. Exemplary heavy chain CDR sequences of EpCAM antibodies.

[0265] Antibody VH- CDR1 VH-CDR2 VH-CDR3

[0266] Murine and Chimeric

[0267] muEpcam23 NYYIH WIYPGNVYIQYNEKFK DGPWFAY

[0268] (SEQ ID NO: 13) G (SEQ ID NO: 15)

[0269] (SEQ ID NO: 14)

[0270]

[0271] Atty. Dkt. No.: 45325-0051WO1 Antibody VH- CDR1 VH-CDR2 VH-CDR3 chEpcam23 NYYIH WIYPGNVYIQYNEKFK DGPWFAY

[0272] (SEQ ID NO: 13) G (SEQ ID NO: 15)

[0273] (SEQ ID NO: 14)

[0274] Humanized Variants

[0275] huEpCAM23 VHGvl NYYIH WIYPGNVYIQYSQKFQ DGPWFAY

[0276] (SEQ ID NO: 13) G (SEQ ID NO: 15)

[0277] (SEQ ID NO:26)

[0278] huEpCAM23 VHGv2 NYYIH WIYPGNVYIQYNEKFK DGPWFAY

[0279] (SEQ ID NO: 13) G (SEQ ID NO: 15)

[0280] (SEQ ID NO: 14)

[0281] huEpCAM23 VHGv3 NYYIH WIYPGNVYIQYNEKFK DGPWFAY

[0282] (SEQ ID NO: 13) G (SEQ ID NO: 15)

[0283] (SEQ ID NO: 14)

[0284] huEpCAM23 VHGv4 NYYIH WIYPGNVYIQYSQKFQ DGPWFAY

[0285] (SEQ ID NO: 13) G (SEQ ID NO: 15)

[0286] (SEQ ID NO: 26)

[0287] Affinity Variants

[0288] huEpCAM23HCGv2a NYYIH WIYPGNVYIQYNEKFK DGPWFAY

[0289] (SEQ ID NO: 13) G(SEQ ID NO: 14) (SEQ ID NO: 15) huEpCAM23HCGv2b SYYIH WIYPGNVYIQYNEKFK DGPWFAY

[0290] (SEQ ID NO: 16) G(SEQ ID NO: 14) (SEQ ID NO: 15) huEpCAM23HCGv2c NYNIH WIYPGNVYIQYNEKFK DGPWFAY

[0291] (SEQ ID NO: 17) G(SEQ ID NO: 14) (SEQ ID NO: 15) huEpCAM23HCGv2d NYYIH WIYPGDVYIQYNEKFK DGPWFAY

[0292] (SEQ ID NO: 13) G (SEQ ID NO: 15)

[0293] (SEQ ID NO:27)

[0294] huEpCAM23HCGv2e NYFIH WIYPGNVYIQYNEKFKG DGPWFAY

[0295] (SEQ ID NO: 18) (SEQ ID NO: 14) (SEQ ID NO: 15) huEpCAM23HCGv2f NYSIH WIYPGNVYIQYNEKFKG DGPWFAY

[0296] (SEQ ID NO: 19) (SEQ ID NO: 14) (SEQ ID NO: 15) huEpCAM23HCGv2g NYWIH WIYPGNVYIQYNEKFKG DGPWFAY

[0297] (SEQ ID NO:20) (SEQ ID NO: 14) (SEQ ID NO: 15)

[0298]

[0299] Atty. Dkt. No.: 45325-0051WO1 Antibody VH- CDR1 VH-CDR2 VH-CDR3 huEpCAM23HCGv2h NYYIH WFYPGNVYIQYNEKFK DGPWFAY

[0300] (SEQ ID NO: 13) G (SEQ ID NO: 15)

[0301] (SEQ ID NO:28)

[0302] huEpCAM23HCGv2i / 2o NYYIH WIYPGNVYIQYNEKFKG DGPWFAY

[0303] (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 15) huEpCAM23HCGv2j NYYIH WINPGNVYTQYNEKFKG DGPWFAY

[0304] (SEQ ID NO: 13) (SEQ ID NO: 29) (SEQ ID NO: 15) huEpCAM23HCGv2k NYYIH WIYPGNVYIQYNEKFKG EGPWFAY

[0305] (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO:30) huEpCAM23HCGv21 NYYIH WIYPGNVYIQYNEKFKG DGPYFAY

[0306] (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO:31) hnEpCAM23HCGv2m / NYYIH WIYPGNVYIQYNEKFKG DGAWFAY huEpCam23 HG2-1565-A (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ IDNO:32) Heavy Chain

[0307] huEpCAM23HCGv2n NYYMH WIYPGNVYIQYNEKFKG DGPWFAY

[0308] (SEQ ID NO:21) (SEQ ID NO: 14) (SEQ ID NO: 15) huEpCAM23HCGv2p NYYIH WIYPGNVYIQYNEKFKG DGPWFAY

[0309] (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 15) huEpCAM23HCGv2q NYYIH WIYPGNVYIQYNEKFKG DGPWFAY

[0310] (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 15) huEpCAM23HCGv2r NYYIH WIYPGNVYIQYNEKFKG DGPWFAY

[0311] (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 15) huEpCAM23HCGv2s NYYIH WIYPGNVYIQYNEKFKG DGPWFAY

[0312] (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 15) huEpCam23HG2-1361-H NYHIH WIYPGNVYIQYNEKFKG DGPWFAY

[0313] Heavy Chain (SEQ ID NO:22) (SEQ ID NO: 14) (SEQ ID NO: 15) huEpCam23HG2-1361-D NYDIH WWPGNVYIQYNEKFKG DGPWFAY

[0314] Heavy Chain (SEQ ID NO:23) (SEQ ID NO: 14) (SEQ ID NO: 15) huEpCam23HG2-1565-Y NYYIH WIYPGNVYIQYNEKFKG DGYWFAY

[0315] Heavy Chain (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 33) huEpCam23HG2-1565-S NYYIH WIYPGNVYIQYNEKFKG DGSWFAY

[0316] Heavy Chain (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 34)

[0317]

[0318] Atty. Dkt. No.: 45325-0051WO1 Antibody VH- CDR1 VH-CDR2 VH-CDR3 huEpCam23HG2-1565-F NYYIH WIYPGNVYIQYNEKFKG DGFWFAY

[0319] Heavy Chain (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO:35) huEpCam23HG2-1565-G NYYIH WIYPGNVYIQYNEKFKG DGGWFAY

[0320] Heavy Chain (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO:36) huEpCam23HG2-1565-T NYYIH WIYPGNVYIQYNEKFKG DGTWFAY

[0321] Heavy Chain (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ ID NO: 37) huEpCam23HG2-1565-V NYYIH WIYPGNVYIQYNEKFKG DGVWFAY

[0322] Heavy Chain (SEQ ID NO: 13) (SEQ ID NO: 14) (SEQ IDNO:38) huEpCam23HG2-1361-I NYIIH WIYPGNVYIQYNEKFKG DGPWFAY

[0323] Heavy Chain (SEQ ID NO: 24) (SEQ ID NO: 14) (SEQ ID NO: 15) huEpCam23HG2-1361-L NYLIH WIYPGNVYIQYNEKFKG DGPWFAY

[0324] Heavy Chain (SEQ ID NO:25) (SEQ ID NO: 14) (SEQ ID NO: 15)

[0325]

[0326]

[0177] In some embodiments, the disclosure provides an EpCAM antibody or EpCAM-bmding antibody fragment thereof in any of the immunoconjugates described herein comprising the heavy chain CDRs of a single row in Table 2. In some embodiments, the disclosure provides an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR1 selected from SEQ ID NOs: 13, and 16-25; a VH-CDR2 selected from SEQ ID NOs: 14, and 26-29; and a VH-CDR3 selected from SEQ ID NOs: 15, and 30-38, In some embodiments, the disclosure provides an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR1 of SEQ ID NO: 13; a VH-CDR2 of SEQ ID NO: 14; and a VH-CDR3 of SEQ ID NO: 15. In some embodiments, the disclosure provides an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDRI of SEQ ID NO: 13; a VH-CDR2 of SEQ ID NO:26; and a VH-CDR3 of SEQ ID NO: 15.

[0327]

[0178] In one embodiment, the disclosure provides immunoconjugates having an EpCAM antibody, EpCAM-bmding antibody fragment thereof, or EpCAM conditionally activatable masked antibody comprising, a VH-CDR1 comprising NYYIH (SEQ ID NO: 13), or a variant thereof comprising 1, 2. 3, or 4, conservative amino acid substitutions; a VH-CDR2 comprising WIYPGNVYIQYNEKFKG (SEQ ID NO: 14), or a variant thereof comprising 1, 2, 3, or 4, amino conservative acid substitutions; and a heavy chain CDR3 comprising DGPWFAY (SEQ ID NO: 15), or a variant thereof comprising 1, 2, 3, or 4, conservative amino acid substitutions.

[0328]

[0179] In some embodiments, the disclosure provides immunoconjugates having an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDRI of SEQ ID NO:22; a VH-CDR2 of SEQ ID NO: 14; and a VH-CDR3 of SEQ ID NO: 15. In some embodiments, the disclosure providesAtty. Dkt. No.: 45325-0051WO1 an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDRI of SEQ ID NO: 13; a VH-CDR2 of SEQ ID NO: 14; and a VH-CDR3 of SEQ ID NO:33. In some embodiments, the disclosure provides an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR1 of SEQ ID NO:23; a VH-CDR2 of SEQ ID NO:14; and a VH-CDR3 of SEQ ID NO:15. In some embodiments, the disclosure provides an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDRI of SEQ ID NO:25; a VH-CDR2 of SEQ ID NO: 14; and a VH-CDR3 of SEQ ID NO: 15.

[0329]

[0180] In one embodiment, the disclosure provides immunoconjugates having an EpCAM antibody, EpCAM-binding antibody fragment thereof, or EpCAM conditionally activatable masked antibody comprising, a VH-CDRI comprising NYHIH (SEQ ID NO:22), or a variant thereof comprising 1. 2, 3, or 4. conservative amino acid substitutions; a VH-CDR2 comprising WIYPGNVYIQYNEKFKG (SEQ ID NO: 14), or a variant thereof comprising 1, 2, 3. or 4, ammo conservative acid substitutions; and a heavy chain CDR3 comprising DGPWFAY (SEQ ID NO: 15), or a variant thereof comprising 1, 2. 3, or 4, conservative amino acid substitutions. In one embodiment, the disclosure provides an EpCAM antibody, EpCAM-binding antibody fragment thereof, or EpCAM conditionally activatable masked antibody comprising, a VH-CDRI comprising NYYIH (SEQ ID NO: 13), or a variant thereof comprising 1, 2, 3, or 4, conservative ammo acid substitutions; a VH-CDR2 comprising WIYPGNVYIQYNEKFKG (SEQ ID NO: 14), or a variant thereof comprising 1. 2, 3, or 4, amino conservative acid substitutions; and a heavy chain CDR3 comprising DGYWFAY (SEQ ID NO:33), or a variant thereof comprising 1, 2, 3, or 4, conservative amino acid substitutions.

[0330]

[0181] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any of the immunoconjugates described herein comprises a set of complementarity determining regions (CDRs): heavy chain variable region (VH)-CDRl. VH-CDR2, VH-CDR3, light chain variable region (VL) CDR1, VL-CDR2 and VL-CDR3, wherein the light chain CDRs are disclosed in Table 3.

[0331] Table 3. Exemplary light chain CDR sequences of EpCAM antibodies.

[0332] Antibody VL- CDR1 VL-CDR2 VL-CDR3

[0333] Murine and Chimeric

[0334] muEpcain23 RSSKSLLHSDGFTYLY QTSNLAS AQNLELPNT (SEQ ID NO: 39) (SEQ ID NO:40) (SEQ ID NO:41) chEpcam23 RS SKSLLHSDGFTYLY QTSNLAS AQNLELPNT (SEQ ID NO: 39) (SEQ ID NO:40) (SEQ ID NO:41) Humanized Variants

[0335] huEpCAM23 VLG vl RSSRSLLHSDGFTYLY QTSNLAS AQNLELPNT (SEQ ID NO:42) (SEQ ID NO:40) (SEQ ID NO:41)

[0336]

[0337] Atty. Dkt. No.: 45325-0051WO1 Antibody VL- CDR1 VL-CDR2 VL-CDR3 huEpCAM23 VLG v2 RS SKSLLHSDGFTYLY QTSNLAS AQNLELPNT (SEQ ID NO:39) (SEQ ID NO: 40) (SEQ ID NO:41) huEpCAM23 VLG v3 RS SKSLLHSDGFTYLY QTSNLAS AQNLELPNT (SEQ ID NO:39) (SEQ ID NO:40) (SEQ ID NO:41) huEpCAM23 VLG v4 RSSRSLLHSDGFTYLY QTSNLAS AQNLELPNT (SEQ ID NO:42) (SEQ ID NO: 40) (SEQ ID NO:41)

[0338] Affinity Variants

[0339] huEpCAM23LCGv4a RSSRSLLHSNGFTYLY QTSNLAS AQNLELPNT (SEQ ID NO:43) (SEQ ID NO:40) (SEQ ID NO:41) huEpCAM23LCGv4b RSSRSLLHSDGITYLY QTSNLAS AQNLELPNT (SEQ ID NO:44) (SEQ ID NO:40) (SEQ ID NO:41) huEpCAM23LCGv4c RSSRSLLHSDGFTYLY QTSNLAS AQNLELPWT (SEQ ID NO:42) (SEQ ID NO:40) (SEQ ID NO:46) huEpCAM23LCGv4e RSSRSLLHSDGFTYLS QTSNLAS AQNLELPNT (SEQ ID NO:45) (SEQ ID NO:40) (SEQ ID NO:41) huEpCAM23LCGv4 f RSSRSLLHSDGFTYLY QTSNLAS AQNLELPNT (SEQ ID NO:42) (SEQ ID NO:40) (SEQ ID NO:41) huEpCAM23LCGv4g RSSRSLLHSDGFTYLY QTSNLAS QQNLELPNT (SEQ ID NO:42) (SEQ ID NO:40) (SEQ ID NO:47) huEpCAM23LCGv4h RSSRSLLHSDGFTYLY QTSNLAS LQNLELPNT (SEQ ID NO:42) (SEQ ID NO:40) (SEQ ID NO:48) huEpCAM23LCGv4 i RSSRSLLHSDGFTYLY QTSNLAS AQYLELPNT (SEQ ID NO:42) (SEQ ID NO:40) (SEQ ID NO:49) huEpCAM23LCGv4 j RSSRSLLHSDGFTYLY QTSNLAS AQGLELPNT (SEQ ID NO:42) (SEQ ID NO:40) (SEQ ID NO:50) huEpCAM23LCGv4k RSSRSLLHSDGFTYLY QTSNLAS AQSLELPNT (SEQ ID NO:42) (SEQ ID NO:40) (SEQ ID NO:51)

[0340]

[0341]

[0182] In some embodiments, the disclosure provides immunoconjugates having an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising the light chain CDRs of a single row in Table 3. In some embodiments, the disclosure provides immunoconjugates having an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VL-CDR1 selected from SEQ ID NOs:39, and 42-45: a VL-CDR2 of SEQ ID NO:40; and a VL-CDR3 selected from SEQ ID NOs:41, and 46-51. InAtty. Dkt. No.: 45325-0051WO1 some embodiments, the disclosure provides immunoconjugates having an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VL-CDR1 of SEQ ID NO:42;a VL-CDR2 of SEQ ID NO:40; and a VL-CDR3 of SEQ ID NO:41. In some embodiments, the disclosure provides immunoconjugates having an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VL-CDR1 of SEQ ID NO:39; a VL-CDR2 of SEQ ID NO:40; and a VL-CDR3 of SEQ ID NO:41.

[0342]

[0183] In one embodiment, the disclosure provides immunoconjugates having an EpCAM antibody, EpCAM-binding antibody fragment thereof, or EpCAM conditionally activatable masked antibody comprising: a VL-CDR1 comprising RSSRSLLHSDGFTYLY (SEQ ID NO:42), or a variant thereof comprising 1, 2, 3. or 4, conservative amino acid substitutions; a VL-CDR2 comprising QTSNLAS (SEQ ID NO:40), or a variant thereof comprising 1, 2. 3. or 4. conservative ammo acid substitutions; and a VL-CDR3 comprising AQNLELPNT (SEQ ID NO:41), or a variant thereof comprising 1, 2, 3. or 4, conservative amino acid substitutions. In one embodiment, the disclosure provides immunoconjugates having an EpCAM antibody, EpCAM-binding antibody fragment thereof, or EpCAM conditionally activatable masked antibody comprising: a VL-CDR1 comprising RSSKSLLHSDGFTYLY (SEQ ID NO:39), or a variant thereof comprising 1, 2. 3, or 4, conservative amino acid substitutions; a VL-CDR2 comprising QTSNLAS (SEQ ID NO:40), or a variant thereof comprising 1, 2, 3, or 4, conservative amino acid substitutions; and a VL-CDR3 comprising AQNLELPNT (SEQ ID NO:41), or a variant thereof comprising 1, 2, 3, or 4, conservative amino acid substitutions.

[0343]

[0184] In some embodiments, the disclosure provides immunoconjugates having an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR1 selected from SEQ ID NOs: 13, and 16-25; a VH-CDR2 selected from SEQ ID NOs: 14, and 26-29; a VH-CDR3 selected from SEQ ID NOs: 15, and 30-38; a VL-CDR1 selected from SEQ ID NOs:39, and 42-45; a VL-CDR2 of SEQ ID NO:40; and a VL-CDR3 selected from SEQ ID NOs:41, and 46-51. In some embodiments, the disclosure provides immunoconjugates having an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL- CDR3 having the sequences of SEQ ID NOs: 13-15, 42, 40. and 41, respectively. In some embodiments, the disclosure provides immunoconjugtes having an EpCAM antibody or EpCAM- binding antibody fragment thereof comprising a VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL- CDR2, and VL-CDR3 having the sequences of SEQ ID NOs: 13-15, and 39-41, respectively. In some embodiments, the disclosure provides immunoconjugates having an EpCAM antibody or EpCAM- binding antibody fragment thereof comprising a VH-CDR1, VH-CDR2. VH-CDR3. VL-CDRL VL- CDR2, and VL-CDR3 having the sequences of SEQ ID NOs: 13, 26, 15, and 39-41, respectively. In some embodiments, the disclosure provides immunoconjugates having an EpCAM antibody orAtty. Dkt. No.: 45325-0051WO1 EpCAM-binding antibody fragment thereof comprising a VH-CDRl, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3 having the sequences of SEQ ID NOs: 13, 26, 15, 42, 40, and 41, respectively.

[0344]

[0185] In some embodiments, the disclosure provides immunoconjugates having an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR1, VH-CDR2. VH-CDR3. VL- CDRL VL-CDR2, and VL-CDR3 having the sequences of SEQ ID NOs: 22, 14, 15, 42, 40, and 41, respectively. In some embodiments, tire disclosure provides immunoconjugates having an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR1, VH-CDR2, VH- CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having the sequences of SEQ ID NOs: 13, 14, 33, 42, 40, and 41, respectively. In some embodiments, the disclosure provides immunoconjugates having an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having the sequences of SEQ ID NOs: 23, 14, 15, 42,

[0345] 40. and 41, respectively. In some embodiments, the disclosure provides immunoconjugates having an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having the sequences of SEQ ID NOs: 25, 14, 15, 42, 40, and 41, respectively.

[0346]

[0186] In some embodiments, immunoconjugates having the EpCAM antibody or EpCAM-binding antibody fragment thereof comprise a heavy chain variable region (VH) sequence disclosed in Table 4. In some embodiments, the EpC AM antibody or EpCAM-binding antibody fragment thereof comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to a VH sequence disclosed in Table 4. In some embodiments, the percent identity' in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0347] Table 4. Exemplary Heavy’ Chain Variable Sequences of EpCAM Antibodies.

