EPCAM Immunoconjugate and Its Use

Abstract

Broadly speaking, the present disclosure relates to immunoconjugates comprising antibodies and antibody fragments that specifically bind to human EPCAM, as well as methods for making and using human EPCAM antibodies, human EPCAM antibody fragments, human EPCAM activatable antibodies, and human EPCAM immunoconjugates for the diagnosis and treatment of diseases such as cancer.

Description

Technical Field

[0001] 1. Cross - reference to related applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 388,486, filed on July 12, 2022, the entire content of which is incorporated herein by reference.

[0002] 2. Sequence Listing This application includes a sequence listing that was electronically submitted in XML format, the entire content of which is incorporated herein by reference. The name of the above XML copy created on July 6, 2023 is CYTX - 097 - PCT_SL.xml, and the size is 336,140 bytes.

Background Art

[0003] 3. Background Art Broadly speaking, the present disclosure relates to antibodies and antibody fragments that specifically bind to human EpCAM, EpCAM - activatable antibodies, and their immunoconjugates, as well as methods for making and using such antibodies, antibody fragments, activatable antibodies, and immunoconjugates for the diagnosis and treatment of diseases such as cancer.

[0004] Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein that includes an extracellular domain, a transmembrane domain, and a single intracellular domain. The expression of EpCAM in humans is epithelial - specific. Most epithelial cells, except for some specific epithelial cell types such as squamous epithelial cells, epidermal keratinocytes, hepatocytes, gastric parietal cells, and myoepithelial cells, express EpCAM (Balzar et al., J. Mol. Med. 77:699 - 712 (1999), Momburg et al., Cancer Res 47:2883 - 2891 (1987)).

[0005] EpCAM is expressed abundantly and homogeneously on human carcinomas of various origins (Went et al., Br. J. Cancer 94:128 - 35 (2006); Herlyn et al., Proc Natl. Acad. Sci. USA 76:1438 - 1442 (1979); Went et al., Hum. Pathol. 35:122 - 128 (2004)). EpCAM is overexpressed in the majority of epithelial cancers, including, for example, ovarian, colorectal, gastric, prostate, and lung cancers. In addition, EpCAM has been shown to be expressed in the majority of primary, metastatic cancer cells, and disseminated NSCLC (non - small cell lung cancer cells) (Passlick, Int. 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, and breast cancers (Szala, Proc. Natl. Acad. Sci. USA 87:3542 - 3546 (1990), Packeisen, Hybridoma 18:37 - 40 (1999)). In another study, immunohistochemical analysis of 108 secondary tumor samples revealed that only 4% lacked EpCAM expression. EpCAM is also overexpressed in cancer - initiating cells or cancer stem cells isolated from colorectal, breast, pancreatic, and prostate cancers (O’Brien et al., Nature 445:106 - 110 (2007); Marhaba et al., Curr. Mol. Med. 8:784 - 804 (2008)).

[0006] Normal cells express EpCAM on the basolateral side of the epithelial membrane, while cancer cells express EpCAM abundantly on the apical membrane side. Since EpCAM in normal cells is less prominent and less exposed, healthy cells may be less likely to be affected by the binding of therapeutic anti - EpCAM antibodies. Antibody - based therapeutic agents are designed to take advantage of this property of EpCAM expression.

[0007] Camptothecin (CPT) is a pentacyclic alkaloid isolated from the bark and stems of Camptotheca acuminata (Camptotheca, happy tree), a tall tree native to China. Camptothecin inhibits topoisomerase I, which leads to cell death. Due to its cytotoxic mechanism and broad-spectrum antitumor activity, great efforts have been made towards the development of clinical analogs of camptothecin. However, due to its poor solubility and inactivity under physiological conditions, the clinical development of suitable camptothecin analogs has been limited. Most of camptothecin and its derivatives are insoluble in aqueous buffers. Furthermore, since camptothecin exists in an equilibrium state between the active lactone form and the inactive hydrolyzed carboxylate form, its therapeutic effect is limited.