[0348] Antibody name Sequence

[0349] Murine and Chimeric

[0350] muEpCAM23 EVKLEESGPALVKPGASVRISCKASGYTFTNYYIHWVKQRPGQGLDYIGWIYPGNVYIQYNEKFKGKATLTADKSSSTAFMQLSSLTSED SAVYFCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:52) chEpCAm23 EVKLEESGPALVKPGASVRISCKASGYTFTNYYIHWVKQRPGQGLDYIGWIYPGNVYIQYNEKFKGKATLTADKSSSTAFMQLSSLTSED SAVYFCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:52)

[0351] Humanized Variants

[0352]

[0353] Atty. Dkt. No.: 45325-0051WO1 huEpCAM23 VHGvl QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEWMGWIYPGNVYIQYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:53) huEpCAM23 VHGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:54) huEpCAM23 VHGv3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEWMGWIYPGNVYIQYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:55) huEpCAM23 VHGv4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEYIGWIYPGNVYIQYSQKFQGRVTITADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:56)

[0354] Affinity Variants

[0355] huEpCAM23HCGv2a QVQLVESGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:57) buEpCAM23HCGv2b QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQRLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:58) huEpCAM23HCGv2c QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNIHWVRQAPGQRLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:59) huEpCAM23HCGv2d QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIirAA7RQAPGQ RLEYIGWIYPGDVYIQYNEKFKGRATLTADKSASTAYMELSSLR SEDTAV’YYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:60) huEpCAM23HCGv2e QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQ RLEYIGW1Y PGNVYIQY N EKFKGRATLTADKSAST AYMELSSLR SEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:61) huEpCAM23HCGv2f QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYSIHWVRQAPGQRLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:62) huEpCAM23HCGv2g QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQRLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:63)

[0356]

[0357] Atty. Dkt. No.: 45325-0051WO1 huEpCAM23HCGv2h QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEYIGWFYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:64) huEpCAM23HCGv2i QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEWIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:65) huEpCAM23HCGv2j QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEYIGWINPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:66) huEpCAM23HCGv2k QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCAREGPWFAYWGQGTLVTVSS (SEQ ID NO:67) huEpCAM23HCGv2l QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPYFAYWGQGTLVTVSS (SEQ ID NO:68) huEpCAM23HCGv2m / QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGAWFAYWGQGTLVTVSS (SEQ ID NO:69) huEpCAM23HCGv2n QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQRLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:70) huEpCAM23HCGv2p QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEYIGWVYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:71) huEpCAM23HCGv2q QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEAIGWIYPGNVYIQYNEKFKGRATITADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:72) huEpCAM23HCGv2r QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEYIGWIYPGNVYIQYNEKFKGRATLTRDKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:73) huEpCAM23HCGv2s QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLEYIGWIYPGNVYIQYNEKFKGRVTLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:74)

[0358]

[0359] Atty. Dkt. No.: 45325-0051WO1 huEpCam23HG2-1361-H QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYHIHWVRQAPGQ Heavy Chain RLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO: 75) huEpCam23HG2-1361-D QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDIHWVRQAPGQ Heavy Chain RLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLR SEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO:76) huEpCani23HG2-1565-Y QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQ Heavy Chain RLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLR SEDTAVYYCARDGYWFAYWGQGTLVTVSS (SEQ ID NO:77) huEpCam23HG2-1565-S QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQ Heavy Chain RLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLR SEDTAVYYCARDGSWFAYWGQGTLVTVSS (SEQ ID NO:78) huEpCam23HG2-1565-F QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQ Heavy Chain RLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLR SEDTAVYYCARDGFWFAYWGQGTLVTVSS (SEQ ID NO: 79 huEpCam23HG2-1565-G QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQ Heavy Chain RLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGGWFAYWGQGTLVTVSS (SEQ ID NO:80) huEpCani23HG2-1565-T QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQ Heavy Chain RLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLR SEDTAVYYCARDGTWFAYWGQGTLVTVSS (SEQ ID NO:81) huEpCam23HG2-1565-V QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQ Heavy Chain RLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAVYYCARDGVWFAYWGQGTLVTVSS (SEQ ID NO:82) huEpCam23HG2-1361-I QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYIIHWVRQAPGQ Heavy' Chain RLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLR SEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO: 83) huEpCam23HG2-1361-L QVQLVQSGAEVKKPGA SVKVSCKA SGYTFTNYLIHWVRQAPGQ Heavy' Chain RLEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLR SEDTAVYYCARDGPWFAYWGQGTLVTVSS (SEQ ID NO: 84)

[0360]

[0361]

[0187] In some embodiments, an immunoconjugate as described herein comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH sequence having a total of one, two, three, four, five, six, seven, eight, nine, ten, fewer than fifteen, or zero, amino acidAtty. Dkt. No.: 45325-0051WO1 substitutions, deletions, and / or insertions from a reference VH sequence selected from SEQ ID NOs: 53-84. In some embodiments, the insertions, substitutions, deletions, and / or insertions are in framework regions(s) of the reference sequence. In some embodiments, the substitutions are conservative. In other embodiments, the substitutions are non-conservative.

[0362]

[0188] In some embodiments, an immunoconjugate as described herein comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH having a sequence selected from SEQ ID NOs:53-84. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH having a sequence selected from SEQ ID NOs:53-56. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH having the sequence of SEQ ID NO:54. In some embodiments, the EpCAM antibody or EpCAM- binding antibody fragment thereof comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94%.

[0363] 95%, 96%. 97%, 98%, or 99%, identical to a sequence selected from SEQ ID NOs: 53-56. In some embodiments, the EpCAM antibody' or EpCAM-binding antibody fragment thereof comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%, identical to the sequence of SEQ ID NO: 54. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0364]

[0189] In some embodiments, the immunoconjugates disclosed herein comprise EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH having a sequence selected from SEQ ID NOs: 75-77, and 84. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH having the sequence of SEQ ID NO:75. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH having the sequence of SEQ ID NO:77. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%. or 99%, identical to a sequence selected from SEQ ID NOs:75-77, and 84. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%, identical to the sequence of SEQ ID NO: 75. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH sequence that is at least 90%. 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, and 99%, identical to the sequence of SEQ ID NO: 77. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0365]

[0190] In some embodiments, the immunoconjugates disclosed herein comprise EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a light chain variable region (VL) sequence disclosed in Table 5. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VL sequence that is at least 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%,Atty. Dkt. No.: 45325-0051WO1 98%, or 99%, identical to a VL sequence disclosed in Table 5. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%. Table 5. Exemplary Light Chain Variable Sequences of EpCAM Antibodies.

[0366] Antibody name Sequence

[0367] Murine and Chimeric

[0368] muEpcam23 DIVLTQTPFSNPVTLGTSASISCRSSKSLLHSDGFTYLYWFLQKPGQ SPHLLIYQTSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCA QNLELPNTFGGGTKLEIK (SEQ ID NO: 85)

[0369] chEpcam23 DIVLTQTPFSNPVTLGTSASISCRSSKSLLHSDGFTYLYWFLQKPGQ SPHLLIYQTSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCA QNLELPNTFGGGTKLEIK (SEQ ID NO: 85)

[0370] Humanized Variants

[0371] huEpCAM23_VLGv1 DIVMTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWYLQKPG QSPQLLIYQTSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CAQNLELPNTFGQGTKLEIK (SEQ ID NO: 86)

[0372] huEpCAM23_VLGv2 DIVLTQTPLSLSVTPGQPASISCRSSKSLLHSDGFIYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKTSRVEAEDVGVYYCA QNLELPNTFGQGTKLEIK (SEQ ID NO: 87)

[0373] huEpCAM23 VLGv3 DIVMTQTPLSLSVTPGQPASISCRSSKSLLHSDGFTYIAAVTLQKPGQ SPQLLIYQTSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC AQNLELPNTFGQGTKLEIK (SEQ ID NO: 88)

[0374] huEpCAM23 VLGv4 DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QNLELPNTFGQGTKLEIK (SEQ ID NO: 89)

[0375] Affinity Variants

[0376] huEpCAM23LCGv4a DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSNGFTYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QNLELPNTFGQGTKLEIK (SEQ ID NO:90)

[0377] huEpCAM23LCGv4b DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGITYLYWFLQKPGQS PQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QNLELPNTFGQGTKLEIK (SEQ ID NO:91)

[0378] huEpCAM23LCGv4c DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QNLELPWTFGQGTKLEIK (SEQ ID NO:92)

[0379]

[0380] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence

[0381] huEpCAM23LCGv4e DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLSWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QNLELPNTFGQGTKLEIK (SEQ ID NO:93)

[0382] huEpCAM23LCGv4f DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWYLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QNLELPN TFGQGTKLEIK (SEQ ID NO: 94)

[0383] huEpCAM23LCGv4g DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCQ QNLELPNTFGQGTKLEIK (SEQ ID NO: 95)

[0384] huEpCAM23LCGv4h DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCL QNLELPNTFGQGTKLEIK (SEQ ID NO: 96)

[0385] huEpCAM23LCGv4i DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQYLELPNTFGQGTKLEIK (SEQ ID NO: 97)

[0386] huEpCAM23LCGv4j DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QGLELPNTFGQGTKLEIK (SEQ ID NO: 98)

[0387] huEpCAM23LCGv4k DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QSLELPNTFGQGTKLEIK (SEQ ID NO: 99)

[0388]

[0389]

[0191] In some embodiments, the immunoconjugates disclosed herein comprise EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VL sequence having a total of one, two, three, four, five, six, seven, eight, nine, ten, fewer than fifteen, or zero, amino acid substitutions, deletions, and / or insertions from a reference VH sequence selected from SEQ ID NOs: 86-99. In some embodiments, the insertions, substitutions, deletions, and / or insertions are in framework regions(s) of the reference sequence. In some embodiments, the substitutions are conservative. In other embodiments, the substitutions are non-conservative.

[0390]

[0192] In some embodiments, the immunoconjugates disclosed herein comprise an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VL having a sequence selected from SEQ ID NOs:86-99. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VL having the sequence of SEQ ID NO: 89. In some embodiments, the EpCAMAtty. Dkt. No.: 45325-0051WO1 antibody or EpCAM-binding antibody fragment thereof comprises a VL having the sequence of SEQ ID NO: 87. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to a sequence selected from SEQ ID NOs: 86-89. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VL sequence that is at least 90%.

[0391] 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, and 99%, identical to the sequence of SEQ ID NO: 89. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%, identical to the sequence of SEQ ID NO: 87. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%,

[0392]

[0193] In some embodiments, the immunoconjugates disclosed herein comprise EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH comprising a sequence selected from SEQ ID NOs:53-84 and a VL comprising a sequence selected from SEQ ID NOs: 86-89. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH comprising the sequence of SEQ ID NO:54 and a VL comprising the sequence of SEQ ID NO: 89, In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH comprising the sequence of SEQ ID NO:54 and a VL comprising the sequence of SEQ ID NO: 87. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH comprising the sequence of SEQ ID NO:55, and a VL comprising the sequence of SEQ ID NO: 87. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH comprising the sequence of SEQ ID NO:56, and a VL comprising the sequence of SEQ ID NO: 88. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH comprising the sequence of SEQ ID NO:55. and a VL comprising the sequence of SEQ ID NO: 89. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH comprising the sequence of SEQ ID NO:56, and a VL comprising the sequence of SEQ ID NO: 89.

[0393]

[0194] In some embodiments, the immunoconjugates disclosed herein comprise EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH comprising the sequence of SEQ ID NO:75, and a VL comprising tire sequence of SEQ ID NO: 89. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH comprising the sequence of SEQ ID NO:77, and a VL comprising the sequence of SEQ ID NO: 89. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH comprising the sequence of SEQ ID NO:76, and a VL comprising the sequence of SEQ ID NO: 89, In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH comprising the sequence of SEQ ID NO: 84, and a VL comprising the sequence of SEQ ID NO: 89.Atty. Dkt. No.: 45325-0051WO1

[0195] In some embodiments, the immunoconjugates disclosed herein comprise EpCAM antibody or EpCAM-binding antibody fragment thereof that competes for binding to human EpCAM with an antibody comprising aVH and a VL sequence disclosed in Tables 4 and 5, respectively.

[0394]

[0196] In one embodiment, the disclosure provides immunoconjugates comprising an EpCAM antibody or EpCAM-bmding antibody fragment thereof that competes for binding to human EpCAM with an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL) selected from:

[0395] (a) a heavy chain variable region (VH) of SEQ ID NO:54 and a light chain variable region (VL) of SEQ ID NO: 89;

[0396] (b) a VH of SEQ ID NO:54 and a VL of SEQ ID NO: 87;

[0397] (c) a VH of SEQ ID NO:75 and a VL of SEQ ID NO: 89; and

[0398] (d) a VH of SEQ ID NO: 77 and a VL of SEQ ID NO: 89.

[0399]

[0197] An EpCAM antibody or EpCAM-binding antibody fragment thereof (or an immunoconjugate having the EpCAM antibody or EpCAM-binding antibody fragment thereof) is said to "compete" with a reference molecule for binding to EpCAM if it binds to human EpCAM to the extent that it blocks, to some degree, binding of the reference molecule to human EpCAM The ability of proteins to compete for binding to EpCAM and thus to interfere with, block or "cross-block" one anothers' binding to EpCAM can be determined by any standard competitive binding assay known in the art including, for example, a competition ELISA assay, surface plasmon resonance (SPR; BIACORE®, Biosensor, Piscataway, N. J.) or according to methods described by Scatchard et al. (Ann. N. Y. Acad. Set. 51:660- 672 (1949)). An antibody can be said to competitively inhibit binding of tire reference EpCAM antibody to human EpCAM, for example, by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0400]

[0198] In some embodiments, immunoconjugates having the EpCAM antibody or EpCAM-binding antibody fragment thereof further includes a heavy chain constant region or fragment thereof. In some aspects, the antibody or antibody fragment comprises a heavy chain immunoglobulin constant region selected from the group consisting of: (a) a human IgA constant region, or fragment thereof; (b) a human IgD constant region, or fragment thereof: (c) a human IgE constant domain, or fragment thereof; (d) a human IgGl constant region, or fragment thereof; (e) a human IgG2 constant region, or fragment thereof; (f) a human IgG3 constant region, or fragment thereof; (g) a human IgG4 constant region, or fragment thereof; and (h) a human IgM constant region, or fragment thereof. In certain embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a heavy chain constant region or fragment thereof, e.g., a human IgG constant region or fragment thereof. In further embodiments, the EpCAM antibody or EpCAM-bmding antibody fragment thereof comprises a heavyAtty. Dkt. No.: 45325-0051WO1 chain immunoglobulin constant domain that has, or has been mutated to have altered effector function and / or half-life.

[0401]

[0199] In some embodiments, immunoconjugates having the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a heavy chain sequence disclosed in Table 6.

[0402] Table 6. Exemplary heavy chain full-length sequences of EpCAM antibodies.

[0403] Antibody name Sequence

[0404] Murine and Chimeric

[0405] muEpCAM-23HC EVKLEESGPALVKPGASVRTSCKASGYTFTNYYIHWVKQRPGQGLD YIGWIYPGNVATQYNEKFKGKATLTADKSSSTAFMQLSSLTSEDSAV YFCARDGPWFAYWGQGTLVTVSSAKTTPPSVYPLAPGCGDTTGSSV TLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTV PSSWPSQrVTCSVAHPASSriYDKKLEPSGPISTINPCPPCKECHKCP APNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWF VNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKV NNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGF NPGDISVEWTSNGHTEENYKDTAPVLDSDGSYF1YSKLD1KTSKWEK TDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID NO: 100) chEpcam23HC EVKLEESGPALVKPGASVRISCKASGYTFTNYYIHWVKQRPGQGLD YIGWIYPGNVATQYNEKFKGKATLTADKSSSTAFMQLSSLTSEDSAV YFCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPFAYCVVVDVSHEDPEVKFNWYVDG VEVFINAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQV7YTLPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 101)

[0406] Humanized variants

[0407] huEpCAM23 VHGv QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRL

[0408] 1 EWMGWIYPGNVYIQYSQKFQGRVTITRDTSASTAYMELSSLRSEDT AVYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNWHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

[0409]

[0410] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence DGVEVHNAKTKPREEQYNSTYRVVSVLWLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 102) huEpCAM23 VHGv QVQLVQSGAEVKKPGASVKVSCKASGV 1 F 1 NYYIHWVRQAPGQRL

[0411] 2 (lysine-linked) EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPS S SLGTQT YICN VNHKP SNTKVDKKVEPKS C * DKTHTC * PPC* P A PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSIYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQWTI. PPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 103)

[0412] * denotes cysteines which, when in a native conformation (e.g not

[0413] reduced), participate in forming interchain disulfide bonds.

[0414] huEpCAM23 VHGv QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRL

[0415] 2-C442 (site-specific) EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV

[0416] TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLWLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLCLSPG SLSPG (SEQ ID NO: 104) huEpCAM23 VHGv QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRL3 EWMGWIYPGNVYIQYNEKFKGRVTITRDTSASTAYMELSSLRSEDT AVYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS

[0417]

[0418] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence NKAI. PAPIEKTISKAKGQPREPQVYTLPPSRDEI. YKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 105) huEpCAM23 VHGv QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRL4 EYIGWIYPGNVYIQYSQKFQGRVTITADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 106)

[0419] Affinity variants

[0420] huEpCAM23HCGv2 QVQLVESGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRL a EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 107) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQRL b EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVI'CVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

[0421]

[0422] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 108) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNIHWVRQAPGQRL c EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 109) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRLd EYIGWIYPGDVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:110) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYFIHWVRQAPGQRL e EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 111)

[0423]

[0424] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence

[0425] huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYSIEIWVRQAPGQRL I EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTV'SSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNY'KTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMFIEALIINIIYTQKSLSLSPG (SEQ ID NO:112) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQRL Q EYIGWIYPGNVYIQYNEKFKGRATI YADK S ASTA YMELS SLR SEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK'l'HTCPPCPAPE IXGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVIINAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNH\'TQKSLSLSPG (SEQ ID NO: 113) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYY1HWVRQAPGQRL 11 EYIGWFYPGNVYIQYNEKFKGRATLTADKSASTAYMEI. SSLRSEDT

[0426] A VYYC ARDGPWFAYWG QGTLVTVS S ASTKGPS VFPL APS SKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK'mTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK. CKVS NKALPAPIEKTTSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSD1AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 114) huEpCAM23HCGv2 Q VQL VQSGAE VKKPGAS VKVSCKASGY 1 F 1 N YY1HWVRQAPGQRL i / 2o EWIGWIYPGNA'YIQYNEKFKGRATLTADKSASTAYMELSSLRSEDT

[0427] A\rYYCARDGPWFAWGQGTIJVT\7SSASTKGPSVFPLAPSSKSTSGG

[0428]

[0429] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQWTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 115) huEpCAM23HCGv2 QVQLVQSGAEVKKPG ASVKVSCKASGY 11 1 NYY1I IWVRQAPGQRL i EY1GWINPGNVY1QYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNAWiKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNM7YVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTX'DKSRWQQ GNVFSCSVMHEALHNFIYTQKSLSLSPG (SEQ ID NO:116) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNWIHUWQAPGQRL k EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCAREGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCL VKD Y FPEP VTV SWNSGALTSG VHTFPA VLQS SGL YSLSS VV TA'PSSSLGTQTYICNWHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYK'ITPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:1 17) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFFNYYIHWVRQAPGQRL L EYIGWIATGNVYTQYNEKFKGRATI. TADKSASTAYMELSSLRSEDTA \AATARDGPYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCL VKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTIA1ISRTPFYTCVVVDVSHEDPEVKFNWYVD

[0430]

[0431] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence GVEVHNAKTKPREEQYNSTYRAWSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 118) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGY 1 F! NYYIHWVRQAPGQRL m / huEpCani23HG2- EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA 1565-A Heavy Chain VYYC ARDG AWF AYWGQGTLVTVS SA STKGPS VFPLAPS SK STSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKHSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 119) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQR n LEYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDT AVYYCARDGPWFAYWGQGTLVrVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVTSSSLGTQTTTCNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSIYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTTSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 120) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGY1F 1NYYIHXWRQ APGQRL P EYIGWVYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDT AVYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVTSSSLGTQTYICNC'NHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEK TISKAKGQPREPQ VYTLPPSRDELTKNQVSL TCLVKGF

[0432]

[0433] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence YPSDLAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMIIEALIINIIYTQKSLSLSPG (SEQ ID NO: 121) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGT IF TNYYIHWVRQ APGQRL q EYIGWIYPGNVYIQYNEKFKGRATITADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVFTKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAV E W ESNGQPENNY KTIPP VLDSDGSFFLY SKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 122) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRL r EYIGWIYPGNVYIQYNEKFKGRATLTRDKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE IXGGPSVFLFPPKPKDTLMISRTPFATCVVVDVSHEDPEVKFNWYVD GVEVFINAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAV EVV ESNGQPENNY KTIPP VLDSDGSFFLY SKL TVDKSRWQQ GNVFSCSVMHEALHNHV'TQKSLSLSPG (SEQ ID NO: 123) huEpCAM23HCGv2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQRL s EYIGWIYPGNVYIQYNEKFKG RVTI YADK S ASTA YMELS SLR SEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVIVSWNSGALTSGVFrrFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVrCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 124)

[0434]

[0435] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence

[0436] huEpCam23HG2- QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYHIEIWVRQAPGQRL 1361 -II Heavy Chain EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNA'KTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMFIEALHNHYTQKSLSLSPG (SEQ ID NO: 125) huEpCam23HG2- QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYD1HWVRQAPGQRL 1361-D Heavy Chain EYIGWIYPGNVYIQYNEKFKGRATI YADK S ASTA YMELS SLR SEDT A VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVIINAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHA'TQKSLSLSPG (SEQ ID NO: 126) huEpCam23HG2- QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYY1HWVRQAPGQRL 1565- Y Heavy Chain EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGYWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLG I’QTYICN VNHKPSNTKVDKKVEPKSCDK'mTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 127) huEpCam23HG2- Q VQL VQSGAE VKKPGAS VKVSCKASGY 1 F 1 N YY1HWVRQAPGQRL 1565-S Heavy Chain EYIGW1YPGNVY1QYNEKFKGRATLTADKSASTAYMELSSLRSEDTA

[0437] VYYCARDGSWFAYWGQGTI. VTVSSASTKGPSVFPLAPSSKSTSGGT

[0438]

[0439] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 128) huEpCam23HG2- QVQLVQSGAEVKKPG ASVKVSCKASGY 11 1 NYYII IWVRQAPGQRL 1565-F Heavy Chain EYIGWIYPGNVY1QYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGFWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTX'DKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 129) huEpCam23HG2- QVQLVQSGAEVKKPGASVKVSCKASGYTFTNWIHUMRQAPGQRL 1565-G Heavy Chain EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGGWFAYWGQGTLVTVS SASTKGPS VFPLAPS SKSTSGGT AALGCL VKD Y FPEP VTV SWNSGALTSG VHTFPA VLQS SGL YSLSS VV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKITPPVLDSDGSFFLYSKUFVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 130) huEpCam23HG2- QVQLVQSGAEVKKPGASVKVSCKASGYTFFNYYIHWVRQAPGQRL 1565-T Heavy Chain EYIGWIA: TGNVYTQYNEKFKGRAn. TADKSASTAYMELSSLRSEDTA VYYCARDGTWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCL VKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD

[0440]

[0441] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence GVEVHNAKTKPREEQYNSTYRNWSVLWLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 13 i) huEpCam23HG2- QVQLVQSGAEVKKPGASVKVSCKASGY 1 F! NYYIHWVRQAPGQRL 1565- V Heavy Chain EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYC ARDG W AYWGQGTI. YTVS SA STKGPS VFPLAPS SK STSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKHSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 132) huEpCam23HG2- QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYIIHWVRQAPGQRLE 1361-1 Heavy Chain YIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTAV YYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNAWVDG VEVHNAKTKPREEQYNSTYRVVSVLl’VLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYT1. PPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNPsTCTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 133) huEpCam23HG2- QVQLVQSGAEVKKPGASVKVSCKASGY I F 1NYLIHWVRQAPGQRL 1361-L Heavy Chain EYIGWIYPGNVYIQYNEKFKGRATLTADKSASTAYMELSSLRSEDTA VYYCARDGPWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNWHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEK1TSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

[0442]

[0443] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 134)

[0444]

[0445]

[0200] In some embodiments, immunoconjugates having the EpCAM antibody or EpCAM-binding antibody fragment thereof comprise a heavy chain (HC) sequence selected from SEQ ID NOs: 102-134. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a heavy chain (HC) sequence selected from SEQ ID NOs: 102-106 In a specific embodiment, the EpCAM antibody or EpCAM-bmding antibody fragment thereof of any one of tire conditionally activatable masked immunoconjugates described herein comprises a HC sequence of SEQ ID NO: 103. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a HC sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to a sequence selected from SEQ ID NOs: 102-134. In some embodiments, tire EpCAM antibody or EpCAM-binding antibody fragment thereof comprises aHC sequence that is at least 90%. 91%. 92%.

[0446] 93%, 94%, 95%, 96%, 97%, 98%. or 99%, identical to a sequence selected from SEQ ID NOs: 102-106. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a HC sequence that is at least 90%, 91%, 92%. 93%, 94%, 95'%, 96%, 97%, 98%, and 99%, identical to the sequence of SEQ ID NO: 103. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0447]

[0201] In alternative embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises HC sequence selected from SEQ ID NOs: 125-127 and 134. In a specific embodiment, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC sequence of SEQ ID NO: 125. In a specific embodiment, the EpCAM antibody or EpCAM-bmding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC sequence of SEQ ID NO: 127. In some embodiments, the EpCAM antibody or EpCAM -binding antibody fragment thereof comprises a HC sequence that is at least 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to a sequence selected from SEQ ID NOs: 125-127 and 134. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises aHC sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%, and 99%. identical to the sequence of SEQ ID NO: 125. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%Atty. Dkt. No.: 45325-0051WO1

[0202] In additional aspects, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein composes a light chain immunoglobulin constant region. In a further aspect, the antibody comprises a human Ig kappa constant region or a human Ig lambda constant region.

[0448]

[0203] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein composes a light chain sequence disclosed in Table 7.

[0449] Table 7. Exemplary light chain full-length sequences of EpCAM antibodies.

[0450] Antibody name Sequence

[0451] Murine and Chimeric

[0452] muEpcam23LC DIVLTQTPFSNPVTLGTSASISCRSSKSLLHSDGFTYLYWFLQKPGQ SPHLLIYQTSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCA QNLELPNTFGGGTKI. EIKRADAAPTVSIFPPSSEQI. TSGGASVVCFI.

[0453] NNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTL TKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 135) chEpCam23LC DIVLTQTPFSNPVTLGTSASISCRSSKSLLHSDGFTYLYWFLQKPGQS PHLLIYQTSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQ NLELPN1TGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 136)

[0454] Humanized variants

[0455] huEpCAM23 VLG DIVMTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWYLQKPGQ

[0456] vl SPQLLIYQTSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCA QNLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCI. LN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 137) huEpCAM23 VLG DIVLTQTPLSLSVTPGQPASISCRSSKSLLHSDGFTYLYWFLQKPGQS

[0457] v2 PQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQ NLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTI. TI. SKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 138) huEpCAM23_VLG DIVMTQTPLSLSVTPGQPASISCRSSKSLLHSDGFTYLYWFLQKPGQS

[0458] v3 PQLL1YQTSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQ

[0459] NLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN

[0460]

[0461] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence FYPRFAKVQWKVDNALQSGNSQESVTEQDSKDSTYSESSTLTLSKA DYEKIIKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 139) buEpCAM23 VLG DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWTLQKPGQ

[0462] v4 SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QNLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID

[0463] NO: 140)

[0464] * denotes cysteine which, when in a native conformation (e.g.. not

[0465] reduced), participate in forming interchain disulfide bond.

[0466] Affinity variants

[0467] huEpCAM23LCGv DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSNGFTYLYWFLQKPGQ

[0468] 4a SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QNLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 141) huEpCAM23LCGv DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGITYLYWFLQKPGQS

[0469] 4b PQLLIY'QTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYAfCA QNLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 142) huEpCAM23LCGv DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQ

[0470] 4c SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QNLELPWTFGQGTKLEIKRl'VAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 143) huEpCAMGvLC4e DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYESWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVAATA QNLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSIYSLSSTLTL SKADYEKHKWACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 144) huEpCAMGvLC4f DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFIYLYWYLQKPGQ

[0471] SPQLL1YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGWYCA

[0472]

[0473] Atty. Dkt. No.: 45325-0051WO1 Antibody name Sequence QNLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 145) huEpCAMGvLC4g DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCQ QNLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKIIKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 146) huEpCAMGvLC4h D1VLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVY>rCL QNLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 147) huEpCAMGvLC4i DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QYLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 148) huEpCAMGvLC4j DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QGLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 149) huEpCAMGvLC4k DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQ SPQLLIYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCA QSLELPNTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 150)

[0474]

[0475]

[0204] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a light chain (EC) sequence selected from SEQ ID NOs: 137-150. In a specific embodiment, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatableAtty. Dkt. No.: 45325-0051WO1 masked immunoconjugates described herein comprises a LC sequence of SEQ ID NO:140. In another embodiment, the EpC AM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a LC sequence of SEQ ID NO: 138. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a LC sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. or 99% identical to a sequence selected from SEQ ID NOs: 137-150. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a LC sequence that is at least 90%, 91%, 92%. 93%, 94%. 95%, 96%, 97%, 98%, and 99%, identical to the sequence of SEQ ID NO: 140. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a LC sequence that is at least 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, and 99%, identical to the sequence of SEQ ID NO: 130. In some embodiments, the percent identity in each instance is 95 In some embodiments, the percent identity in each instance is 98%.

[0476]

[0205] In additional embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises aHC having a sequence selected from SEQ ID NOs: 102-134 and an LC having a sequence selected from SEQ ID NOs: 137-150. In additional embodiments, the EpCAM antibody or EpCAM- binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 103 and an LC having the sequence of SEQ ID NO: 140. In additional embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 103 and an LC having the sequence of SEQ ID NO: 138. In additional embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 105 and an LC having the sequence of SEQ ID NO: 138. In additional embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatablc masked immunoconjugates described herein composes a HC having the sequence of SEQ ID NO: 106 and an LC having the sequence of SEQ ID NO: 139. In additional embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 105 and an LC having the sequence of SEQ ID NO: 140. In additional embodiments, the EpCAM antibody7or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 106 and an LC having the sequence of SEQ ID NO: 140. In some embodiments, the EpCAM antibody or EpCAM- binding antibody fragment thereof of any one of the conditionally activatable maskedAtty. Dkt. No.: 45325-0051WO1 immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 125 and an LC having the sequence of SEQ ID NO: 140. In additional embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 127 and an LC having the sequence of SEQ ID NO: 140. In additional embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 126 and an LC having the sequence of SEQ ID NO: 140. In additional embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatablc masked immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 134 and an LC having the sequence of SEQ ID NO: 140.

[0477]

[0206] In some embodiments, the EpCAM antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises an altered (e g., mutated or engineered) Fc region. For example, in some aspects, the Fc region has been altered to reduce or enhance the effector functions of the antibody, alter serum half-life or other functional properties of the antibody. Reduction or elimination of effector function is desirable in certain cases, for example in the case of antibodies whose mechanism of action involves blocking or antagonism, but not killing of the cells bearing a target antigen. Increased effector function is generally desirable when directed to undesirable cells, such as tumor and foreign cells, where the FcyRs are expressed at low levels, for example, tumor-specific B cells with low levels of FcyRIIB (e.., non-Hodgkin’s lymphoma, CLL, and Burkitt’s lymphoma). Immunoconjugates of the invention possessing such conferred or altered effector function activity are useful for the treatment and / or prevention of a disease, disorder or infection in which an enhanced efficacy of effector function activity is desired. In some aspects, the Fc region is an isotype selected from IgM, IgA, IgG, IgE, or other isotype.

[0478]

[0207] Although the Fc Region of the EpCAM antibodies and EpCAM-binding antibody fragment thereof can possess the ability to bind to one or more Fc receptors (e.g,, FcyR(s)), in certain embodiments the antibody or antibody fragment comprises a variant Fc region having an altered binding to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD 16a) or FcyRIIIB (CD 16b) (relative to the binding exhibited by a wild-type Fc Region), e.g. has enhanced binding to an activating receptor and / or has substantially reduced or no ability to bind to inhibitory receptor(s). Tirus, the Fc region of the EpCAM antibody or EpCAM-binding antibody fragment thereof can include some or all of the CH2 domain and / or some or all of the CH3 domain of a complete Fc region, or can comprise a variant CH2 and / or a variant CH3 sequence (that can include, for example, one or more insertions and / or one or more deletions with respect to the CH2 or CH3 domains of a complete Fc Region). Such Fc regions can comprise non-Fc polypeptide portions, or can comprise portions of non-Atty. Dkt. No.: 45325-0051WO1 naturally complete Fc regions, or can comprise non-naturally occurring orientations of CH2 and / or CH3 domains (such as, for example, two CH2 domains or two CH3 domains, or in the N-terminal to C- terminal direction, a CH3 domain linked to a CH2 domain, etc.).

[0479]

[0208] Fc Region modifications identified as altering effector function are known in the art, including modifications that increase binding to activating receptors (e.g., FcyRlIA (CD16A) and reduce binding to inhibitory receptors (e.g, FcyRIIB (CD32B) (see, e.g., Stavenhagen, et al., Cancer Res.

[0480] 57(18): 8882-8890 (2007)). Table 8 lists exemplary single, double, triple, quadruple and quintuple substitutions (numbering is that of the EU index as in Kabat. and substitutions are relative to the amino acid sequence of SEQ ID NO:304) of exemplary modification that increase binding to activating receptors and / or reduce binding to inhibitory receptors.

[0481] Table 8. Variations of Particular Activating Fc Regions

[0482] Single-Site Variations

[0483] F243L R292G D270E R292P

[0484] Y300L P396L

[0485] Double-Site Variations

[0486] F243L and R292P F243L and Y300L F243L and P396L R292P and Y300L D270E and P396L R292P and V305I P396L and Q419H P247L and N421K R292P and P396L Y300L and P396L R255L and P396L R292P and P305I K392T and P396L

[0487] Triple-Site Variations

[0488] F243L, P247L and N421K P247L, D270E andN421K

[0489] F243L, R292P and Y300L R255L, D270E and P396L

[0490] F243L, R292P and V305I D270E, G316D and R416G

[0491] F243L. R292P and P396L D270E, K392T and P396L

[0492] F243L, Y300L and P396L D270E, P396L and Q419H

[0493] V284M, R292L and K370N R292P, Y300L and P396L

[0494] Quadruple-Site Variations

[0495] L234F. F243L. R292P and Y300L F243L. P247L. D270E and N421K

[0496] L234F, F243L, R292P and Y300L F243L, R255L, D270E and P396L

[0497] L235I, F243L, R292P and Y300L F243L, D270E. G316D and R416G

[0498] L235Q, F243L, R292P and Y300L F243L, D270E, K392T and P396L

[0499] P247L. D270E, Y300L and N421K F243L. R292P, Y300L, and P396L

[0500] R255L, D270E, R292G and P396L F243L, R292P, V3051 and P396L

[0501] R255L, D270E, Y300L and P396L F243L, D270E, P396L and Q419H

[0502]

[0503] Atty. Dkt. No.: 45325-0051WO1 D270E, G316D, P396L and R416G

[0504] Quintuple-Site Variations

[0505] L235V, F243L, R292P, Y300L and P396L F243L, R292P, V305I, Y300L and P396L L235P. F243L. R292P. Y300L and P396L

[0506]

[0507]

[0209] Exemplar)' variants of human IgGl Fc Regions with reduced binding to CD32B and / or increased binding to CD16A contain F243L, R292P, Y300L. V3051 or P396L substitutions, wherein the numbering is that of the EU index as in Kabat. These amino acid substitutions can be present in a human IgGl Fc Region in any combination. In one embodiment, the variant human IgGl Fc Region contains a F243L, R292P and Y300L substitution. In another embodiment, the variant human IgGl Fc Region contains a F243L, R292P, Y300L, V305I and P396L substitution.

[0508]

[0210] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises an immunoglobulin heavy chain constant region containing a modification that decreases effector function (see, e.g., Idusogie etal., J. Immunol. 166:2571-2575 (2001); Sazinsky et al., PNAS USA 105:20167- 20172 (2008); Davis et al.. J Rheumatol. 34:2204-2210 (2007); Bolt et al., Eur. J Immunol. 23:403- 411 (1993); Alegre et al.. Transplantation 57: 1537-1543 (1 94); Xu et al.. Cell Immunol. 200:16-26 (2000); Cole et l., Transplantation 68:563-571 (1999); Hutchins et al, PNAS USA 92:11980-11984 (1995); Reddy etal., J. Immunol. 164:1925-1933 (2000): WO97 / 11971, and WOO 7 / 106585; U. S. Appl. Publ. 2007 / 0148167A1; McEarchem et al.. Blood 109:1185-1192 (2007); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Kumagai et al.. J. Clin. Pharmacol. 47: 1489-1497 (2007), the contents of each of which is herein incorporated by reference in its entirety).

[0509]

[0211] In some embodiments, the Fc region of the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein to exhibit decreased (or substantially no) binding to an effector receptor selected from the group consisting of: FcyRIA (CD64), FcyRIIA (CD32A)(allotypes R 131 and Hl 31), FcyRTIB (CD32B), FcyRTIIA (CD 16a) (allotype VI 58 and Fl 58) and FcyRIIIB (CD 16b)(allotype FcbllTb-NAl and Fci.jIIlb-NA2); relative to the binding exhibited by the wild-type IgG Fc Region (SEQ ID NO:304). In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof Fc region variant effector receptor binding affinity has been reduced to 1 / 10 or less, 1 / 50 or less, or 1 / 100 or less as. compared to the binding affinity of the corresponding antibody or antibody binding fragment comprising the wildtype Fc region of the corresponding immunoglobulin.

[0510]

[0212] In a specific embodiment, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatablc masked immunoconjugates described herein comprises anAtty. Dkt. No.: 45325-0051WO1 IgG Fc region that exhibits reduced effector function (e.g.. reduced ADCC) and comprise a modification at one or more amino acid positions selected from the group consisting of 233, 234, 235, 236, 237, 238, 239, 265, 266, 267, 269, 270, 271, 295, 296, 297, 298, 300, 324, 325, 327, 328, 329, 331, and 332, wherein the amino acid position numbering is according to the EU index as set forth in Kabat. In one embodiment, the CH2-CH3 domain of the EpCAM antibody include any 1, 2, 3, or 4 of the substitutions: L234A, L235A. D265A, N297Q, N297A. and N297G, wherein the numbering is that of the EU index as in Kabat. In another embodiment, the CH2-CH3 domains contain an N297Q substitution, an N297A substitution, or L234A and L235A substitutions, as these mutations abolish FcR binding. Alternatively, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a CH2-CH3 domain of a naturally occurring Fc region that inherently exhibits decreased (or substantially no) binding to FcyRlIIA (CD 16a) and / or reduced effector function (relative to the binding and effector function exhibited by the wild-type IgGl Fc region (SEQ ID NO:304). In a specific embodiment, the Fc constant region of the EpCAM antibody comprises an IgG2 Fc region (SEQ ID NO:305) or an IgG4 Fc region (SEQ ID NO:306). Since the N297A, N297G, N297Q, L234A, L235A and D265A substitutions abolish effector function, in circumstances in which effector function is desired, these substitutions would in some instances not be employed.

[0511]

[0213] A particular IgGl sequence for the CH2 and CH3 domains of the Fc region-containing EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein that has reduced or abolished effector function comprises the substitutions L234A / L235A (shown underlined) (SEQ ID NO:307):

[0512] APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTTSKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG

[0513]

[0214] A particular IgGl sequence for the CH2 and CH3 Domains of the Fc region -containing EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein that has reduced or abolished effector function comprises the substitution N297A (shown underlined) (SEQ ID NO:308):

[0514] APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYASTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG

[0515]

[0215] A particular IgGl sequence for the CH2 and CH3 Domains of the Fc region -containing EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatableAtty. Dkt. No.: 45325-0051WO1 masked immunoconjugates described herein that has reduced or abolished effector function comprises the substitution N297Q (shown underlined) (SEQ ID NO:309):

[0516] APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYQSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPG

[0517]

[0216] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises one or more modifications corresponding to: IgGl-C220S, C226S, C229S, P238S; IgGl-C226S, C229S; IgGl- C226S, C229S, E233P, L234V, L235A; IgGl-E234A, L235A; IgGl-L234F, L235E. P331S; IgGl- L234F, L235E, P331S; TgGl-H268Q. A330S. P331S; IgGl-G236R, L328R; IgGl-L235G, G236R. IgGl-N297A; IgGl-N325A, L328R: IgGl-N325L. L328R; IgGl-K326W, E333S; IgG2-V234A.

[0518] G237A; IgG2-E333S; IgG2 H268Q, V309L, A330S, A331S; IgG4-S228P, L236E; IgG4-F234A, L235A; IgG4-F234A, G237A, E318A; IgG4-L235A, G237A, E318A; IgG4-L236E: IgG2-EU sequence 118-260; and IgG4-EU sequence 261-447: wherein the position numbering is according to the EU index as in Kabat.

[0519]

[0217] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a heavy chain immunoglobulin constant domain that has reduced CDC activity. In particular aspects, EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises an IgGl heavy chain constant region containing a mutation that decreases CDC activity (see. e.g., WO 1997 / 11971 and WO 2007 / 106585; U. S. Appl. Publ. 2007 / 0148167A 1; McEarchem etal., Blood 109: 1185-1192 (2007); Hayden -Ledbetter etal, Clin. Cancer 15:2739-2746 (2009); Lazar el al., PNAS USA 103:4005-4010 (2006); Bruckheimer el al.. Neoplasia 11:509-517 (2009); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Sazinsky el al., PNAS USA 105:20167-20172 (2008); each of which is herein incorporated by reference in its entirety). Examples of heavy chain constant domain sequence modifications that decrease CDC include one or more modifications corresponding to: IgGl-C226S, C229S, E233P, L234V, L235A; IgGl-C226S, P230S; IgGl-L234F, L235E, P331S; IgGl-S239D, A330L, I332E; IgG2 EU sequence 118-260; IgG4- EU sequence 261-447; and IgG2-H268Q, V309L, A330S, A331S, according to the EU index

[0218] In some embodiments, the provided EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a heavy chain immunoglobulin constant domain that contains one or more half-life extending amino acid modifications (e.g., substitutions). Numerous mutations capable of increasing the half-life of an Fc region-containing molecule are known in the art and are encompassed as components of theAtty. Dkt. No.: 45325-0051WO1 EpCAM antibodies and EpCAM-binding antibody fragment thereof provided herein. See. e.g. U. S. Patent Nos. 6,277,375; 7,083,784; 7,217,797, and 8,088,376; U. S. Publ. Nos. 2002 / 0147311; and 2007 / 0148164; and PCT Publication Nos. WO 1998 / 23289; WO 2009 / 058492; and WO 2010 / 033279, the contents of each of which is herein incorporated by reference in its entirety.

[0520]

[0219] The serum half-life of proteins comprising Fc regions can be increased by increasing the binding affinity of the Fc Region for FcRn. The term “half-life’' as used herein means a pharmacokinetic property of a molecule that is a measure of the mean survival time of the molecules following their administration. Half-life can be expressed as the time required to eliminate fifty percent (50%) of a known quantity of the molecule from a subject’s (e.g., a human patient or other mammal) body or a specific compartment thereof, for example, as measured in serum, i.e., circulating half-life, or in other tissues. In general, an increase in half-life results in an increase in mean residence time (MRT) in circulation for the administered molecule.

[0521]

[0220] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a half- life extending amino acid substitution at one or more positions selected from the group consisting of: 238. 250, 252. 254, 256. 257, 256.265, 272. 286, 288. 303, 305. 307, 308. 309, 3 l l. 312, 317. 340, 356, 360, 362, 376, 378. 380, 382. 413, 424, 428, 433, 434, 435, and 436. wherein the amino acid position numbering is according to the EU index In some embodiments, the EpCAM antibody or EpCAM- binding antibody fragment thereof contains one or more amino acid substitutions of amino acid residues at positions 251 -257, 285-290, 308-314, 385-389, and 428-436, wherein the amino acid position numbering is according to the EU index. In some embodiments, the EpCAM antibody or EpCAM- binding antibody fragment thereof contains one or more of a substitution of the amino acid at Kabat position 252 with Tyr, Phe. Trp, or Thr; a substitution of the amino acid at Kabat position 254 with Thr; a substitution of the amino acid at Kabat position 256 with Ser, Arg, Gin, Glu, Asp, or Thr; a substitution of the amino acid at Kabat position 257 with Leu; a substitution of the amino acid at Kabat position 309 with Pro; a substitution of the amino acid at Kabat position 311 with Ser; a substitution of the amino acid at Kabat position 428 with Thr, Leu. Phe, or Ser; a substitution of the amino acid at Kabat position 433 with Arg. Ser, Iso. Pro. or Gin; or a substitution of the amino acid at Kabat position 434 with Trp, Met, Ser, His, Phe, or Tyr More specifically, the EpCAM antibody or EpCAM-binding antibody fragment thereof domain can contain amino acid substitutions relative to a wild-type human IgG constant domain including a substitution of the amino acid at Kabat position 252 with Tyr, a substitution of the amino acid at Kabat position 254 with Thr, and a substitution of the amino acid at Kabat position 256 with Glu.