[0008] Several antibodies against EpCAM have been used clinically but have failed for various reasons. The tested EpCAM antibodies take several formats, including naked antibodies, immunotoxins, and bispecific or trispecific antibodies (Baeuerle, Br. J. Cancer, 96:417-423 (2007)). For example, adecatumumab (MT201), a naked anti-EpCAM antibody, is being tested in clinical trials for the treatment of colorectal cancer, prostate cancer, and breast cancer. Safety issues faced by current anti-EpCAM antibody-based approaches include systemic intolerance and acute pancreatitis. Thus, several attempts have been made to develop therapeutic antibodies against EpCAM, but there is a great need for the development of novel therapeutic EpCAM immunoconjugates that overcome the drawbacks and limitations of the antibodies or antibody derivatives developed so far. SUMMARY OF THE INVENTION

[0009] 4. Summary of the Invention In one aspect, the present disclosure provides an activatable antibody comprising (a) a full-length human IgG1 EpCAM antibody having eight cysteines that form four inter-chain disulfide bonds when in its native higher-order structure, wherein the full-length human IgG1 EpCAM 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, and (b) a structure: [Chemical formula] or eight linker payloads illustrated by its pharmaceutically acceptable salt, wherein each of the eight linker payloads is individually covalently bound to the activatable antibody via one of the eight cysteines, and provides an immunoconjugate comprising the eight linker payloads.

[0010] In another aspect, the present disclosure provides an activatable antibody comprising: (a) an activatable antibody, wherein (i) the activatable antibody is an EpCAM antibody or an EpCAM-binding fragment thereof, and comprises: (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); (ii) a cleavable moiety coupled to the EpCAM antibody or an EpCAM-binding fragment thereof, the cleavable moiety comprising the amino acid sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 168) or the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169); and (iii) a masking moiety coupled to the EpCAM antibody or an EpCAM-binding fragment thereof, the masking moiety comprising the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein when the activatable antibody is in an uncleaved state, the masking moiety inhibits the binding of the antibody or antibody fragment to EpCAM; and the activatable antibody has a structural sequence from the N-terminus to the C-terminus of: (masking moiety)-(cleavable moiety)-(antibody or antibody fragment), or (antibody or antibody fragment)-(cleavable moiety)-(masking moiety) when in an uncleaved state; and (b) a structure: [Chemical Structure] and at least one linker payload represented by or a pharmaceutically acceptable salt thereof.

[0011] In some embodiments, the immunoconjugate has a drug-to-antibody ratio (DAR) of 8.

[0012] In some embodiments, the activatable antibody is a full-length human IgG1 EpCAM antibody having eight cysteines that form four inter-chain disulfide bonds when in its native higher-order structure, and each linker payload is covalently attached individually to the activatable antibody via one of the eight cysteines.

[0013] In yet another aspect, the present disclosure provides an activatable antibody comprising: (a) an activatable antibody that is a full-length human IgG1 EpCAM antibody having eight cysteines that form four inter-chain disulfide bonds when in its native higher-order structure, and comprising: (i) a heavy-chain complementarity-determining region 1 (VH-CDR1) comprising the amino acid sequence NYYIH (SEQ ID NO: 13); (ii) a heavy-chain complementarity-determining region 2 (VH-CDR2) comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14); (iii) a heavy-chain complementarity-determining region 3 (CDR3) (VH-CDR3) comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15); (iv) a light-chain complementarity-determining region 1 (VL-CDR1) comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO: 42); (v) a light-chain complementarity-determining region 2 (VL-CDR2) comprising the amino acid sequence QTSNLAS (SEQ ID NO: 40); and (vi) a light-chain complementarity-determining region 3 (VL-CDR3) comprising the amino acid sequence AQNLELPNT (SEQ ID NO: 41); (ii) a cleavable moiety coupled to the EpCAM antibody or an EpCAM-binding fragment thereof and comprising the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169); and (iii) a masking moiety coupled to the EpCAM antibody or an EpCAM-binding fragment thereof and comprising 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 the uncleaved state. The activatable antibody has a structural sequence from the N-terminus to the C-terminus of: (masking moiety)-(cleavable moiety)-(antibody) or (antibody)-(cleavable moiety)-(masking moiety) when in the uncleaved state; (b) a structure:

Chemical Structure

[0014] In some embodiments, the full-length human IgG1 EpCAM antibody 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. In other embodiments, the full-length human IgG1 EpCAM 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.

[0015] In another aspect, the present specification provides an immunoconjugate obtainable by the method of the section "Preparation of huEpCAM23Gv4.2-LalaLalaLala-CPT66" of Example 4 provided herein.

[0016] In another aspect, the present specification (a) mixes an activatable EpCAM antibody consisting of a heterotetramer of two heavy chains each consisting of the amino acid sequence of SEQ ID NO: 103 and two light chains each consisting of the amino acid sequence of SEQ ID NO: 179 with a mixture of an EPPS buffer and an aqueous EDTA solution to form an activatable EpCAM reaction mixture, (b) mixes an activatable EpCAM antibody consisting of a heterotetramer of two heavy chains each consisting of the amino acid sequence of SEQ ID NO: 103 and two light chains each consisting of the amino acid sequence of SEQ ID NO: 179 with a mixture of an EPPS buffer and an aqueous EDTA solution to form an activatable EpCAM reaction mixture, (c) mixes the reduced activatable EpCAM reaction mixture with a stock solution of a camptothecin linker toxin containing a linker toxin represented by the following structure,

Chemical formula

[0017] In another aspect, the present specification provides a nucleic acid comprising one or more codings of the activatable antibody of the immunoconjugate described herein. Vectors containing such nucleic acids are also provided. In some embodiments, the vector is a viral vector.

[0018] In another aspect, the present specification provides a method for producing an immunoconjugate, comprising: (a) culturing a host cell containing a vector of the present disclosure; (b) isolating an activatable antibody from the host cell; and (c) conjugating at least one linker-payload reactant containing camptothecin or a derivative thereof to the activatable antibody.

[0019] In another aspect, the present specification provides a method for producing an immunoconjugate, comprising conjugating at least one linker-payload reactant containing camptothecin or a derivative thereof to the activatable EpCAM antibody described herein.

[0020] In some embodiments, the linker-payload reactant comprises a compound of Formula I, E-A-L-D (Formula I) wherein E-A-Z’-L 1 -D (Formula I) wherein D is represented by the following structural formula,

Chemical formula

Chemical formula

[0021] In some embodiments, the linker-payload reactant is

Chemical formula

[0022] In some embodiments, the linker-payload reactant is

Chemical formula

[0023] In certain embodiments, the linker-payload reactant comprises exetecan. In other embodiments, the linker-payload reactant comprises deluxtecan.

[0024] Compositions comprising the immunoconjugates of the present disclosure and a pharmaceutically acceptable carrier are also provided.

[0025] In some aspects, the immunoconjugates or compositions of the present disclosure can be used for the treatment of cancers that express EpCAM. In some embodiments, the cancer that expresses EpCAM is colorectal cancer.

Brief Description of the Drawings

[0026] 5. Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0027] 6. Modes for Carrying Out the Invention The present disclosure provides an immunoconjugate having an antibody and an antigen-binding antibody fragment that specifically binds to human EpCAM and includes an activatable antibody (an activatable form of an EpCAM antibody or its EpCAM-binding antibody fragment). In some cases, the immunoconjugate includes a camptothecin derivative linker payload having the following structure (where the following structure represents the linker payload prior to conjugation to the antibody, i.e., the linker payload reactant). [Chem.] Accordingly, in some cases, the immunoconjugate comprises a camptothecin derivative linker payload having the following structure (wherein ** indicates the position of covalent attachment to the EpCAM antibody or activatable antibody): [Chem.]