[0522]

[0221] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprises a leastAtty. Dkt. No.: 45325-0051WO1 one substitution selected from: T250Q, M252Y S254T, T256E, K288D, T307Q. V308P, A378V, M428L, N434A, N434S, N434H, N4.34Y, H435K, and Y436I, wherein the numbering is that of the EU index as in Kabat. In further embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises substitutions selected from: (a) M252Y, S254T and T256E, (b) M252Y and S254T; (c) M252Y and T256E; (d) T250Q and M428L; (e) T307Q and N434A; (f) A378 V and N434A: (g) N434A and Y436L (h) V308P and N434A; and (i) K288D and H435K

[0523]

[0222] In a particular embodiment, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein contains a variant IgG Fc Region comprising any 1, 2, or 3 of the substitutions: M252Y, S254T and T256E. The disclosure further provides EpCAM antibody or EpCAM-binding antibody fragment thereof possessing vanant Fc regions comprising: (a) one or more mutations which alter effector function and / or FcyR: and (b) one or more mutations which extend serum half-life.

[0524]

[0223] In additional embodiments, the disclosure provides immunoconjugates comprising EpCAM conditionally activatable masked antibody (e.g. conditionally activatable masked EpCAM antibodies and conditionally activatable masked EpCAM-binding antibody fragments thereof). In some embodiments, the EpCAM conditionally activatable masked antibody comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof that specifically binds EpCAM (e.g.. human EpCAM) coupled to a masking moiety (MM), such that coupling of the MM reduces the ability of the EpCAM antibody or EpCAM-binding antibody fragment thereof to bind EpCAM. In some embodiments, the MM is coupled via a sequence that includes a substrate for a protease, for example, a protease that is active in diseased tissue and / or a protease that is co-localized with EpCAM at a treatment site in a subject. In some embodiments, the immunoconjugates comprising an EpCAM conditionally activatable masked antibody includes an EpCAM antibody described in Section 0 coupled to a MM via a protease substrate.

[0525]

[0224] Tire EpCAM conditionally activatable masked antibodies are in some instances stable in circulation, activated at intended sites of therapy and / or diagnosis but not in normal, e g., healthy tissue or other tissue not targeted for treatment and / or diagnosis, and. when activated, exhibit binding to EpCAM that is at least comparable to the corresponding, unmodified antibody Immunoconjugates comprising the EpCAM conditionally activatable masked antibody are also provided, as are nucleic acids or sets of nucleic acids encoding the EpCAM conditionally activatable masked antibodies, and vectors and host cells comprising the nucleic acids. Pharmaceutical compositions comprising tire activatable conditionally activatable masked antibodies, immunoconjugates, nucleic acids, vectors, and host cells, are also provided.Atty. Dkt. No.: 45325-0051WO1

[0225] In some embodiments, immunoconjugates having the EpCAM conditionally activatable masked antibody or antibody fragment comprise:

[0526] (a) a cleavable moiety coupled to the antibody or antibody fragment, wherein the cleavable moiety is a polypeptide that functions as a substrate for a protease; and

[0527] (b) a masking moiety coupled to the antibody or antibody fragment, wherein the masking moiety inhibits the binding of the antibody or antibody fragment to EpCAM when the conditionally activatable masked antibody is in an uncleaved state,

[0528] wherein the conditionally activatable masked antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: (masking moiety)-(cleavable moiety) -(anti body or antibody fragment) or (antibody or antibody fragment) -(cleavable moi ety)-(m asking moiety).

[0529]

[0226] In some instances, an immunoconjugate disclosed herein comprises an EpCAM conditionally activatable masked antibody comprising:

[0530] (a) an EpCAM antibody or EpCAM -binding antibody fragment thereof comprising a VH-CDR1, VH- CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having the sequences of a member selected from the group:

[0531] (i) SEQ ID NOs: 13-15, 42, 40, and 41. respectively:

[0532] (ii) SEQ ID NOs: 13-15, and 39-41, respectively;

[0533] (iii) SEQ ID NOs: 13, 26, 15, and 39-41, respectively; and

[0534] (iv) SEQ ID NOs: 13, 24, 15, 42, 40, and 41, respectively;

[0535] (b) a masking moiety coupled to the EpCAM antibody or EpCAM-binding antibody fragment thereof, wherein the masking moiety inhibits the binding of the antibody or antibody fragment to EpCAM when the conditionally activatable masked antibody is m an uncleaved state; and

[0536] (c) a cleavable moiety coupled to the EpCAM antibody or EpCAM-binding antibody fragment thereof, wherein the cleavable moiety is a polypeptide that functions as a substrate for a protease; wherein the conditionally activatable masked antibody in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: (masking moiety)-(cleavable moiety) -(antibody or antibody fragment) or (antibody or antibody fragment) -(cleavable moiety)-(masking moiety).

[0537]

[0227] The EpCAM conditionally activatable masked antibodies of the immunoconjugates described herein in an activated state bind human EpCAM and include (i) an EpCAM antibody or a EpCAM- binding antibody fragment thereof (AB) of any one of the conditionally activatable masked immunoconjugates described herein that specifically binds to human EpCAM (as disclosed herein in, e.g., Section 0); (ii) a masking moiety (MM) that, when the EpCAM conditionally activatable masked antibody, is in an uncleaved state, inhibits the binding of the EpCAM conditionally activatable masked antibody to EpCAM: and (c) a cleavable moiety (CM) coupled to the EpCAM antibody or EpCAM- binding antibody fragment thereof, wherein the CM is a polypeptide that functions as a substrate for aAtty. Dkt. No.: 45325-0051WO1 protease. In some embodiments, the EpCAM conditionally activatable masked antibody, in the uncleaved state has the structural arrangement from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM In some embodiments, the EpCAM conditionally activatable masked antibody, comprises a linking peptide between the MM and the CM. In some embodiments, the EpCAM conditionally activatable masked antibody, comprises a linking peptide between the CM and the AB.

[0538]

[0228] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein, in an uncleaved state specifically binds to mammalian EpCAM with a dissociation constant less than or equal to 1 nM, less than or equal to 5 nM, less than or equal to 10 nM, less than or equal to 15 nM, less than or equal to 20 nM, less than or equal to 25 nM. less than or equal to 50 nM, less than or equal to 100 nM, less than or equal to 150 nM. less than or equal to 250 nM, less than or equal to 500 nM, less than or equal to 750 nM, less than or equal to 1000 nM. and 122. / or less than or equal to 2000 nM.

[0539]

[0229] In some embodiments, the EpCAM conditionally activatable masked antibody of the immunoconjugates described herein, in an uncleaved state specifically binds to mammalian EpCAM (e.g.. human EpCAM or cynomolgus EpCAM) with a dissociation constant greater than or equal to 1 nM, greater than or equal to 5 nM. greater than or equal to 10 nM. greater than or equal to 15 nM. greater than or equal to 20 nM. greater than or equal to 25 nM, greater than or equal to 50 nM. greater than or equal to 100 nM, greater than or equal to 150 nM, greater than or equal to 250 nM, greater than or equal to 500 nM, greater than or equal to 750 nM, greater than or equal to 1000 nM, and 122. / or greater than or equal to 2000 nM.

[0540]

[0230] In some embodiments, the EpCAM conditionally activatable masked antibody of the immunoconjugates described herein, in an uncleaved state specifically binds to the mammalian EpCAM (e.g., human EpCAM or cynomolgus EpCAM) with a dissociation constant in the range of 1 nM to 2000 nM, 1 nM to 1000 nM. 1 nM to 750 nM, 1 nM to 500 nM, 1 nM to 250 nM, 1 nM to 150 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 25 nM, 1 nM to 15 nM, 1 nM to 10 nM, 1 nM to 5 nM, 5 nM to 2000 nM, 5 nM to 1000 nM. 5 nM to 750 nM, 5 nM to 500 nM, 5 nM to 250 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 25 nM. 5 nM to 15 nM. 5 nM to 10 nM. 10 nM to 2000 nM, 10 nM to 1000 nM, 10 nM to 750 nM. 10 nM to 500 nM, 10 nM to 250 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 25 nM, 10 nM to 15 nM, 15 nM to 2000 nM, 15 nM to 1000 nM, 15 nM to 750 nM, 15 nM to 500 nM, 15 nM to 250 nM, 15 nM to 150 nM, 15 nM to 100 nM, 15 nM to 50 nM, 15 nM to 25 nM, 25 nM to 2000 nM, 25 nM to 1000 nM, 25 nM to 750 nM, 25 nM to 500 nM, 25 nM to 250 nM, 25 nM to 150 nM, 25 nM to 100 nM, 25 nM to 50 nM, 50 nM to 2000 nM, 50 nM to 1000 nM, 50 nM to 750 nM. 50 nM to 500 nM, 50 nM to 250 nM. 50 nM to 150 nM, 50 nM to 100 nM.

[0541] 100 nM to 2000 nM. 100 nM to 1000 nM, 100 nM to 750 nM, 100 nM to 500 nM. 100 nM to 250 nM, 100 nM to 150 nM, 150 nM to 2000 nM. 150 nM to 1000 nM, 150 nM to 750 nM. 150 nM to 500 nM,Atty. Dkt. No.: 45325-0051WO1 150 nM to 250 nM, 250 nM to 2000 nM, 250 nM to 1000 nM, 250 nM to 750 nM, 250 nM to 500 nM, 500 nM to 2000 nM, 500 nM to 1000 nM. 500 nM to 750 nM, 500 nM to 500 nM, 500 nM to 250 nM, 500 nM to 150 nM, 500 nM to 100 nM, 500 nM to 50 nM, 750 nM to 2000 nM, 750 nM to 1000 nM, or 1000 nM to 2000 nM.

[0542]

[0231] In some embodiments, the EpCAM conditionally activatable masked antibody, in an activated state specifically binds to mammalian EpCAM (e.g., human EpCAM or cynomolgus EpCAM) with a dissociation constant that is less than or equal to 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, or 10 nM. In some embodiments, the EpCAM conditionally activatable masked antibody, in an activated state specifically binds to mammalian EpCAM with a dissociation constant is greater than or equal to 0.01 nM, 0,05 nM, 0,1 nM, 0.5 nM, 1 nM. 5 nM, or 10 nM.

[0543]

[0232] In some embodiments, the EpCAM conditionally activatable masked antibody, in an activated state specifically binds to the mammalian EpCAM e.g., human EpCAM or cynomolgus EpCAM) with a dissociation constant in the range of 0.01 nM to 100 nM, 0.01 nM to 10 nM, 0.01 nM to 5 nM, 0.01 nM to 1 nM, 0.01 to 0.5 nM, 0.01 nm to 0.1 nM, 0.01 nm to 0.05 nM, 0.05 nM to 100 nM, 0.05 nM to 10 nM, 0.05 nM to 5 nM, 0.05 nM to 1 nM, 0.05 to 0.5 nM, 0,05 nm to 0.1 nM, 0.1 nM to 100 nM, 0.1 nM to 10 nM, 0.1 nM to 5 nM, 0.1 nM to 1 nM. 0.1 to 0.5 nM, 0.5 nM to 100 nM. 0.5 nM to 10 nM, 0.5 nM to 5 nM, 0.5 nM to 1 nM, 1 nM to 100 nM. 1 nM to 10 nM, 1 nM to 5 nM, 5 nM to 100 nM, 5 nM to 10 nM, or 10 nM to 100 Nm.

[0544]

[0233] In some embodiments, the EpCAM conditionally activatable masked of the immunoconjugates described herein specifically binds to human EpCAM with a dissociation constant of less than 1 nM. In some embodiments, the EpCAM conditionally activatable masked antibody specifically binds to cynomolgus EpCAM with a dissociation constant of less than 1 nM. In some embodiments, the EpCAM conditionally activatable masked antibody specifically binds to human EpCAM and cynomolgus EpCAM with a dissociation constant of less than 1 nM.

[0545]

[0234] In some embodiments, the serum half-life of the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein is longer than that of the corresponding antibody; e.g., the pK of the EpCAM conditionally activatable masked antibody is longer than that of the corresponding antibody. In some embodiments, the serum half-life of the EpCAM conditionally activatable masked antibody is similar to that of the corresponding antibody.

[0546]

[0235] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR 1, a VH-CDR2, and a VH-CDR3 having the sequences set forth in one row of Table 2. In some embodiments, the EpCAM conditionallyAtty. Dkt. No.: 45325-0051WO1 activatable masked antibody comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VL-CDR1. aVL-CDR2, and a VL-CDR3 having the sequences set forth in one row of Table 3.

[0547]

[0236] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR 1 selected from SEQ ID NOs: 13, and 16-25; a VH-CDR2 selected from SEQ ID NOs: 14, and 26-29; a VH-CDR3 selected from SEQ ID NOs: 15, and 30-38; a VL-CDR1 selected from SEQ ID NOs:39, and 42-45; a VL-CDR2 of SEQ ID NO:40; and a VL-CDR3 selected from SEQ ID NOs:41, and 46-51.

[0548]

[0237] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of tire conditionally activatable masked immunoconjugates described herein comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR1, VH-CDR2, VH-CDR3, VL- CDR1, VL-CDR2, and VL-CDR3 having the sequences selected from the group consisting of: (i) SEQ ID NOs: 13-15, 42, 40, and 41, respectively: (ii) SEQ ID NOs: 13-15, and 39-41, respectively; (lii) SEQ ID NOs: 13, 26, 15, and 39-41, respectively: and (iv) SEQ ID NOs: 13, 26, 15, 42, 40. and 41. respectively. In some embodiments, the EpCAM conditionally activatable masked antibody comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR1, VH- CDR2. VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having the sequences of SEQ ID NOs: 13-15, 42, 40, and 41, respectively,

[0549]

[0238] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of tire conditionally activatable masked immunoconjugates described herein comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR1. VH-CDR2, VH-CDR3. VL- CDR1, VL-CDR2, and VL-CDR3 having the sequences selected from the group consisting of: (i) SEQ ID NOs: 22, 14, 15, 42, 40, and 41, respectively; (ii) SEQ ID NOs: 13, 14, 33, 42, 40, and 41, respectively; (hi) SEQ ID NOs: 23, 14, 15, 42, 40, and 41, respectively, and; (iv) SEQ ID NOs: 25, 14, 15, 42. 40. and 41. respectively. In some embodiments, the EpCAM conditionally activatable masked antibody comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH-CDR 1, VH-CDR2. VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having the sequences of SEQ ID NOs: 22, 14, 15, 42, 40, and 41, respectively. In some embodiments, the EpCAM conditionally activatable masked antibody comprises an EpCAM antibody or EpCAM -binding antibody fragment thereof comprising a VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having the sequences of SEQ ID NOs: 13, 14. 33, 42, 40, and 41, respectively.

[0550]

[0239] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a VH disclosed inAtty. Dkt. No.: 45325-0051WO1 Table 4. In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a VL disclosed in Table 5. In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a VH having the sequence of SEQ ID NO: 54 and a VL having the sequence of SEQ ID NO: 89. In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof of any one of the conditionally activatable masked immunoconjugates described herein comprising a VH having the sequence of SEQ ID NO: 75 and a VL having tire sequence of SEQ ID NO: 89. In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH having the sequence of SEQ ID NO: 77 and a VL having the sequence of SEQ ID NO: 89.

[0551]

[0240] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a VH sequence that is at least 90%. 91%. 92%. 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from SEQ ID NO: 54, 75, and 77. In some embodiments, the EpCAM conditionally activatable masked antibody comprises a VL sequence that is at least 90%, 91 %, 92%. 93%, 94%, 95%. 96%, 97%, 98% or 99% identical to a sequence comprising SEQ ID NO: 89. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0552]

[0241] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC disclosed in Table 6. In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a LC disclosed in Table 7. In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 103 and a LC having the sequence of SEQ ID NO: 140, In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 125 and a LC having the sequence of SEQ ID NO: 140. In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 127 and a light chain having the sequence of SEQ ID NO: 140.

[0553]

[0242] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises an EpCAM antibodyAtty. Dkt. No.: 45325-0051WO1 or EpCAM-binding antibody fragment thereof that specifically binds to an epitope within the extracellular region of human EpCAM (SEQ ID NO: 1). In certain embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises an EpCAM antibody or EpCAM-binding antibody fragment thereof that specifically binds to an epitope within the first extracellular domain (DI) of human EpCAM (SEQ ID NO:2).

[0554]

[0243] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a VH-CDR1 comprising X1YX3X4H, wherein Xi is selected from and S, X3is selected from Y, N, F, S, H, D, L, I, and W. and X4is selected from I and M; a VH-CDR2 comprising WX2X3PGX6VYIQYX12X13KFX17G, wherein X2is selected from I and F, X3is selected from Y and N, X6is selected from N and D, X12is selected from N and S, X13is selected from E and Q, and X17is selected from K and Q (SEQ ID NO:7); and a VH-CDR3 comprising X1GX3X4FAY, wherein Xi is selected from D and E, X3is selected from P, A, S. Y, F, G, T, and V, and X4is selected from Y and W (SEQ ID NO:8). In some embodiments, the EpCAM conditionally activatable masked antibody comprises a VL-CDRI comprising RSSX4SLLHSX10G X12TYLX16, wherein X4is selected from R and K. X;o is selected from N and D, X12is selected from F and I, and X16is selected from Y and S (SEQ ID NO: 10); a light chain VL-CDR2 comprising QTSNLAS (SEQ ID NO:40); and a VL-CDR3 comprising XiQX3LELPXsT, wherein Xi is selected from A, L, and Q, X3is selected from S, G. Y, and N, and X8is selected from N and W (SEQ ID NO: 11). In some embodiments, the EpCAM conditionally activatable masked antibody comprises a VH-CDR1 comprising the sequence of SEQ ID NO: 13; a VH-CDR2 comprising the sequence of SEQ ID NO: 14; a VH-CDR3 comprising the sequence of SEQ ID NO: 15; a VL-CDRI comprising the sequence of SEQ ID NO:42: a VL-CDR2 comprising the sequence of SEQ ID NO:40; and a VL-CDR3 comprising the sequence of SEQ ID NO:41.

[0555]

[0244] In some embodiments, the VH-CDR1 of the EpCAM conditionally activatable masked antibody of any one of tire conditionally activatable masked immunoconjugates described herein comprises the sequence NYX3IH, wherein X3is selected from Y, N, F, S. H, D. L, I, and W (SEQ ID NO: 6). In some embodiments, the VH-CDR3 of the EpCAM conditionally activatable masked antibody composes the sequence DGPX₄FAY, wherein X4is selected from Y and W (SEQ ID NON) In some embodiments, the VL-CDR3 of the EpCAM conditionally activatable masked antibody comprises the sequence AQX₃LELPNT, wherein X3is selected from S, G, Y, and N (SEQ ID NO: 12). In some embodiments, the EpCAM conditionally activatable masked antibody comprises a VH-CDR1 comprising the sequence of SEQ ID NO: 13; a VH-CDR2 comprising the sequence of SEQ ID NO: 14; a VH-CDR3 comprising the sequence of SEQ ID NO: 15; a VL-CDR1 comprising the sequence of SEQ ID NO:42; a VL-CDR2Atty. Dkt. No.: 45325-0051WO1 comprising the sequence of SEQ ID NO:40; and a VL-CDR3 comprising the sequence of SEQ ID NO:41.

[0556]

[0245] Suitable components of the disclosed EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein also include an EpCAM antibody or EpCAM-binding antibody fragment thereof, that cross-competes for binding to human EpCAM and / or cynomolgus EpCAM with an EpCAM antibody comprising a VH having the sequence of SEQ ID NO: 54 and a VL having the sequence of SEQ ID NO: 89. Additional suitable EpCAM conditionally activatable masked antibodies cross-compete for binding to human EpCAM and / or cynomolgus EpCAM with an EpCAM antibody comprising a VH having the sequence of SEQ ID NO: 75 and a VL having the sequence of SEQ ID NO: 89. Additional suitable EpCAM conditionally activatable masked antibodies cross-compete for binding to human EpCAM and / or cynomolgus EpCAM with an EpCAM antibody comprising a VH having the sequence of SEQ ID NO: 77 and a VL having the sequence of SEQ ID NO: 89.

[0557]

[0246] The EpCAM conditionally activatable masked antibodies of any one of the conditionally activatable masked immunoconjugates described herein provided herein include a masking moiety (MM). In some embodiments, the masking moiety (or "mask”) is an amino acid sequence that is coupled or otherwise attached to the EpCAM antibody and is positioned within the EpCAM conditionally activatable masked antibody construct such that the masking moiety reduces the ability of the EpCAM antibody to specifically bind EpCAM. Suitable masking moieties are identified using any of a variety of known techniques. For example, peptide masking moieties are identified using the methods described in WO 2009 / 025846. the contents of which is herein incorporated by reference in its entirety.

[0558]

[0247] In some embodiments, the MM of the conditionally activatable masked antibody has a dissociation constant for binding to the AB which is greater than the dissociation constant of the AB to EpCAM. In some embodiments, the MM has a dissociation constant for binding to the AB which is no more than the dissociation constant of the AB to EpCAM.

[0559]

[0248] In some embodiments, the MM has a dissociation constant for binding to the AB which is less than the dissociation constant of the AB to EpCAM.

[0560]

[0249] In some embodiments, the dissociation constant (KQ of the MM towards the AB is no more than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000,

[0561] 1,000,000. 5,000,000, 10,000,000. 50,000.000 times or greater, or between 1-5, 5-10, 10-100, 10-1,000, 10-10.000, 10-100,000, 10-1,000.000. 10-10,000,000. 100-1,000, 100-10.000. 100-100.000. 100- 1,000,000, 100-10,000,000, 1,000- 10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-Atty. Dkt. No.: 45325-0051WO1 100,000, 10,000-1,000,000, 1,000- 10,000,000, 100.000-1,000,000, or 100,000-10,000,000 times or greater than the dissociation constant of the AB towards the target.

[0562]

[0250] In some embodiments, the MM does not interfere or compete with the AB for binding to EpCAM when the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein is in a cleaved state. In some embodiments, the MM is a polypeptide of about 2 to 40 amino acids in length. In some embodiments, the MM is a polypeptide of up to about 40 amino acids in length.

[0563]

[0251] In some embodiments, the MM polypeptide sequence is different from that of EpCAM. In some embodiments, the MM polypeptide sequence is no more than 50% identical to any natural binding partner of the AB, In some embodiments, the MM polypeptide sequence is different from that of EpCAM and is no more than 40%. 30%, 25%, 20%, 15%, or 10% identical to any natural binding partner of the AB.

[0564]

[0252] In some embodiments, the coupling of the MM to the AB reduces the ability of the AB to bind EpCAM such that tire dissociation constant (Kd) of the AB when coupled to the MM towards EpCAM is at least 2, 5, 10, 20, 40, 100. 1.000, 10,000 greater than the Kd of the AB when not coupled to the MM towards EpCAM.

[0565]

[0253] In some embodiments, in the presence of EpCAM, the MM reduces the ability of the AB to bind EpCAM by at least 90% when the CM is uncleaved, as compared to when the CM is cleaved when assayed in vitro using a target displacement assay such as, for example, the assay described in WO 2010 / 081173, the contents of which are hereby incorporated by reference in its entirety.

[0566]

[0254] When the AB is modified with a MM and is in the presence of human EpCAM, specific binding of the AB to human EpCAM is reduced or inhibited, as compared to the specific binding of the AB not modified with an MM or the specific binding of the parental AB to human EpCAM.

[0567]

[0255] The Kd of the AB modified with a MM towards human EpCAM is at least 5, 10, 25, 50, 100, 250.500, 1,000.2.500. 5,000, 10,000.50.000. 100,000, 500,000, 1,000.000. 5,000,000, 10,000.000. 50.000.000 or greater, or between 5-10, 10-100, 10-1,000, 10-10.000, 10-100,000, 10-1,000.000. 10- 10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000.000, 1,000-10,000, 1,000-100,000, 1,000-1,000.000, 1000-10.000,000, 10,000-100,000, 10,000-1,000,000,

[0568] 10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times greater than the Kd of tire AB not modified with an MM or of the parental AB towards human EpCAM. Conversely, the binding affinity of the AB modified with a MM towards human EpCAM is at least 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1.000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000,Atty. Dkt. No.: 45325-0051WO1 1,000-1,000,000, 1000-10,000,000, 10,000-100,000. 10,000-1,000,000, 10,000-10.000,000, 100,000- 1,000,000, or 100,000-10,000,000 times lower than the binding affinity of the AB not modi fied with an MM or of the parental AB towards human EpCAM.

[0569]

[0256] In some embodiments, the dissociation constant (Kd) of the MM towards the AB is approximately equal to the Kd of the AB towards human EpCAM. In some embodiments, the dissociation constant (Kd) of the MM towards the AB is no more than the dissociation constant of the AB towards human EpCAM. In some embodiments, the dissociation constant (Kd) of the MM towards the AB is less than the dissociation constant of the AB towards human EpCAM. In some embodiments, the dissociation constant (Kd) of the MM towards the AB is greater than the dissociation constant of the AB towards human EpCAM,

[0570]

[0257] In some embodiments, the MM has a Kd for binding to the AB that is no more than the Kd for binding of the AB to human EpCAM.

[0571]

[0258] In some embodiments, the MM has a Kd for binding to the Bb that is no less than the Kd for binding of the AB to human EpCAM. In some embodiments, the MM has a Kd for binding to the AB that is approximately equal to the Kd for binding of the AbBto human EpCAM. In some embodiments, the MM has a Kd for binding to the AB that is less than the Kd for binding of the AB to human EpCAM. In some embodiments, the MM has a Kd for binding to the AB that is greater than the Kd for binding of the AB to human EpCAM. In some embodiments, the MM has a Kd for binding to the AB that is no more than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 fold greater than the Kd for binding of the AB to human EpCAM. In some embodiments, the MM has a Kd for binding to the AB that is between 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100. 10-1.000, 20-100, 20-1000, or 100-1.000 fold greater than the Kd for binding of the AB to human EpCAM.

[0572]

[0259] In some embodiments, the MM has an affinity for binding to the AB that is less than the affinity of binding of the AB to human EpCAM. In some embodiments, tire MM has an affinity for binding to the AB that is no more than the affinity of binding of the AB to human EpCAM. In some embodiments, the MM has an affinity for binding to the AB that is approximately equal of the affinity of binding of the AB to human EpCAM. In some embodiments, the MM has an affinity for binding to the AB that is no less than the affinity of binding of the AB to human EpCAM. In some embodiments, the MM has an affinity for binding to the AB that is greater than tire affinity of binding of the AB to human EpCAM.

[0573]

[0260] In some embodiments, the MM has an affinity for binding to the AB that is 2, 3. 4, 5, 10, 25, 50, 100, 250, 500. or 1,000 less than the affinity of binding of the AB to human EpCAM. I In some embodiments, the MM has an affinity for binding to the AB that is between 1-5. 2-5, 2-10, 5-10. 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1000, or 100- 1,000 fold less than the affinity of binding of the AB to human EpCAM. In some embodiments, the MM has an affinity for binding to the AB that isAtty. Dkt. No.: 45325-0051WO1 2 to 20 fold less than the affinity of binding of the AB to human EpCAM. In some embodiments, a MM not covalently linked to the AB and at equimolar concentration to the EpCAM conditionally activatable masked antibody does not inhibit the binding of tire AB to human EpCAM.

[0574]

[0261] When the AB is modified with a MM and is in the presence of human EpCAM specific binding of the AB to human EpCAM is reduced or inhibited, as compared to the specific binding of the AB not modified with an MM or the specific binding of the parental AB to human EpCAM. When compared to the binding of the AB not modified with an MM or the binding of the parental AB to human EpCAM, the AB's ability’ to bind human EpCAM when modified with an MM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36. 48. 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45. 60. 90, 120, 150. or 180 days, or 1.

[0575] 2, 3. 4, 5, 6, 7. 8, 9, 10. 11, or 12 months or more w hen measured in vivo or in an in vitro assay.

[0576]

[0262] The MM inhibits the binding of the AB to human EpCAM. Tire MM binds the antigen binding domain of the AB and inhibits binding of the AB to human EpCAM. Hie MM can sterically inhibit the binding of the AB to human EpCAM. The MM can allosterically inhibit the binding of the AB to its target. In these embodiments, when the AB is modified or coupled to a MM and in the presence of target there is no binding or substantially no binding of the AB to human EpCAM, or no more than 0.001%, 0.01%, 0.1%, 1%. 2%, 3%, 4%, 5%, 6%, 7%. 8%, 9%, 10%, 15%, 20%, 25%, 30%. 35%. 40%, or 50% binding of the AB to human EpCAM, as compared to the binding of the AB not modified with an MM, the parental AB, or the Bb not coupled to an MM to human EpCAM, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 11, or 12 months or longer when measured in vivo or in an in vitro assay.

[0577]

[0263] When an AB is coupled to or modified by a MM, the MM 'masks' or reduces or otherwise inhibits the specific binding of the AB to human EpCAM. When an AB is coupled to or modified by a MM, such coupling or modification can effect a structural change that reduces or inhibits the ability of the AB to specifically bind its target.

[0578]

[0264] An AB coupled to or modified with an MM can be represented by the following formulae (in order from an amino (N) terminal region to carboxyl (C) terminal region:

[0579] (MM)-(AB)

[0580] (AB)-(MM)

[0581] (MM)-L-(AB)

[0582] (AB)-L-(MM)

[0583] where MM is a masking moiety, the AB is an EpCAM antibody or EpCAM-binding antigen fragment thereof, and the L is a linker. In many embodiments, it can be desirable to insert one or more linkers, e.g.. flexible linkers, into the composition so as to provide for flexibility.Atty. Dkt. No.: 45325-0051WO1

[0265] In certain embodiments, the MM is not a natural binding partner of the AB. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%.

[0584] 50%, 55%, 60%, 65%, 70%. 75%, or 80% similar to any natural binding partner of the AB. In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%. 50%. 55%.

[0585] 60%, 65%. 70%, 75%, or 80% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 25% identical to any’ natural binding partner of the AB. In some embodiments, the MM is no more than 50% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 20% identical to any natural binding partner of the AB. In some embodiments, the MM is no more than 10% identical to any natural binding partner of the AB.

[0586]

[0266] In some embodiments, the MM comprises a sequence disclosed in Table 9. In some embodiments, the MM comprises a sequence selected from SEQ ID NOs:151-157. In some embodiments, the MM comprises a sequence selected from SEQ ID NOs: 158-161. In some embodiments, the MM comprises a sequence selected from SEQ ID NOs: 162-167. In some embodiments, the MM comprises the sequence of SEQ ID NO: 155.

[0587] Table 9. Exemplary Masking Moiety Sequences.

[0588] Mask name Sequence

[0589] Masks for EpCAM23Gv4.2 humanized antibody

[0590] Ep01-2 PLMTCSDYYTCLNNL (SEQ ID NO: 151)

[0591] Ep02 LSCTHSRYDMHCPHM (SEQ ID NO: 152)

[0592] Ep03 HYCHSRTDTITHCNA (SEQ ID NO: 153)

[0593] Ep04 WCPRLFDRPSMGCPT (SEQ ID NO: 154)

[0594] Ep05 WW PPCQGGAWCEQRI (SEQ ID NO: 155)

[0595] Ep07 HSGCPRLFDRCSAPA (SEQ ID NO: 156)

[0596] Ep11 FICPTLYDRPHCMHT (SEQ ID NO: 157)

[0597] Masks for 1565- Y affinity variant

[0598] Ep101 SWCHSATDTILPCSN (SEQ ID NO: 158)

[0599] Ep102 SPACSDRYYQTCVLN (SEQ ID NO: 159)

[0600] Ep103 MSCVVDRFDRQCPHL (SEQ ID NO: 160)

[0601] Ep104 TTRCEIIYWFTCPLSP (SEQ ID NO: 161)

[0602] Masks for 1361-H affinity variant

[0603] Ep105 DCTGYSPSVLPACRV (SEQ ID NO: 162)

[0604] Ep106 FCSGYSPSVLPSCLM (SEQ ID NO: 163)

[0605] Ep107 SKPCSYMHPYCFYNS (SEQ ID NO: 164)

[0606]

[0607] Atty. Dkt. No.: 45325-0051WO1 Mask name Sequence

[0608] Ep108 LTRCTIAHPYCYYNY (SEQ ID NO: 165)

[0609] Ep109 PNTCMSERRICSLTY (SEQ ID NO: 166)

[0610] Ep110 PRPHCAILRQCLAAT (SEQ ID NO: 167)

[0611]

[0612]

[0267] The EpCAM conditionally activatable masked antibodies of any one of the conditionally activatable masked immunoconjugates described herein provided herein include a cleavable moiety. In some embodiments, the cleavable moiety (or “substrate") includes an amino acid sequence that is a substrate for a protease, usually an extracellular protease. Suitable substrates are identified using any of a variety of known techniques. For example, peptide substrates are identified using the methods described in US Patent Nos. 7,666,817 and 8,563,269; and WO 2014 / 026136, the contents of each of which is herein incorporated by reference in its entirety. (See also, Boulware et al., Biotechnol Bioeng.

[0613] 106(3):339-346 (2010)).

[0614]

[0268] In some embodiments, the EpCAM conditionally activatable masked antibodies of any one of the conditionally activatable masked immunoconjugates described herein include an AB that is modified by an MM and also includes one or more cleavable moieties (CM). Such EpCAM conditionally activatable masked antibodies exhibit activatable binding, to human EpCAM, Tire EpCAM conditionally activatable masked antibodies generally include an antibody or antigen-binding antibody fragment (AB), modified by or coupled to a masking moiety (MM) and a modifiable or cleavable moiety (CM). In some embodiments, the CM contains an amino acid sequence that serves as a substrate for at least one protease. In some embodiments, the MM and the cleavable moiety are coupled to the EpCAM conditionally activatable masked antibody directly. In other embodiments, the MM and cleavable moiety are coupled to the EpCAM conditionally activatable masked antibody indirectly (e.g., via one or more linkers).

[0615]

[0269] The elements of the EpCAM conditionally activatable masked antibodies of any one of the conditionally activatable masked immunoconjugates described herein are arranged so that the MM and CM are positioned such that in a cleaved (or relatively active) state and in the presence of human EpCAM, the EpCAM conditionally activatable masked antibody binds human EpCAM, but when the EpCAM conditionally activatable masked antibody is in an uncleaved (or relatively inactive) state in the presence of human EpCAM. specific binding of the EpCAM conditionally activatable masked antibody to human EpCAM is reduced or inhibited. The specific binding of the EpCAM conditionally activatable masked antibody to human EpCAM can be reduced due to the inhibition or masking of the EpCAM conditionally activatable masked antibody's ability to specifically bind human EpCAM by the MM.Atty. Dkt. No.: 45325-0051WO1

[0270] The Kd of the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein modified with a MM and a CM towards human EpCAM is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10.000,000, 50,000,000 or greater, or between 5-10, 10-100, 10- 1,000. 10-10,000, 10-100.000. 10-1.000,000, 10-10.000.000, 100-1.000. 100-10,000, 100- 100,000.

[0616] 100-1,000,000, 100-10,000,000, 1,000-10.000, 1,000-100,000, 1,000-1,000.000. 1000-10.000.000. 10,000-100,000, 10,000-1,000,000, 10,000-10.000,000, 100,000- 1,000,000, or 100,000-10,000,000 times greater than the Kd of the EpCAM conditionally activatable masked antibody not modified with an MM and a CM or of the parental AB towards human EpCAM. Conversely, the binding affinity of the AB modified with a MM and a CM towards human EpCAM is at least 5, 10. 25, 50, 100, 250, 500, 1,000. 2.500. 5,000, 10,000. 50.000. 100,000, 500.000. 1,000.000. 5,000,000, 10,000.000. 50.000.000 or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10- 100,000, 10-1.000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100- 1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000- 10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000- 1,000,000. or 100,000-10,000,000 times lower than the binding affinity of the EpCAM conditionally activatable masked antibody not modified with an MM and a CM or of the parental AB towards human EpCAM.

[0617]

[0271] When the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein is modified with a MM and a CM and is in the presence of human EpCAM but not in the presence of a modifying agent (for example at least one protease), specific binding of the EpCAM conditionally activatable masked antibody to human EpCAM is reduced or inhibited, as compared to the specific binding of the EpCAM conditionally activatable masked antibody not modified with an MM and a CM or of the parental AB to human EpCAM. When compared to the binding of the parental AB or the binding of an EpCAM conditionally activatable masked antibody not modified with an MM and a CM to human EpCAM, the EpCAM conditionally activatable masked antibody's ability to bind human EpCAM when modified with an MM and a CM can be reduced by at least 50%, 60%, 70%, 80%, 90%. 92%, 93%, 94%. 95%. 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12. 28, 24, 30, 36, 48, 60, 72, 84. or 96 hours or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured in vivo or in an in vitro assay.

[0618]

[0272] As used herein, the term “cleaved state” refers to the condition of the EpCAM conditionally activatable masked antibodies of any one of the conditionally activatable masked immunoconjugates described herein following modification of the CM by at least one protease. The term uncleaved or intact state, as used herein, refers to the condition of the EpCAM conditionally activatable masked antibodies in the absence of cleavage of the CM by a protease. As discussed above, the termAtty. Dkt. No.: 45325-0051WO1 ‘“conditionally activatable masked antibody,"’ ‘‘activatable antibody” or "activatable antibody" is used herein to refer to an EpCAM conditionally activatable masked antibody, in both its uncleaved (native or intact) state, as well as in its cleaved state. It will be apparent to ordinarily skilled artisan that in some embodiments, a cleaved EpCAM conditionally activatable masked antibody, can lack an MM due to cleavage of the CM by protease, resulting in release of at least the MM (e.g., where the MM is not joined to the EpCAM conditionally activatable masked antibody), by a covalent bond (e.g., a disulfide bond between cysteine residues).

[0619]

[0273] In some instances, the linker-payload is covalently bound to tire AB. In some instances, two or more linker-payloads (e.g., 2, 3, 4, 5, 6, 7, 8, or more) are covalently bound to the AB.

[0620]

[0274] In some instances, bound to the activatable antibody refers to an interaction i.e., a binding) to the AB via the 8 interchain cysteines that form disulfide bond.

[0621]

[0275] By activatable it is meant that the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein, exhibits a first level of binding to a target when the EpCAM conditionally activatable masked antibody, is in a inhibited, masked, intact or uncleaved state (i.e., a first conformation), and a second level of binding to human EpCAM in the uninhibited, unmasked and / or cleaved state (i.e., a second conformation), where the second level of target binding is greater than the first level of binding In general, the access of human EpCAM to the AB of the EpCAM conditionally activatable masked antibody, is greater in the presence of a cleaving agent capable of cleaving the CM, i.e.. a protease, than in the absence of such a cleaving agent. Thus, when the EpCAM conditionally activatable masked antibody is in the uncleaved state, the AB is inhibited from binding human EpCAM and can be masked from human EpCAM -binding (i.e., the first conformation is such that the AB cannot bind human EpCAM), and in the cleaved state the AB is not inhibited or is unmasked to target binding.

[0622]

[0276] The CM and AB of the EpCAM conditionally activatable masked antibodies of any one of the conditionally activatable masked immunoconjugates described herein are selected so that the AB represents a binding moiety for a given target, and the CM represents a substrate for a protease. In some embodiments, the protease is co-localized with human EpCAM at a treatment site or diagnostic site in a subject. As used herein, co-localized refers to being at the same site or relatively close nearby. In some embodiments, a protease cleaves a CM yielding an conditionally activatable masked antibody that binds to a target located nearby the cleavage site. The EpCAM conditionally activatable masked antibodies disclosed herein find particular use where, for example, a protease capable of cleaving a site in the CM. i.e.. a protease, is present at relatively higher levels in target-containing tissue of a treatment site or diagnostic site than in tissue of non-treatment sites (for example in healthy tissue). In some embodiments, a CM of the disclosure is also cleaved by one or more other proteases. In someAtty. Dkt. No.: 45325-0051WO1 embodiments, it is the one or more other proteases that is co-localized with human EpCAM and that is responsible for cleavage of the CM in vivo.

[0623]

[0277] In some embodiments, EpCAM conditionally activatable masked antibodies of any one of the conditionally activatable masked immunoconjugates described herein provide for reduced toxicity and / or adverse side effects that could otherwise result from binding of the EpCAM conditionally activatable masked antibodies at non- treatment sites if the EpCAM conditionally activatable masked antibodies were not masked or otherwise inhibited from binding to human EpCAM.

[0624]

[0278] In general, an EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein, can be designed by selecting an AB of interest and constructing the remainder of the EpCAM conditionally activatable masked antibody, so that, when conform ationally constrained, the MM provides for masking of the EpCAM conditionally activatable masked antibodies or reduction of binding of the EpCAM conditionally activatable masked antibodies to human EpCAM. Structural design criteria can be to be taken into account to provide for this functional feature.

[0625]

[0279] EpCAM conditionally activatable masked antibodies of any one of the conditionally activatable masked immunoconjugates described herein exhibiting a conditionally activatable masked phenotype of a desired dynamic range for target binding in an inhibited versus an uninhibited conformation are provided. Dynamic range generally refers to a ratio of (a) a maximum detected level of a parameter under a first set of conditions to (b) a minimum detected value of that parameter under a second set of conditions. For example, in the context of an EpCAM conditionally activatable masked antibody, the dynamic range refers to the ratio of (a) a maximum detected level of target protein binding to an EpCAM conditionally activatable masked antibody, in the presence of at least one protease capable of cleaving the CM of the EpCAM conditionally activatable masked antibodies to (b) a minimum detected level of target protein binding to an EpCAM conditionally activatable masked antibody, in the absence of the protease. The dynamic range of an EpCAM conditionally activatable masked antibody, can be calculated as the ratio of the dissociation constant of an EpCAM conditionally activatable masked antibody, cleaving agent (e.g., enzyme) treatment to the dissociation constant of the EpCAM conditionally activatable masked antibodies cleaving agent treatment. Tire greater the dynamic range of an EpCAM activatable antibody; the better the activatable phenotype of tire EpCAM conditionally activatable masked antibody.

[0626]

[0280] EpCAM conditionally activatable masked antibodies of any one of the conditionally activatable masked immunoconjugates described herein having relatively higher dynamic range values (e.g., greater than 1) exhibit more desirable switching phenotypes such that target protein binding by the EpCAM conditionally activatable masked antibodies occurs to a greater extent (e.g., predominantly occurs) inAtty. Dkt. No.: 45325-0051WO1 the presence of a cleaving agent

[0627]

[0628] enzyme) capable of cleaving the CM of the EpCAM conditionally activatable masked antibodies than in the absence of a cleaving agent.

[0629]

[0281] EpCAM conditionally activatable masked antibodies of any one of the conditionally activatable masked immunoconjugates described herein can be provided in a variety of structural configurations. Exemplary formulae for EpCAM conditionally activatable masked antibodies are provided below. It is specifically contemplated that the N- to C-temnnal order of the AB. MM and CM can be reversed within an EpCAM conditionally activatable masked antibody. It is also specifically contemplated that the CM and MM can overlap in amino acid sequence, e.g., such that tire CM is contained within the MM.

[0630]

[0282] For example, EpCAM conditionally activatable masked antibodies of any one of the conditionally activatable masked immunoconjugates described herein can be represented by the following formula (in order from an amino (N) terminal region to carboxyl (C) terminal region:

[0631] (MM)-(CM)-(AB)

[0632] (AB)-(CM)-(MM)

[0633] where MM is a masking moiety, CM is a cleavable moiety, and AB is an EpCAM antibody or an EpCAM-binding antibody fragment thereof. It should be noted that although MM and CM are indicated as distinct components in the formulae above, in all exemplary embodiments, (including formulae) disclosed herein it is contemplated that the amino acid sequences of the MM and the CM could overlap, e.g. such that the CM is completely or partially contained within the MM. In addition, the formulae above provide for additional amino acid sequences that can be positioned N-terminal or C-terminal to the EpCAM conditionally activatable masked antibodies elements.

[0634]

[0283] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a CM that is cleavable by a protease. In some embodiments, the protease that cleaves the CM is active, e.g., up- regulated or otherwise unregulated, in diseased tissue, and the protease cleaves the CM in the EpCAM conditionally activatable masked antibody, when the EpCAM conditionally activatable masked antibody, is exposed to the protease.