[0028] EpCAM is involved in cell-cell adhesion in epithelia and is known to be involved in cell signaling, differentiation, proliferation, and migration. Overexpression of EpCAM is involved in the etiology of diseases and disorders such as cancer. For example, EpCAM is highly expressed in various cancer types such as, for example, breast cancer, lung cancer, liver cancer, gastric cancer, head and neck cancer, prostate cancer, pancreatic cancer, ovarian cancer, and colon cancer, as well as most cancers (and metastases) of epithelial origin. EpCAM is also highly expressed in tumor-initiating cells / cancer stem cells. The provided EpCAM immunoconjugates have uses including the treatment of such diseases and cancers.

[0029] 6.1 Definitions To facilitate understanding, a number of terms and phrases are defined below.

[0030] As used herein, the term "epithelial cell adhesion molecule" or "EpCAM" refers to any native human EpCAM, unless otherwise indicated. This term also encompasses naturally occurring variants of EpCAM, such as splice variants, allelic variants, and isoforms. EpCAM polypeptides can be isolated from various sources, such as human or cynomolgus monkey tissues or other biological samples, or can be 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, MIC18, HNPCC8, and ESA. Examples of EpCAM sequences include, but are not limited to, NCBI reference number NP_002345.2 (amino acid residues 24 to 314 correspond to mature EpCAM, and amino acids 24 to 265 correspond to the extracellular region of mature EpCAM (SEQ ID NO: 1)). The extracellular region of mature EpCAM can be further divided into three domains (D1 (amino acids 1 to 36 of SEQ ID NO: 1 (SEQ ID NO: 2)), D2 (amino acids 43 to 112 of SEQ ID NO: 1 (SEQ ID NO: 3)), and D3 (amino acids 113 to 243 of SEQ ID NO: 1 (SEQ ID NO: 4))).

[0031] As used herein, terms such as "antibody" and "antigen-binding antibody fragment" include any protein or peptide-containing molecule that includes 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 an antibody optionally further affects at least one EpCAM activity, for example, but not limited to, such an antibody regulates, reduces, increases, antagonizes, stimulates, partially stimulates, partially antagonizes, alleviates, mitigates, blocks, inhibits, suppresses, 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 an 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.

[0032] An antibody is a heterotetrameric glycoprotein composed of two identical light chains (LCs) and two identical heavy chains (HCs). Typically, each light chain is linked to the heavy chain by one covalent disulfide bond, although the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has intrachain disulfide bridges that are spaced apart. Each heavy chain has a variable region (VH) at one end, followed by several constant domains. Each light chain has a variable region (VL) at one end and a constant domain at the other end. The constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable region of the light chain aligns with the variable region of the heavy chain. Antibody light chains from all vertebrate species can be assigned to one of two distinct types, namely kappa and lambda, based on the amino acid sequence of their constant domains. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG, and IgM, depending on the amino acid sequence of the heavy chain constant domains. IgA and IgG are further subclassified as isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4.

[0033] The term "antibody" includes its fragments, specific portions, and variants, and includes antibody mimetic substances, or portions of an antibody that mimic the structure and / or function of an antibody or its designated fragment or portion, including single-chain antibodies and antigen (e.g., EpCAM)-binding antibody fragments. Functional fragments include antigen-binding fragments that bind to mammalian antigens such as EpCAM alone or in combination with other antigens. For example, 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 plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction, and reassembly), Fv or scFv (e.g., by molecular biological techniques) fragments, among others, antibody fragments capable of binding to an antigen or a portion thereof are encompassed by the present disclosure (see, e.g., Colligan, Immunology).

[0034] Such fragments can be produced by enzymatic cleavage, synthetic, or recombinant techniques known in the art and / or disclosed herein. Antibodies can also be generated in various truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural termination site. For example, combinations of genes encoding the F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and / or hinge region of the heavy chain. Various portions of an antibody can also be chemically linked to each other by conventional techniques, or prepared as a continuous protein using genetic modification techniques.