[0635]

[0284] In some embodiments, the protease is co-localized with EpCAM in a tissue, and the protease cleaves the CM tn the EpCAM conditionally activatable masked antibody, when the EpCAM conditionally activatable masked antibody, is exposed to the protease.

[0636]

[0285] In some embodiments, the CM is positioned in the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein, such that when the EpCAM conditionally activatable masked antibody, is in the uncleaved state, binding of the EpCAM conditionally activatable masked antibody, to EpCAM is reduced to occur with a dissociation constant that is at least 2, 5, 10, 20, 40, 50, 100, or 200, greater than the dissociationAtty. Dkt. No.: 45325-0051WO1 constant of an unmodified AB binding to EpCAM, whereas in the cleaved state (i.e., when the EpCAM conditionally activatable masked antibody, is in the cleaved state), the AB binds EpCAM.

[0637]

[0286] In some embodiments, the CM is a polypeptide of up to 15 amino acids in length.

[0638]

[0287] In some embodiments, the CM is a polypeptide that includes a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). In some embodiments, each of the CM 1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate is independently a polypeptide of up to 15 amino acids in length.

[0639]

[0288] In some embodiments, the CM is a substrate for at least one protease that is or is believed to be up-regulated or otherwise unregulated in cancer. In some embodiments, the CM is a substrate for at least one protease that is or is believed to be up-regulated in inflammation. In some embodiments, the CM is a substrate for at least one protease that is or is believed to be up-regulated or otherwise unregulated in autoimmunity.

[0640]

[0289] In some embodiments, the CM is a substrate for at least one protease selected from a matrix metalloprotease (MMP), thrombin, a neutrophil elastase, a cysteine protease, legumain, and a serine protease, such as matriptase (MT-SP1), and urokinase (uPA). Without being bound by theory / , it is believed that these proteases are up-regulated or otherwise unregulated in at least one of cancer, inflammation, and / or autoimmunity.

[0641]

[0290] Exemplary substrates include but are not limited to substrates cleavable by one or more of the following enzymes or proteases: an ADAMS / ADAMTS. (e.g.. ADAM8, ADAM9. ADAM 10, ADAM 12. ADAM 15, ADAM 17 / TACE, AD AMDEC1, ADAMTS1. AD AMTS4, ADAMTS5 ); an aspartate protease (e.g, BACE, Renin); an aspartic cathepsin (e g., Cathepsin D and Cathepsin E); a caspase (e.g.. Caspase 1-10, and Caspase 14); a cysteine cathepsin (e.g., Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L, Cathepsin S, Cathepsin V / L2, and Cathepsin X / Z / P); a cysteine proteinase (e.g..

[0642] Cruzipain. Legumain, and Otubain-2); a KLK (e.g., KLK4-8, KLK10. KLK 11, KLK 13, and KLK14 ); a metalloproteinase (e.g., Meprm, Neprilysm, PSMA, and BMP1); an MMP (e.g., MMP1-3, MMP 7-17, MMP 19,, MMP 20, MMP 23, MMP 24. MMP 26, and MMP 27); a serine protease (e.g.. activated protein C, Cathepsin A, Cathepsin C, Chymase, and a coagulation factor protease such as FVIIa. FIXa, FXa, FXIa, and FXIIa), an Elastase (e.g., human Neutrophil Elastase); Granzyme B;

[0643] Guanidinobenzoatase; HtrAl; Lactoferrin; Marapsin; NS3 / 4A; PACE4; Plasmin; PSA, tPA; Thrombin; Tryptase; uPA; a Type II Transmembrane Serine Protease (TTSP) (e g.. DESCI. DPP -4. FAP, Hepsin, Matnptase-2, MT-SPl / Matriptase. and TMPRSS2-4).Atty. Dkt. No.: 45325-0051WO1

[0291] In some embodiments, the CM is selected for use with a specific protease, for example a protease that is known to be co-localized with the target of the EpCAM conditionally activatable masked antibody.

[0644]

[0292] In some embodiments, the CM is a substrate for at least one MMP. Examples of MMPs include MMPI-3, MMP 7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27. In some embodiments, the CM is a substrate for a protease selected from MMP 9, MMP 14, MMP1, MMP3, MMP 13, MMP 17, MMP11, and MMP19. In some embodiments, the CM is a substrate for MMP9. In some embodiments, the CM is a substrate for MMP 14.

[0645]

[0293] Suitable CM that can routinely be incorporated into the provided activatable antibodies are known in the art. See. for example, WO 2016 / 179285, e.g, pages 40-47, the contents of which is herein incorporated by reference in its entirety.

[0646]

[0294] In some embodiments, tire CM is a substrate for a neutrophil elastase. In some embodiments, the CM is a substrate for a serine protease. In some embodiments, the CM is a substrate for legumain. In some embodiments, the CM is a substrate for matriptase. In some embodiments, the CM is a substrate for a cysteine protease. In some embodiments, the CM is a substrate for a cysteine protease, such as a cathepsin. Tn other embodiments, the CM is a substrate for a uPA.

[0647]

[0295] In particular embodiments, tlie CM is a substrate for uPA. In some embodiments, tire CM comprises a sequence disclosed m Table 10.

[0648] Table 10. Exemplary CM Sequences.

[0649] Substrate name Sequence

[0650] 3014 AVGLLAPPGGLSGRSDNI (SEQ ID NO: 168)

[0651] 2014 ISSGLLSGRSDNI (SEQ ID NO: 169)

[0652]

[0653]

[0296] In some embodiments, the CM comprises the sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 168). In some embodiments, the CM comprises the sequence ISSGLLSGRSDNI (SEQ ID NO: 169).

[0654]

[0297] In some embodiments, the CM is a substrate for at least two proteases. In some embodiments, each protease is selected from an ADAMS / ADAMTS. (e.g, ADAM8, ADAM9. ADAM 10, ADAM 12, ADAM 15, ADAM 17 / TACE. ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5); an aspartate protease (e.g., BACE, Renin); an aspartic cathepsin (e.g.. Cathepsin D and Cathepsin E); a caspase (e.g.. Caspase 1 - 10, and Caspase 14); a cysteine cathepsin (e.g., Cathepsin B, Cathepsin C, Cathepsin K, Cathepsin L. Cathepsin S, Cathepsin V / L2, and Cathepsin X / Z / P); a cysteine proteinase (e.g., Cruzipain, Legumain, and Otubain-2); a KLK (e.g., KLK4-8, KLK 10, KLK 11, KLK 13, and KLK14 ); a metalloproteinase (e.g., Meprin, Neprilysin, PSMA, and BMP1); an MMP (e.g., MMP1-3. MMP 7-17,Atty. Dkt. No.: 45325-0051WO1 MMP 19„ MMP 20. MMP 23, MMP 24, MMP 26, and MMP 27); a serine protease (e.g., activated protein C, Cathepsin A, Cathepsin C, Chymase, and a coagulation factor protease such as FVTIa, FIXa, FXa, FXIa, and FXIIa), an Elastase (e.g., human Neutrophil Elastase); Granzyme B;

[0655] Guanidinobenzoatase; HtrAl; Lactoferrin: Marapsin; NS3 / 4A; PACE4; Plasmin; PSA, tPA; Thrombin; Tryptase; uPA; a Type II Transmembrane Serine Protease (TTSP) (e g., DESCI. DPP -4. FAP, Hepsin, Matriptase-2, MT-SPl / Matriptase. and TMPRSS2-4. Tn some embodiments, the CM is a substrate for at least two proteases, wherein one of the proteases is selected from: a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain and matriptase and the other protease is selected from those listed above. In some embodiments, the CM is a substrate for at least two proteases selected from the group: a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain and matriptase.

[0656]

[0298] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein includes at least a first CM and a second CM. In some embodiments, the first CM and the second CM are each polypeptides of no more than 15 amino acids long. In some embodiments, the first CM and the second CM in the EpCAM conditionally activatable masked antibody, in the uncleaved state have the structural arrangement from N-terminus to C -terminus as follows: MM-CM1-CM2-AB or AB-CM2-CM1-MM. In some embodiments, at least one of the first CM and the second CM is a polypeptide that functions as a substrate for a protease selected from a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase. In some embodiments, the first CM is cleaved by a first cleaving agent selected from MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase in a target tissue and the second CM is cleaved by a second cleaving agent in a target tissue. In some embodiments, the other protease is selected from the list presented in the preceding paragraph. In some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from a MMP, thrombin, a neutrophil elastase, a cysteine protease, uPA, legumain, and matriptase, and the first CM and the second CM are different substrates for the enz me. In some embodiments, tire first cleaving agent and the second cleaving agent arc the same protease selected from the list in the preceding paragraph. In some embodiments, the first cleaving agent and the second cleaving agent are different proteases. In some embodiments, the first cleaving agent and the second cleaving agent are co-localized in the target tissue. In some embodiments, the first CM and the second CM are cleaved by at least one cleaving agent in the target tissue.

[0657]

[0299] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein, also includes a signal peptide. In some embodiments, the signal peptide is conjugated to the EpCAM conditionally activatable masked antibody, via a spacer, in some embodiments, the spacer is conjugated to the EpCAM conditionally activatable masked antibody, in the absence of a signal peptide In some embodiments, the spacer isAtty. Dkt. No.: 45325-0051WO1 joined directly to the MM of the EpCAM conditionally activatable masked antibody In some embodiments, the spacer is joined directly to the MM of the EpCAM conditionally activatable masked antibody, in the structural arrangement from N-tenninus to C- terminus of spacer-MM-CM-AB.

[0658]

[0300] Suitable spacers and spacer technology is known in the art and can routinely be used to incorporate spacers in some embodiments of the provided conditionally activatable masked antibodies. See, for example, WO 2016 / 179285 (e.g, at pages 52-53), the contents of which is herein incorporated by reference in its entirety.

[0659]

[0301] In many embodiments, it can be desirable to insert one or more linkers, e.g., flexible linkers, into the EpCAM conditionally activatable masked antibody, construct so as to provide for flexibility at one or more of the MM-CM junction, the CM- AB junction, or both. For example, the AB, MM, and / or CM cannot contain a sufficient number of residues (e.g., Gly, Ser, Asp. Asn, especially Gly and Ser, particularly Gly) to provide the desired flexibility. As such, the conditionally activatable masked phenotype of such EpCAM conditionally activatable masked antibody, constructs can benefit from introduction of one or more amino acids to provide for a flexible linker. In addition, as described below, where the EpCAM conditionally activatable masked antibody, is provided as a conformationally constrained construct, a flexible linker can be operably inserted to facilitate formation and maintenance of a cyclic structure in the uncleaved EpCAM conditionally activatable masked antibody.

[0660]

[0302] For example, in certain embodiments, an EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein, comprises one of the following formulae (where die formula below represent an amino acid sequence in either N- to C- terminal direction or C- to N-terminal direction):

[0661] (MM)-L1-(CM)-(AB)

[0662] (MM)-(CM)-L2-(AB)

[0663] (MM)-L1-(CM)-L2-(AB)

[0664] wherein MM, CM, and AB are as defined above; wherein L1 and L2 are each independently and optionally present or absent, are the same or different flexible linkers that include at least 1 flexible amino acid (e.g, Gly). In addition, the formulae above provide for additional amino acid sequences that can be positioned N-termmal or C-terminal to the EpCAM conditionally activatable masked antibodies elements. Examples include, but are not limited to, targeting moieties (e.g., a ligand for a receptor of a cell present in a target tissue) and serum halflife extending moieties (e.g., polypeptides that bind serum proteins, such as immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HSA)).

[0665]

[0303] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein is exposed to and cleaved by a protease such that, in the conditionally activatable masked or cleaved state, the conditionally activatableAtty. Dkt. No.: 45325-0051WO1 masked antibody includes a light chain sequence that includes at least a portion of LP2 and / or CM sequence after the protease has cleaved the CM.

[0666]

[0304] The CM is specifically cleaved by at least one protease at a rate of about 0.001-1500 x 104M’ ’S’1or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5. 5, 7.5, 10, 15, 20, 25, 50. 75, 100, 125, 150, 200, 250. 500, 750. 1000. 1250, or 1500 x 104M^S’1. In some embodiments, the CM is specifically cleaved at a rate of about 100,000

[0667]

[0668] In some embodiments, the CM is specifically cleaved at a rate from about 1 x 102to about 1 x 106M’'S’’ ( / .<?., from about 1 xl02to about 1 x 106M"!S’1).

[0669]

[0305] For specific cleavage by an enzyme, contact between the enzyme and CM is made. When the EpCAM conditionally activatable masked antibody, comprising an AB (e.g., an EpCAM antibody or EpCAM-binding antibody fragment thereof) coupled to a MM and a CM is in the presence of EpCAM and sufficient enzyme activity, the CM can be cleaved. Sufficient enzyme activity can refer to the ability' of the enz me to make contact with the CM and effect cleavage. It can readily' be envisioned that an enzyme can be in the vicinity of the CM but unable to cleave because of other cellular factors or protein modification of the enzyme.

[0670]

[0306] Linkers suitable for use in EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein, compositions disclosed herein are generally ones that provide flexibility of the modified AB (e.g.. an EpCAM antibody or EpCAM- binding antibody fragment thereof) or the EpCAM conditionally activatable masked antibody', to facilitate the inhibition of the binding of the EpCAM conditionally activatable masked antibody to human EpCAM. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 ammo acids to 15 amino acids, from 3 ammo acids to 12 ammo acids, including 4 amino acids to 10 amino acids. 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.

[0671]

[0307] Exemplary flexible linkers for the conditionally activatable masked antibodies, antibodies, and antibody' fragments provided herein include, glycine polymers (G)n, glycine-serme polymers (including, for example, (GS)n. Suitable linkers and linker technology are known in the art and can routinely' be used to incorporate spacers in some embodiments of tire provided conditionally activatable masked antibodies. See, for example, W'O 2016 / 179285 (e.g., at pages 26, 113-116), the contents of which is herein incorporated by reference in its entirety. The ordinarily skilled artisan recognizes that design of an EpCAM conditionally activatable masked antibodies can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure to provide for a desired EpCAM conditionally activatable masked antibodies structure.Atty. Dkt. No.: 45325-0051WO1

[0308] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein tire EpCAM conditionally activatable masked antibody, in the uncleaved state has the structural arrangement from N -terminus to C -terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the two linking peptides need not be identical to each other,

[0672]

[0309] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of tire conditionally activatable masked immunoconjugates described herein, comprises a first linking peptide (LP1) and a second linking peptide (LP2), and wherein tlie EpCAM conditionally activatable masked antibody, in the uncleaved state has the structural arrangement from N-temnnus to C -terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the two linking peptides need not be identical to each other,

[0673]

[0310] In some embodiments, at least one of LP1 or LP2 of the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a flexible linker. Suitable linkers and linker technology are known in the art and can routinely be used to incorporate spacers in some embodiments of the provided conditionally activatable masked antibodies. See, for example, WO 2016 / 179285 (e.g., at pages 26. 113-116), the contents of which is herein incorporated by reference in its entirety.

[0674]

[0311] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a light chain having a sequence disclosed in Table 11. In some embodiments, the conditionally activatable masked antibody comprises a light chain having the sequence of SEQ ID NO: 174. In some embodiments, the conditionally activatable masked antibody comprises a light chain having the sequence of SEQ ID NO: 179.

[0675] Table 11. Exemplary light chain conditionally activatable masked antibody sequences.

[0676] Conditionally Sequence

[0677] actiyalab.le

[0678] masked antibody

[0679] name

[0680] For use with heavy chain of HuEpCAM23Gv4.2, 1361-H, or 1565- Y

[0681] Ep01-23014 Lv QGQSGQGPLMTCSDYYTCLNNLGGGSSGGAVGLLAPPGGLSGRSDNIGGS DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI YQTSNLASGVPDRESSSGSGTDFFLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRIWAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV

[0682]

[0683] Atty. Dkt. No.: 45325-0051WO1 DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 170)

[0684] Ep02 3014 Lv QGQSGQGLSCTHSRYDMHCPHMGGGSSGGAVGLLAPPGGLSGRSDNIGG SDIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLL IYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTF GQGTKLEJKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC (SEQ ID NO: 171)

[0685] Ep03 3014 Lv QGQSGQGFIYCHSRTDTITHCNAGGGSSGGAVGLLAPPGGLSGRSDNIGGS DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKWACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 172)

[0686] Ep043014 Lv QGQSGQGWCPRLFDRPSMGCPTGGGSSGGAVGLLAPPGGLSGRSDNIGGS DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 173)

[0687] Ep053014 Lv QGQSGQGWWPPCQGGAWCEQRIGGGSSGGAVGLLAPPGGLSGRSDNIGG SDIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLL

[0688] 1YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTF GQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSIYSLSSTI. TL. SKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC (SEQ ID NO: 174)

[0689] Ep073014 Lv QGQSGQGHSGCPRLFDRCSAPAGGGSSGGAVGLLAPPGGLSGRSDNIGGS DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYIAAVFLQKPGQSPQLLI YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 175)

[0690] Ep11 3014 Lv QGQSGQGFICPTLYDRPHCMHTGGGSSGGAVGLLAPPGGLSGRSDNIGGS

[0691] DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI

[0692]

[0693] Atty. Dkt. No.: 45325-0051WO1 YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV1HQG LSSPVTKSFNRGEC (SEQ ID NO: 176)

[0694] Ep01-22014 Lv QGQSGQGPLMTCSDYYTCLNNLGGGSSGGSISSGLLSGRSDN1GGSDIVLT QTPLSLSVTPGQPASISCRSSRSLLHSDGFWLWFLQKPGQSPQLLIYQTS NLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFGQGTK LE1KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 177)

[0695] Ep042014 Lv QGQSGQGWCPRLFDRPSMGCPTGGGSSGGSISSGLLSGRSDNIGGSDIVLT QTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLIYQTS NLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 178)

[0696] Ep052014 Lv QGQSGQGWWPPCQGGAWCEQRIGGGSSGGSISSGLLSGRSDNIGGSD1VL TQTPLSLSVTPGQPASISCRSSRSLLHSDGFIYLYWFLQKPGQSPQLLIYQTS NLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC* (SEQ ID NO: 179)

[0697] * denotes cysteine which, when in a native conformation (e.g., not reduced), participate in forming interchain disulfide bond.

[0698] Ep072014 Lv QGQSGQGHSGCPRLFDRCSAPAGGGSSGGSISSGLLSGRSDNIGGSDIVLTQ TPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLIYQTSNL ASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSIYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC (SEQ ID NO: 180)

[0699] For use with heavy chain of 1S65-Y

[0700] Ep101 2014 Lv QGQSGQGSWCHSATDTILPCSNGGGSSGGSISSGLLSGRSDNIGGSDIVLTQ TPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLIYQTSNL ASGVPDRFSSSGSGTDFTLKISRVEAEDVGV’YV'CAQNLELPNTFGQGTKLE

[0701]

[0702] Atty. Dkt. No.: 45325-0051WO1 IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC (SEQ ID NO: 181)

[0703] Ep102 2014 Lv QGQSGQGSPACSDRYYQTCVLNGGGSSGGSISSGLLSGRSDNIGGSD1VLT QTPLSLSVTPGQPAS1SCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLIYQTS NLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 182)

[0704] Ep103 2014 Lv QGQSGQGMSCVVDRFDRQCPHLGGGSSGGSISSGLLSGRSDNIGGSDIVLT QTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLIYQTS NLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKFIKVYACEVTFIQGLSSP VTKSFNRGEC (SEQ ID NO: 183)

[0705] Ep104 2014 Lv QGQSGQGTTRCEHYWFTCPLSPGGGSSGGSISSGLLSGRSDNIGGSDIVLTQ TPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLIYQTSNL ASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC (SEQ ID NO: 184)

[0706] Ep101 3014 Lv QGQSGQGSWCHSATDTILPCSNGGGSSGGAVGLLAPPGGLSGRSDNIGGS DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 185)

[0707] Ep102 3014 Lv QGQSGQGSPACSDRYYQTCVLNGGGSSGGAVGLLAPPGGLSGRSDNIGGS DIVLTQTPLSLSVTPGQPASTSCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTT / n, SKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 186)

[0708]

[0709] Atty. Dkt. No.: 45325-0051WO1 Ep103 3014 Lv QGQSGQGMSCVVDRFDRQCPHLGGGSSGGAVGLLAPPGGLSGRSDNIGG SDIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLL IYQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTF GQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC (SEQ ID NO: 187)

[0710] fEp104 3014 Lv QGQSGQGTTRCEHYWFTCPLSPGGGSSGGAVGLLAPPGGLSGRSDNIGGS D1VLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 188)

[0711] For use with heavy chain of 1361-H

[0712] Ep105 2014 Lv QGQSGQGDCTGYSPSVLPACRVGGGSSGGS1SSGLLSGRSDN1GGSDIVLT QTPLSLSVTPGQPASISCRSSRSLLHSDGFTY'LYWFLQKPGQSPQLLIYQTS NLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVl'EQDSKDSl’YSLSSTLTLSKADYEKHKVYACEVTOQGLSSP VTKSFNRGEC (SEQ ID NO: 189)

[0713] Ep106 2014 Lv QGQSGQGFCSGYSPSVLPSCLMGGGSSGGSISSGLLSGRSDNIGGSDIVLTQ TPLSLSVTPGQPASISCRSSRSLI. HSDGFTYLYAVFL. QKPGQSPQI. LIYQTSNL ASGVPDRFSSSGSGTOFTLKISRVEAEDVGVYYCAQNLELPNTFGQGTKLE

[0714] 1KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVIHQGLSSPVT KSFNRGEC (SEQ ID NO: 190)

[0715] Ep107 2014 Lv QGQSGQGSKPCSYMHPYCFYNSGGGSSGGSISSGLLSGRSDNIGGSDIVLT QTPLSLSVITGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLIYQTS NLASGVPDRFSSSGSGTDFT1. KISRVEAEDVGVYYCAQNLELPNTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 191)

[0716] Ep108 2014 Lv QGQSGQGLTRCTIAHPYCYYNYGGGSSGGSISSGLLSGRSDNIGGSD1VLT QTPLSLSVTPGQPAS1SCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLIYQTS NLASGVPDRFSSSGSGTDFTLKTSRVEAEDVGVYYCAQNLELPNTFGQGTK

[0717]

[0718] Atty. Dkt. No.: 45325-0051WO1 LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDS1YSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC (SEQ ID NO: 192)

[0719] Ep109 2014 Lv QGQSGQGPNTCMSERRICSLTYGGGSSGGSISSGLLSGRSDNIGGSDIVLTQ TPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLIYQTSNL ASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC (SEQ ID NO: 193)

[0720] Ep110 2014 Lv QGQSGQGPRPHCAILRQCLAATGGGSSGGSISSGLLSGRSDNIGGSDIVLTQ TPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLIYQTSNL ASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNIYTREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTESKADYEKFIKVYACEVTIIQGLSSPVT KSFNRGEC (SEQ ID NO: 194)

[0721] Ep105 3014 Lv QGQSGQGDCTGYSPSVLPACRVGGGSSGGAVGLLAPPGGLSGRSDNIGGS DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 195)

[0722] Ep106 3014 Lv QGQSGQGFCSGYSPSVLPSCLMGGGSSGGAVGLLAPPGGLSGRSDNIGGS DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKWACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 196)

[0723] Ep107 3014 Lv QGQSGQGSKPCSYMHPYCFYNSGGGSSGGAVGLLAPPGGLSGRSDNIGGS DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSH / n, SKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 197)

[0724]

[0725] Atty. Dkt. No.: 45325-0051WO1 Ep108 3014 Lv QGQSGQGLTRCTIAHPYCYYNYGGGSSGGAVGLLAPPGGLSGRSDNIGGS DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 198)

[0726] Ep109 2014 Lv QGQSGQGPNTCMSERRICSLTYGGGSSGGAVGLLAPPGGLSGRSDNIGGS D1VLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO: 199)

[0727] Ep110 3014 Lv QGQSGQGPRPHCAILRQCLAATGGGSSGGAVGLLAPPGGLSGRSDNIGGS DIVLTQTPLSLSVTPGQPASISCRSSRSLLHSDGFTYLYWFLQKPGQSPQLLI YQTSNLASGVPDRFSSSGSGTDFTLKISRVEAEDVGVYYCAQNLELPNTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQ ID NO:200)

[0728]

[0729]

[0312] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a light chain having a sequence selected from SEQ ID NOs: 170-180 and a heavy chain having a sequence selected from SEQ ID NOs: 103, 125, and 127. In some embodiments, the EpCAM conditionally activatable masked antibody comprises a light chain having the sequence selected from SEQ ID NOs: 170-180 and a heavy chain having the sequence of SEQ ID NO: 103. In some embodiments, the EpCAM conditionally activatable masked antibody comprises a light chain having the sequence of SEQ ID NO: 174 and a heavy chain having the sequence of SEQ ID NO: 103. In some embodiments, the EpCAM conditionally activatable masked antibody comprises a light chain having the sequence of SEQ ID NO: 179 and a heavy chain having the sequence of SEQ ID NO: 103.

[0730]

[0313] In some embodiments, the EpCAM conditionally activatable masked antibody comprises a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. or 99% identical to a sequence selected from SEQ ID NOs: 170-180 and a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, identical to a sequence selected from SEQ ID NOs: 103, 125, and 127. In some embodiments, the EpCAMAtty. Dkt. No.: 45325-0051WO1 conditionally activatable masked antibody comprises a light chain having an amino acid sequence that is at least 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 170- 180 and a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, or 99%, identical to a from SEQ ID NO: 103. In some embodiments, the EpCAM conditionally activatable masked antibody comprises a light chain having an amino acid sequence that is at least 90%. 91%, 92%, 93%. 94%, 95%. 96%. 97%, 98%. or 99% identical SEQ ID NO: 174 and a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%. 94%, 95%. 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 103. In some embodiments, the EpCAM conditionally activatablc masked antibody comprises a light chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, or 99% identical SEQ ID NO: 179 and a heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%. 94%.

[0731] 95%, 96%, 97%, 98%, or 99%, identical to SEQ ID NO: 103. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0732]

[0314] In some embodiments, the EpCAM conditionally activatable masked antibody of any one of the conditionally activatable masked immunoconjugates described herein comprises a light chain having a sequence selected from SEQ ID NOs: 181-188 and a heavy chain having the sequence of SEQ ID NO: 127. In some embodiments, the EpCAM activatable antibody comprises a light chain having a sequence selected from SEQ ID NOs: 189-200 and a heavy chain having the sequence of SEQ ID NO: 125.

[0733]

[0315] In certain embodiments, the huEpCAM23 antibody is encoded by the plasmids deposited with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Va.

[0734] 20110 on October 4. 2018, under the terms of the Budapest Treaty and having ATCC deposit nos. PTA- 125343 and PTA-125344 or PTA-125345. Examples of EpCAM antibody, EpCAM-binding antibody fragments thereof, and EpCAM conditionally activatable masked antibody, immunoconjugates are provided herein.

[0735]

[0316] Polynucleotides, vectors, host cells, and recombinant methods are described in WO 2024 / 015830. WO 2024 / 015830 is incorporated by reference in its entirety.

[0736] IMMUNOCONJUGATES

[0737]

[0317] The EpCAM immunoconjugates as disclosed herein comprise camptothecin and / or derivatives thereof. Camptothecin and derivatives thereof have been disclosed in US Appl. Publ. No.

[0738] 2021 / 0077482 A1 and U.S. Pat. No. 11,229,639 B2, each of which is incorporated by reference herein in its entirety.Atty. Dkt. No.: 45325-0051WO1

[0318] In some instances, the EpCAM immunoconjugates are generated by conjugating at least one linker-payload reactant comprising a compound of Formula I or a pharmaceutically acceptable salt thereof with an EpCAM antibody as described in Section 0 or an EpCAM conditionally activatable masked antibody:

[0739] E-A-Z -L’-D (Formula 1)

[0740] wherein:

[0741] D is represented by the following structural formula:

[0742]

[0743] R1is F;

[0744] R2is methyl,

[0745] -L’-Z’-* is -(C1-C4 alkylene)-O-CH2-NR8-*, -(C1-C4 alkylene)-NR8, or-(Ci-Cs alkylene)-NR’C(=O)-(Ci-C<i alkylene)-O-CH2-NR8*, where * is a site covalently attached to A;

[0746] each R5is independently H, methyl, or benzyl;

[0747] each R8is independently H, methyl, or benzyl;

[0748] A is a peptide comprising 2 to 4 amino acids; and

[0749] E is C(=O)-(C₁-C₁₀ alkylene)-X³ wherein X³ is

[0750]

[0751]

[0319] In some instances, tire linker-payload reactant comprises:Atty. Dkt. No.: 45325-0051WO1

[0752]

[0753] or a pharmaceutically acceptable salt thereof.

[0754]

[0320] In some instances, the linker-payload reactant comprises:

[0755]

[0756] or a pharmaceutically acceptable salt thereof.

[0757]

[0321] Where the linker-payload reactant comprises a compound of Formula I or a pharmaceutically acceptable salt thereof, the maleimide group X3reacts with a thiol (-SH) group in the EpCAM antibody or conditionally activatable masked antibody to form a covalent bond in the immunoconjugate, as shown below.:

[0758]

[0759] Atty. Dkt. No.: 45325-0051WO1 where * * indicates the atachment to the EpCAM antibody or activatable antibody via the sulfur of the thiol group.

[0760]

[0322] In some embodiments, the thiol (-SH) group of the EpCAM antibody or conditionally activatable masked antibody is a cysteine thiol group. Those of skill in the art recognizes that once the maleimide of group X3in Formula I of the linker-payload reactant above is reacted with a thiol of the EpCAM antibody or conditionally activatable masked antibody, the maleimide group is reduced to succinimide. The linker-payload, when conjugated to the EpCAM antibody or conditionally activatable masked antibody, thus comprises succinimide.

[0761]

[0323] In some embodiments, the EpCAM antibody or conditionally activatable masked antibody is an IgGl antibody having eight cysteines which, when in a native conformation (e.g., not reduced), are involved in forming four interchain disulfide bonds. See, e.g., SEQ ID NOs: 103, 140, and 179 in Tables 6, 7, and 11, respectively for exemplary identification of such cysteine residues. In an IgGl antibody, each light chain is linked to a heavy chain by one covalent disulfide bond, while the two heavy chains are linked together via two disulfide bonds. As provided herein, these disulfide bonds can be reduced, resulting in up to eight free cysteines and cysteine thiol groups. One linker-payload reactant can react with one cysteine’s thiol group, with up to eight linker-payload reactants reacting with each IgGl EpCAM antibody or conditionally activatable masked antibody. In some embodiments, each EpCAM antibody or conditionally activatable masked antibody reacts with up to eight linker-payload reactants.

[0762]

[0324] In some instances, the EpCAM immunoconjugates comprise at least one linker-payload comprising a compound of Formula I or a pharmaceutically acceptable salt thereof (where the compound of Formula I or a pharmaceutically acceptable salt thereof represents the linker-payload reactant, i.e. the linker-payload prior to conjugation to the EpCAM antibody or conditionally activatable masked antibody):

[0763] E-A-Z’-L'-D (Formula I)

[0764] wherein:

[0765] D is represented by tire following structural formula:

[0766] R2is methyl;

[0767]

[0768] Atty. Dkt. No.: 45325-0051WO1 -L’-Z’-* is -(C1-C4 a1kylene)-O-CH2-NR8-*, - (C1-C4 alkylene)-NRs, or -(C1-C5 alky1ene)-NR5C(=O)-(Ci-C5 alkylene)-O-CH2-NR8*, where * is a site covalently atached to A;

[0769] each R5is independently H, methyl, or benzyl;

[0770] each R8is independently H, methyl, or benzyl;

[0771] A is a peptide comprising 2 to 4 amino acids; and

[0772] E is C(=0)-(Ci-Cio alkylene)-X;’ wherein XJis

[0773] O

[0774]

[0775] O

[0776]

[0325] Thus, in some instances, the EpCAM immunoconjugates comprise at least one linker-payload comprising the structure of Formula (la) or a pharmaceutically acceptable salt thereof:

[0777] E-A-Z’-L'-D (Formula (la))

[0778] wherein:

[0779] D is represented by the following structural formula:

[0780]

[0781] R1is F;

[0782] R2is methyl;

[0783] -L’-Z’-* is --(C1-C4 alkylene)-O-CH2-NR8-*, -(C1-C4 alkylene)-NR°, or -(C1-C5 alkylene)-MR5C(=O)-(Ci-C5alkylene)-O-CH2-NR8*, where * is a site covalently attached to A;

[0784] each R5is independently H, methyl, or benzyl;

[0785] each R8is independently H, methyl, or benzyl;

[0786] A is a peptide comprising 2 to 4 amino acids; and

[0787] E is C(=O)-(Ci-Cic> alkylene)-X3wherein X3isAtty. Dkt. No.: 45325-0051WO1

[0788]

[0789] wherein * * is a site covalently attached to the EpCAM antibody or conditionally activatable masked antibody.

[0790]

[0326] In some instances, A in Formula (I) or A in Formula (la) is substituted with one or more polyol.

[0791] In some instances, the polyol is -(C1-C6 alkylene)-X5-Y3. wherein X5is -NR12C(=O)- or -C(=O)NR12-; Y3is C6 alkyl substituted with 5 OH groups; and R12is -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroary l, or benzy l.

[0792]

[0327] In some instances, the polyol of the immunoconjugate comprises

[0793]

[0794] wherein R 12 is H or methyl.

[0795]

[0328] In some instances, the immunoconjugate comprises the following linker-payload conjugate (i.e.

[0796] linker-payload prior to conjugation with the EpCAM antibody or conditionally activatable masked antibody):

[0797]

[0798] a pharmaceutically acceptable salt thereof.

[0799]

[0329] Thus, in some instances, the immunoconj ugate comprises the following linker-payload:Atty. Dkt. No.: 45325-0051WO1

[0800]

[0801] or a pharmaceutically acceptable salt thereof.

[0802]

[0330] In some instances, the immunoconjugate comprises the following linker-payload conjugate (z.e., linker-payload prior to conjugation with the EpCAM antibody or conditionally activatable masked antibody):

[0803]

[0804] a pharmaceutically acceptable salt thereof.

[0805]

[0331] Thus, in some instances, the immunoconjugate comprises the following linker-payload:

[0806]

[0807] Atty. Dkt. No.: 45325-0051WO1 or a pharmaceutically acceptable salt thereof.

[0808]

[0332] In the various embodiments disclosing -I. / -Z’-* herein, * is the site covalently attached to A. In some embodiments, A is a peptide comprising 2 to 8 amino acids. In some embodiments, A is a peptide comprising 2 to 4 amino acids. In some embodiments, at least one amino acid in said peptide is a L ammo acid. In some embodiments, each ammo acid in said peptide is a L ammo acid. In some embodiments, at least one amino acid in said peptide is a D ammo acid. In some embodiments, A is - (A A‘)-(AA2)ai-*, where * is the site covalently attached to E; AA1and AA2are each independently an amino acid residue; and al is an integer from 1-9. In some embodiments, -AA1-(AA2)a1-* is -Gly-Gly-Gly-*, -Ala- Vai-*, -Vai- Ala-*, - Val-Cit-*, -Val-Lys-*, -Lys-Val-*, -Phe-Lys-*,-Lys-Phe-*. -Lys-Lys-*, - Ala-Lys-*. -Lys- Ala-*, -Phe-Cit-*,-Cit-Phe-*. -Leu-Cit-*.- Cit-Leu-* -Ile-Cit-*, -Phe-Ala-*,-Ala-Phe-*, -Phe- Ns-tosyl-Arg-*, -N9-tosyl-Arg-Phe-*, -Phe-N9-nitro-Arg-*, -N9-nitro-Arg-Phe *, -Phe-Phe- Lys-*, -Lys-Phe-Phe-*, -Gly-Phe-Lys-*. Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Ile-Ala-Leu-*. - Leu-Ala-Ile-*, - Val-Ala-Val-*, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -^-Ala-Leu-Ala-Leu-*, -Gly-Phe-Leu-Gly- *,-Gly-Leu-Phe-Gly-*, -Val-Arg-*, -Arg- Vai-*, -Arg-Arg-*, -Ala- Ala-*, -Ala-Met-*, -Met-Ala-*, -Thr- Thr-*, -Thr-Met-*, -Met-Thr-*, -Leu-Ala-*, -Ala-Leu- *, -Cit-Val-*, -Gln-Val-*, -Val-Gln-*, -Ser- Vai-*, -Vai-Ser-*, -Ser-Ala-*, -Ser-Gly-*, -Ala- Ser-*, -Gly-Ser-*, -Leu-Gin-*, -Gin-Leu-*, -Phe-Arg-*, -Arg- Phe-*, -Tyr-Arg-*, -Arg-Tyr-*, -Phe-Gln-*, -Gln-Phe-*, -Val-Thr-*, -Thr-Val-*. -Met-Tyr-*, and -Tyr- Met-*. In some embodiments, -AA1-(AA2)al-* is -Val-D-Lys-*, -Val-D-Arg-*, -L- Val-Cit- *, -L- Val- Lys-*, -L-Val-Arg-*, -L-Val-D-Cit-*, -L-Phe-Phe-Lys-*, -L- Val-D-Lys-*, -L-Val- D-Arg-*, -L-Arg-D- Arg-*, -L-Ala-Ala-*, -L-Ala-D-Ala-*, -Ala-D-Ala-*, -Val-D-Cit-*, -L- Ala-L-Ala-*, -L-Ala-L-Val-*, - L-Gln-L-Val-*, -L-Gln-L-Leu-*, or -L-Ser-L- Vai-*. In some embodiments, -AA‘-(AA2)al-* is: -Ala- Ala-*, -Ala- Vai-*. -Vai -Ala-*. -Gin- Leu-*, -Leu-Gin-*. -Ala-Ala-Ala-*, -Ala- Ala-Ala- Ala-*. -Gly-Ala-Gly-Gly-*, -Gly-Gly-Ala-Gly-*, -Gly-Val-Gly-Gly-*, -Gly-Gly-Val-Gly-*, -Gly-Phe-Gly-Gly-*, or -Gly-Gly-Phe-Gly-*. In some embodiments, -AA1-(AA2)al-* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-*, -L-Ala-L-Val-*, -L-Ala-D-Val-*, -L-Val-L-Ala-*, -L-Val-D-Ala-*, -L-Gln-L-Leu-*, -L-Gln-D-Leu- *, -L- Lcu-L-Gln-*, -L-Lcu-D-Gln-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala- L-Ala-D-Ala-*, -L-Ala-L-Ala-L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-L-Ala-*, -L-Ala-L-Ala- D-Ala-L-Ala-*, -L-Ala-L- Ala-L-Ala-D-Ala-*, -Gly-L-Ala-Gly-Gly-*, -Gly-Gly-L-Ala-Gly-*, -Gly-D-Ala-Gly-Gly-*, -Gly-Gly-D-Ala-Gly-*, -Gly-L-Val-Gly-Gly-*, -Gly-Gly-L-Val-Gly-*, -Gly-D-Val-Gly-Gly-*, -Gly-Gly-D-Val-

[0809]

[0810] Gly-*, -Gly-L-Phe-Gly-Gly-*, or -Gly-Gly-L-Phe-Gly-*. In some embodiments, -AA1-(AA2)al-* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-L-Ala-*, or -L-Ala-L-Ala-L-Ala-L-Ala-*.

[0811]

[0333] In some embodiments, -AA’-(AA2)ai-* is -L-Ala-L-Ala-L-AIa-*.

[0812]

[0334] In the various embodiments disclosing -AA’-(AAJ)al-* herein, * is the site covalently attached to E.Atty. Dkt. No.: 45325-0051WO1

[0335] In some embodiments, the EpCAM antibody or conditionally activatable masked antibody is an IgGl antibody having eight cysteines which, when in a native conformation (e.g., not reduced), are involved in forming four disulfide bonds. See, e.g., SEQ ID NOs:103, 140, and 179 in Tables 6, 7, and 11, respectively for exemplary identification of such cysteine residues. As provided herein, these disulfide bonds can be reduced, resulting in eight free cysteines and cysteine thiol groups. One linker¬ payload reactant can react with one cysteine’s thiol group, with up to eight linker-payload reactants reacting with each EpCAM antibody or conditionally activatable masked antibody. In some embodiments, each EpCAM antibody or conditionally activatable masked antibody reacts with up to eight linker-payload reactants resulting in a linker-payload to antibody ratio (i.e., a drug-to-antibody ratio (DAR)) ranging from 2 to 8. In some instances, the DAR ranges from 4 to 8. In some instances, the DAR ranges from 5 to 8. In some instances, the DAR ranges from 6 to 8 In some instances, the DAR ranges from 7 to 8. In certain embodiments, the DAR represents the average number of cytotoxic linker-payload agents per antibody or conditionally activatable masked antibody molecule. In some embodiments the DAR is 2. In some embodiments the DAR is 3. In some embodiments the DAR is 4. In some embodiments the DAR is 5. In some embodiments the DAR is 6. In some embodiments the DAR is 7. In some embodiments the DAR is 8.

[0813]

[0336] In some instances, the linker-payload to antibody ratio (i.e., a drug-to-antibody ratio (DAR)) ranges from 2 to 12. In some instances, the DAR is at least 2, at least 3, at least 4, 4, at least 5, 5, at least 6, 6, at least 7, 7, at least 8, 8, at least 9, 9, at least 10, 10, at least 11, 11, at least 12, or 12. In certain embodiments, the DAR represents the average number of cytotoxic linker-payload agents per antibody or conditionally activatable masked antibody molecule. In some embodiments, the DAR is 8.

[0814]

[0337] In some embodiments, the disclosure provides an EpCAM immunoconjugate comprising at least one linker-payload comprising a compound of the following formula, or a pharmaceutically acceptable salt thereof:

[0815] Z-L'-D

[0816] wherein D is represented by the following structural formula:

[0817]

[0818] R1is —F, —CH3, or —CF3;

[0819] R2is H, —F, —OR3, —SR3, —S(O)R4, —S(O)2R4, C1-C6alkyl, or C1-C6fluoroalkyl; or R1and R2taken together with the carbon atoms to which they are attached form a methylenedioxy or a

[0820] difluoromethylenedioxy ring;Atty. Dkt. No.: 45325-0051WO1 R is H or Ci-Ce alkyl;

[0821] R4is Ci-Cs alkyl;

[0822] L1is absent, — (Ci-Ce alkylene)-, — (Ci-Csalkylene)-X1— (Ct-Ce alkylene)-, — X1' — (C1-C5 alkylene)-*, or —-(Ci-Ce alkylene)-X‘-L2-*: where * is the site covalently attached to Z;

[0823] X1is —O—, —S—, —S(O)—, —S(O)2—, —C(=O)—, —NR5—, —NR5C(=O)— or —C(=O)NR5—

[0824] X is —0— — S—, — S(0)—, or — S(0)2—;

[0825] L2is phenylene;

[0826] each R5is independently —H, C1-C6alkyl, C1-C6fluoroalkyl, C3-C6cycloalkyl, aryl, heteroaryl, or benzyl;

[0827] Z is — II or — X2;

[0828] X2is —OR6, —SR6. — S(O)R6, — S(O)2R6, --SSR6. or — N(R6)2;

[0829] each R6is independently —H, C1-C6alkyl, C1-C6fluoroalkyl, C3-C6cycloalkyl, aryl, heteroaryl, or benzyl;

[0830] L1and L2are each independently optionally substituted with 1 -4 substituents selected from halogen, — CN, — SR7, — N(R7)2, CI -Ce alkyl, Ci-Cg fluoroalkyl, Ci-Ce heteroalkyl, C3-C6 cycloalkyl, C2- C10 heterocycloalkyl, aryl, or heteroaryl; and

[0831] each R7is independently H, C1-C6alkyl, C1-C6fluoroalkyl, C3-C6cycloalkyl, aryl, heteroaryl, or benzyl; with the proviso that if R1is F, then L1is —(C1-C6alkylene)-, —(C1-C6alkylene)-X1—(C1-C6alkylene)-, —X1'—(C1-C6alkylene)-*, or —(C1-C6alkylene)-X1-L2-*; where * is the site covalently attached to Z; and Z is --X2;

[0832] with the proviso that if R1is F and R2is — OMe, then cannot be NH2; and

[0833] with the proviso that if R1is F and R2is -Me, then -L1-Z cannot be —CH2OH.

[0834]

[0338] Additional camptothecin derivatives are disclosed in US Appl. Publ. No. 2021 / 0077482 Al and U. S. Pat. No. 11,229,639 B2, each of which is incorporated by reference herein in its entirety.

[0835]

[0339] In some embodiments, the linker-pay load of the immunoconjugate comprises exatecan or a derivative thereof. In some embodiments, the linker-payload of the immunoconjugate comprises reduced deruxtecan, in which the maleimide group of deruxtecan is reduced to succinimide during the conjugation reaction thereby linking deruxtecan to the EpCAM antibody or conditionally activatable masked antibody via a thiol group. Deruxtecan comprises an exatecan derivative and a maleimide- GGFG linker (SEQ ID NO:311). Exatecan, its derivatives, and deruxtecan are described in, e.g., W02015 / 115091, the contents of which are herein incorporated by reference in its entirety

[0836]

[0340] In a specific embodiment, the conditionally activatable masked immunoconjugate comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179, wherein the one or more linker-payloads of Formula (X) areAtty. Dkt. No.: 45325-0051WO1 each covalently bound to an interchain cysteine of the AB (e.g., via a thioether bond) at a DAR of 8 (“EpCAM-CPT66”). In a further embodiment, the activatable masked immunoconjugate comprises an activatable antibody having a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179, wherein the one or more linkerpayloads of Formula (X) are each covalently bound to an interchain cysteine of the AB (e.g., via a thioether bond) at a DAR of 8 (“EpCAM-CPT66”).

[0837] THERAPEUTIC APPLICATIONS

[0838]

[0341] Also included are methods for inhibiting the growth of cells expressing EpCAM (e.g, human EpCAM or cynomolgus EpCAM). As provided herein, the disclosed EpCAM immunoconjugates have the ability to bind EpCAM present on the surface of a cell (e.g., a human cell or a cynomolgus cell) and mediate cell killing. In particular embodiments, the immunoconjugates comprise a cytotoxic payload, e.g., a camptothecin derivative linker-payload, are internalized and mediate cell killing via the activity of the cytotoxic payload. Such cell killing activity can be augmented by the immunoconjugate inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC).

[0839]

[0342] As used herein the terms ‘’inhibit” and “inhibiting” include any inhibitory effect on cell growth, including cell death. The inhibitory? effects include temporary? effects, sustained effects and permanent effects.

[0840]

[0343] The method involves treating, alleviating symptoms of, or delaying the progression of a disorder or disease in a subject in which diseased cells express epithelial cell adhesion molecule (EpCAM). This is achieved by administering to tlie subject a therapeutically effective amount of an immunoconjugate that consists of an conditionally activatable masked antibody and at least one linkerpayload. The conditionally activatable masked antibody? comprises an EpCAM antibody or an EpCAM- binding fragment that contains specific heavy and light chain complementarity de...

Claims

1. Atty. Dkt. No.: 45325-0051WO1WHAT IS CLAIMED IS:

1. A method of treating, alleviating a symptom of. or delaying the progression of a disorder or disease in a subject in which diseased cells express epithelial cell adhesion molecule (EpCAM), the method comprising administering to the subject a dose of about 1.0 mg / kg to about 20 mg / kg of a conditionally activatable masked immunoconjugate comprising:(a) an activatable antibody comprising:(i) an EpCAM antibody or EpCAM -binding fragment thereof ( AB), comprising:(1) a heavy chain complementarity determining region 1 (VH-CDR1) comprising the amino acid sequence NYYIH (SEQ ID NO: 13); (2) a heavy chain complementarity determining region 2 (VH-CDR2) comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14);(3) a heavy chain complementarity determining region 3 (VH-CDR3) comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15); (4) a light chain complementarity determining region 1 (VL-CDR1) comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO:42);(5) a light chain complementarity determining region 2 (VL-CDR2) comprising the amino acid sequence QTSNLAS ( SEQ ID NO:40); and(6) a light chain complementarity determining region 3 (VL-CDR3) comprising the amino acid sequence AQNLELPNT (SEQ ID NO:41);(li) a cleavable moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169): and(iii) a masking moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155). wherein the masking moiety inhibits the binding of the antibody or antibody fragment to EpCAM when the activatable antibody is in an uncleaved state,wherein the activatable antibody in an uncleaved state has a structural arrangement from N- terminus to C-terminus of: (masking moiety )-(cleavable moiety)-(antibody or antibody fragment) or (antibody or antibody fragment)-(cleavable moiety)-(masking moiety); and (b) at least one linker-payload depicted by the structure of Formula (X):Atty. Dkt. No.: 45325-0051WOIFormula (X)or a pharmaceutically acceptable salt thereof (Formula (X)), wherein the at least one linker-payload is covalently bound to the AB.2, A method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease in a subject in which diseased cells express EpCAM, the method comprising administering to the subject a dose of about 1.0 mg / kg to about 20,0 mg / kg of an conditionally activatable masked immunoconj ugate comprising:(a) an activatable antibody comprising:(i) an EpCAM antibody or EpCAM-binding fragment thereof (AB), comprising:(1) a VH-CDR1 comprising the amino acid sequence NYYIH (SEQ ID NO: 13);(2) a VH-CDR2 comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14);(3) a VH-CDR3 comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15);(4) a VL-CDR1 comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO:42);(5) a VL-CDR2 comprising the amino acid sequence QTSNLAS (SEQ ID NO:40); and(6) a VL-CDR3 comprising the amino acid sequence AQNLELPNT (SEQ ID NO:41);Atty. Dkt. No.: 45325-0051WOI(ii) a cleavable moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 168): and(iii) a masking moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein the masking moiety inhibits the binding of the antibody or antibody fragment to EpCAM when the activatable antibody is in an uncleaved state.wherein the activatable antibody in an uncleaved state has a structural arrangement from N-terminus to C -terminus of: (masking moiety)-(cleavable moiety)-(antibody or antibody fragment) or (antibody or antibody fragment)-(cleavable moiety) -(masking moiety); and(b) at least one linker-payload depicted by the structure of Formula (X):Formula (X)or a pharmaceutically acceptable salt thereof (Formula (X)), wherein the at least one linker-pavload is covalently bound to the AB.

3. The method of claim 1 or 2. wherein the conditionally activatable masked immunoconjugate comprises a drug-to-antibody ratio (DAR) of 8.

4. The method of claim 1 or 2. wherein the conditionally activatable masked immunoconjugate comprises a drug-to-antibody ratio (DAR) of 4.

5. The method of any one of claims 1-4, wherein the EpCAM antibody or EpCAM-binding fragment thereof comprises a heavy chain variable domain (VH) comprising the amino acid sequenceAtty. Dkt. No.: 45325-0051WOIof SEQ ID NO:54 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 89,6. The method of claim 1, wherein the activatable antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179.

7. The method of claim 1, wherein the activatabie antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179, wherein the linker-payload of Formula (X) is conjugated to an interchain Cys of the AB via a thioether bond, and wherein the conditionally activatable masked immunoconjugate has a drug-to-antibody ratio (D R) of 8.

8. The method of claim 1, wherein the activatabie antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179, wherein the linker-payload of Formula (X) is conjugated to an interchain Cys of the AB via a thioether bond, and wherein the conditionally activatable masked immunoconjugate has a drug-to-antibody ratio (DAR) of 4.

9. The method of any one of claims 1-8, wherein the disease is cancer.

10. The method of claim 9. wherein the cancer is a solid tumor cancer.

11. The method of claim 9, wherein the cancer is advanced or metastatic colorectal cancer.

12. The method of claim 9, wherein the cancer is advanced or metastatic gastric cancer.

13. The method of claim 9, wherein the cancer is gastroesophageal junction carcinoma, triple¬ negative breast cancer, non-small cell lung cancer, or pancreatic ductal adenocarcinoma.

14. The method of claim 9, wherein the cancer is advanced or metastatic ovarian cancer.

15. The method of claim 9. wherein the cancer is advanced or metastatic endometrial cancer.

16. The method of any one of claims 1 -15, wherein the subject is human.Atty. Dkt. No.: 45325-0051WOI17. The method of any one of claims 1-16, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 2.4 mg / kg to about 11 mg / kg.

18. The method of any one of claims 1-17. wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 1.2 mg / kg.

19. The method of any one of claims 1-17, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 2.4 mg / kg.

20. The method of any one of claims 1-17, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 4.8 mg / kg.

21. The method of any one of claims 1-17, wherein the conditionally activatable masked immunoconj ugate is administered at a dose of about 7.2 mg / kg.

22. The method of any one of claims 1-17, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 8.6 mg / kg.

23. The method of any one of claims 1-17, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 10.0 mg / kg.

24. The method of any one of claims 1-17, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 11.0 mg / kg.

25. The method of any one of claims 1-17, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 11 mg / kg to about 20 mg / kg.

26. The method of claim 25, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 12.0 mg / kg.

27. The method of any one of claims 1-26, wherein the dose is based on actual body weight (ABW) of the subject.

28. The method of any one of claims 1-26, wherein the dose is based on adjusted ideal body weight (AlBW) of the subject.Atty. Dkt. No.: 45325-0051WOI29. The method of any one of claims 1-27, wherein, if ABW of the subject is less than AIBW, then the dose administered is based on the ABW of the subject.

30. The method of any one of claims 1-26 or 28, wherein, if the height of the subject is less than 60 inches, then the A1BW is used and is calculated as follows:AIBW’ ” {IBW + [0.4 x (ABW-IBW)]} - (5 pounds / inch)(60 inches - the subject's height in inches).

31. The method of any one of claims 1-27 or 29. wherein the weight of the subject is greater than 100 kg, and wherein the dose for the subject is based on an ABW that is fixed at 100 kg.

32. The method of any one of claims 1-31, wherein the administering occurs once every week.

33. The method of any one of claims 1-31, wherein the administering occurs once every two weeks,34. The method of any one of claims 1-31, wherein the administering occurs once every three weeks.

35. The method of any one of claims 1-31, wherein the administering occurs once every four weeks.

36. The method of any one of claims 1-35, wherein the administering is via intravenous infusion.

37. The method of any one of claims 1 -36, wherein the administering is via intravenous infusion for about 30 minutes to about 2 hours.

38. The method of any one of claims 1-37, wherein the administering is via intravenous infusion for about 60 minutes.

39. The method of any one of claims 1-38, further comprising administering one or more additional agents,40. The method of claim 39, wherein the one or more additional agents comprise an anti-VEGF agent, an anti-epidermal growth factor receptor agent, an immune checkpoint inhibitor, a v-Raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor, a mitogen-activated proteinAtty. Dkt. No.: 45325-0051WOIkinase / ERK Kinase (MEK) inhibitor, a human epidermal growth factor receptor 2 (HER2) inhibitor, or a Kirsten rat sarcoma viral oncogene homolog (KRAS) inhibitor.

41. The method of claim 40, wherein the anti-VEGF agent is bevacizumab, wherein the bevacizumab is administered at a dose of about 5 mg / kg ABW once every two weeks or wherein the bevacizumab is administered at a dose of about 7.5 mg / kg ABW once every three weeks.

42. The method of any one of claims 1 -41, wherein the conditionally activatable masked immunoconjugate is formulated as a pharmaceutical composition.

43. The method of claim 42, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

44. A method of treating, alleviating a symptom of, or delaying the progression of a disorder or disease in a subject in which diseased cells express EpC AM, the method comprising administering to the subject:(1) a dose of about 1.0 mg / kg to about 20 mg / kg ABW or AIBW of a conditionally activatable masked immunoconjugate comprising:(a) an activatable antibody comprising:(i) an EpCAM antibody or EpCAM-binding fragment thereof (AB), comprising:(1) a VH-CDR1 comprising the amino acid sequence NYYIH (SEQ ID NO: 13);(2) a VH-CDR2 comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14);(3) a VH-CDR3 comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15);(4) a VL-CDR1 comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO:42);(5) a VL-CDR2 comprising the amino acid sequence QTSNLAS (SEQ ID NO:40); and(6) a VL-CDR3 comprising the amino acid sequence AQNLELPNT (SEQ ID NO:41);(ii) a cleavable moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 168); andAtty. Dkt. No.: 45325-0051WOI(iii) a masking moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein the masking moiety’ inhibits the binding of the antibody or antibody fragment to EpCAM when the activatable antibody is in an uncleaved state.wherein the activatable antibody in an uncleaved state has a structural arrangement from N-terminus to C -terminus of: (masking moiety)-(cleavable moiety) -(antibody or antibody fragment) or (antibody' or antibody fragment)-(cleavable moiety)-(masking moiety); and(b) at least one linker-payload depicted by the structure of Formula (X):Formula (X)or a pharmaceutically acceptable salt thereof (Formula (X)), wherein the at least one linker-payload is covalently bound to the AB; and(II) bevacizumab.

45. A method of treating, alleviating a symptom of. or delaying the progression of a disorder or disease in a subject in which diseased cells express epithelial cell adhesion molecule (EpCAM). the method comprising administering to the subject(I) a dose of about 1.0 mg / kg to about 20 mg / kg ABW or AIBW of a conditionally activatable masked immunoconjugate comprising:(a) an activatable antibody’ comprising:(i) an EpCAM antibody’ or EpCAM-binding fragment thereof (AB), comprising:(1) a VH-CDR1 comprising the amino acid sequence NYYIH (SEQ ID NO: 13);Atty. Dkt. No.: 45325-0051WOI( 2) a VH-CDR2 comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14);(3) a VH-CDR3 comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15):(4) a VL-CDR 1 comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO:42);(5) a VL-CDR2 comprising the amino acid sequence QTSNLAS (SEQ ID NO:40); and(6) a VL-CDR3 comprising the amino acid sequence AQNLELPNT (SEQ ID NO:41):(ii) a cleavable moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169); and(iii) a masking moiety coupled to the EpCAM antibody or EpCAM-binding fragment thereof that comprises the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein the masking moiety inhibits the binding of the antibody or antibody fragment to EpC AM when the activatable antibody is in an uncleaved state.wherein the activatable antibody in an uncleaved state has a structural arrangement from N-terminus to C -terminus of: (masking moiety )-(cleavable moiety)-(antibody or antibody fragment) or (antibody or antibody fragment) -(cleavable moiety)-(masking moiety); and(b) at least one linker-payload depicted by the structure of Formula (X):H H N NN' N N H HFormula (X)Atty. Dkt. No.: 45325-0051WOIor a pharmaceutically acceptable salt thereof (Formula (X)), wherein the at least one linker-payload is covalently bound to the AB; and(II) bevacizumab.

46. The method of claim 44 or 45, wherein the conditionally activatable masked immunoconjugate has a drug-to-antibody ratio (DAR) of 8.

47. The method of claim 44 or 45, wherein the conditionally activatable masked immunoconjugate has a drug-to-antibody ratio (DAR) of 4.

48. The method of any one of claims 44-47, wherein the EpCAM antibody or EpCAM-binding fragment thereof comprises a heavy chain variable domain (VH) comprising the amino acid sequence of SEQ ID NO:54 and a light chain variable domain (VL) comprising the amino acid sequence of SEQ ID NO: 89.

49. The method of claim 44, wherein the activatable antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179.

50. The method of claim 44, wherein the activatable antibody comprises a heavy' chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179, wherein the linker-payload of Formula (X) is conjugated to an interchain Cys of the AB via a thioether bond, and wherein the conditionally activatable masked immunoconjugate has a drug-to-antibody ratio (DAR) of 8.

51. The method of claim 44, wherein the activatable antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 179, wherein the linker-payload of Formula (X) is conjugated to an interchain Cys of the AB via a thioether bond, and wherein the conditionally activatable masked immunoconjugate has a drug-to-antibody ratio (DAR) of 4.

52. The method of any one of claims 44-51. wherein the disease is cancer53. The method of claim 52, wherein the cancer is a solid tumor cancer.

54. The method of claim 52, wherein the cancer is advanced or metastatic colorectal cancer.Atty. Dkt. No.: 45325-0051WOI55. The method of claim 52. wherein the cancer is advanced or metastatic gastric cancer.

56. The method of claim 52, wherein the cancer is gastroesophageal junction carcinoma, triplenegative breast cancer, non-small cell lung cancer, or pancreatic ductal adenocarcinoma.

57. The method of claim 52, wherein the cancer is advanced or metastatic ovarian cancer.

58. The method of claim 52, wherein the cancer is advanced or metastatic endometrial cancer.

59. The method of any one of claims 44-58, wherein the subject is human.

60. The method of any one of claims 44-59, wherein the dose is based on actual body weight (ABW) of the subject.

61. The method of any one of claims 44-59, wherein the dose is based on adjusted ideal body weight (A1BW) of the subject.

62. The method of any one of claims 44-60. wherein, if ABW of the subject is less than AIBW. then the dose administered is based on the ABW of the subject.

63. The method of any one of claims 44-59 or 61, wherein, if the height of the subject is less than 60 inches, then the AIBW is used and is calculated as follows:AIBW7’ = [IBW + [0.4 x (ABW-IBW)]} - (5 pounds / inch)(60 inches - the subject’s height in inches).

64. The method of any one of claims 44-60 or 62, wherein the weight of the subject is greater than 100 kg, and wherein the dose for the subject is based on an ABW that is fixed at 100 kg.

65. 'The method of any one of claims 44-64, wherein the subject is administered the conditionally activatable masked immunoconjugate once every week66. The method of any one of claims 44-64, wherein the subject is administered the conditionally activatable masked immunoconjugate once every 2 weeks.Atty. Dkt. No.: 45325-0051WOI67. The method of any one of claims 44-64. wherein the subject is administered the conditionally activatable masked immunoconjugate once every 3 weeks.

68. The method of any one of claims 44-64, wherein the subject is administered the conditionally activatable masked immunoconjugate once every 4 weeks.

69. The method of any one of claims 44-68, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 1.2 mg / kg.

70. The method of any one of claims 44-68, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 2.4 mg / kg.

71. The method of any one of claims 44-68, wherein the conditionally activatable masked immunoconj ugate is administered at a dose of about 4.8 mg / kg.

72. The method of any one of claims 44-68, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 6.0 mg / kg.

73. The method of any one of claims 44-68. wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 7.2 mg / kg.

74. The method of any one of claims 44-68, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 8.6 mg / kg.

75. The method of any one of claims 44-68. wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 10.0 mg / kg.

76. The method of any one of claims 44-68, w herein the conditionally activatable masked immunoconjugate is administered at a dose of about 11.0 mg / kg.

77. The method of any one of claims 44-68, wherein the conditionally activatable masked immunoconjugate is administered at a dose of about 12 mg / kg.

78. The method of any one of claims 44-77, wherein the conditionally activatable masked immunoconjugate is administered via intravenous infusion.Atty. Dkt. No.: 45325-0051WOI79. The method of any one of claims 44-78. wherein the conditionally activatable masked immunoconjugate is administered via intravenous infusion for about 30 minutes to about 2 hours.

80. The method of any one of claims 44-79, wherein the conditionally activatable masked immunoconjugate is administered via intravenous infusion for about 60 minutes.

81. The method of any one of claims 44-80, wherein the conditionally activatable masked immunoconjugate is formulated as a pharmaceutical composition.

82. The method of claim 81, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

83. The method of any one of claims 44-82, wherein the subject is administered the bevacizumab once every 2 weeks.

84. The method of any one of claims 44-82, wherein the subject is administered the bevacizumab once every 3 weeks.

85. The method of any one of claims 44-82. wherein the subject is administered the bevacizumab once every 4 weeks.

86. The method of any one of claims 44-85, wherein the bevacizumab is administered at a dose of about 5 mg / kg.

87. The method of any one of claims 44-85. wherein the bevacizumab is administered at a dose of about 7.5 mg / kg.

88. The method of any one of claims 44-87, wherein the bevacizumab is administered via intravenous infusion.

89. The method of any one of claims 1-88, further comprising administering a prophylactic agent to the subject.

90. The method of claim 89, wherein the prophylactic agent is an anti-diarrheal agent.

91. The method of claim 90, wherein the anti-diarrheal agent is loperamide.Atty. Dkt. No.: 45325-0051WOI92. The method of claim 91. wherein the loperamide is administered orally.

93. The method of claim 91 or 92, wherein the loperamide is administered at a dose of about 1 mg to about 15 mg94. The method of any one of claims 91-93, wherein the loperamide is administered at a dose of about 4 mg to about 12 mg.

95. The method of any one of claims 91-94, wherein the loperamide is administered at a dose of about 4 mg.

96. The method of any one of claims 91-94, wherein the loperamide is administered one time daily.

97. The method of any one of claims 91-94, wherein the loperamide is administered two times daily.

98. The method of any one of claims 91-94. wherein the loperamide is administered three times daily.

99. The method of any one of claims 91-98. wherein the loperamide is administered for seven days.

100. The method of claim 90, wherein the anti-diarrheal agent is budesonide.

101. The method of claim 100, wherein the budesonide is administered orally, rectally, nasally, or via inhalation.

102. The method of claim 100 or 101, wherein the budesonide is administered at a dose of about 1 mg to about 15 mg.

103. The method of any one of claims 100-102, wherein the budesonide is administered at a dose of about 4 mg to about 12 mg.Atty. Dkt. No.: 45325-0051WOI104. The method of any one of claims 100-103, wherein the budesonide is administered at a dose of about 9 mg.

105. The method of any one of claims 100-104. wherein the budesonide is administered one time daily106 The method of any one of claims 100-104. wherein the budesonide is administered two times daily.

107. The method of any one of claims 100-104. wherein the budesonide is administered three times daily.

108. The method of any one of claims 100-107, wherein the budesonide is administered for seven days.

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