[0035] The term "antibody fragment" refers to a portion of an intact antibody, and broadly, to the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include, among others, Fab, Fab’, F(ab’)2, single-chain (scFv) and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single Fab arm "one-arm" antibodies, and multispecific antibodies formed from antibody fragments (each having VH and VL), but are not limited thereto.

[0036] An antibody fragment includes any protein or peptide-containing molecule that includes at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one heavy or light chain complementarity-determining region (CDR) or ligand-binding portion thereof, heavy or light chain variable region, heavy or light chain constant region, framework region or any portion thereof, or at least a portion of an antigen or antigen receptor or binding protein, that can be incorporated into an EpCAM antibody provided herein.

[0037] The term "full-length antibody" refers to an antibody in a substantially intact form, as opposed to the antibody fragments defined above. This term particularly refers to antibodies having heavy chains that contain an Fc region, including the hinge region and a modified Fc region (e.g., a mutated and / or clipped hinge region).

[0038] The term "variable" refers to the fact that the sequences of certain portions of the variable regions of antibodies vary widely between antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the entire variable region of the antibody. The variability is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both the light chain variable region and the heavy chain variable region. The more highly conserved portions of the variable region are called the framework (FR). Each of the variable regions of the native heavy and light chains contains four FR regions, which mainly assume a β-sheet conformation connected by three CDRs, and this conformation forms loops that connect the β-sheet structures and, in some cases, form part of the β-sheet structure. The CDRs of each chain are held together in proximity by the FR regions and, together with the CDRs from other chains, contribute to the formation of the antigen-binding site of the antibody. The constant domains do not directly participate in binding the antibody to the antigen but exhibit various effector functions such as the involvement of the antibody in antibody-dependent cell cytotoxicity. There are at least the following two techniques for determining CDRs. (1) A method based on interspecies sequence variation (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991, National Institutes of Health, Bethesda Md.)), and (2) A method based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., J. Molec. Biol. 273:927-948 (1997)). Furthermore, in the art, the CDRs may sometimes be determined using a combination of these two methods.

[0039] When referring to residues in the variable region (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0040] The numbering of amino acid positions as in Kabat refers to the numbering system used for the heavy chain variable region or the light chain variable region of the antibody compilation 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 may contain fewer or additional amino acids corresponding to shortening or insertion into the FR or CDR of the variable region. For example, the heavy chain variable region can contain a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2, and insertion residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues for a given antibody can be determined by alignment with the "standard" Kabat-numbered sequence in the homologous region of the antibody's sequence. Chothia, instead, refers to the positions of structural loops (Chothia et al., J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop when numbered using the Kabat numbering rules (this is because the Kabat numbering scheme places insertions at H35A and H35B, and if neither 35A nor 35B is present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM high frequency variable region is a compromise between the Kabat CDR and the Chothia structural loop and is used by Oxford Molecular's AbM antibody modeling software.

[0041] The terms "EpCAM antibody", "EpCAM antibody", "antibody that specifically binds to EpCAM", "EpCAM-binding antibody fragment thereof", and "antibody fragment that specifically binds to EpCAM" refer to antibodies that can bind to EpCAM with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting EpCAM. The degree of binding of an EpCAM antibody to unrelated non-EpCAM proteins is, for example, measured by radioimmunoassay (RIA) and is less than about 10% of the binding of the antibody to EpCAM.

[0042] The numbering of amino acid positions, as in Kabat, refers to the numbering system used for the heavy chain variable domain or the light chain variable domain of the antibody compilation 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 shortening or insertion into the FR or CDR of the variable domain. For example, the heavy chain variable domain can contain a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2, and insertion residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues for a given antibody can be determined by alignment with the "standard" Kabat-numbered sequence in the homologous region of the antibody sequence. Chothia, instead, refers to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), where the loops are also identified as light chain and heavy chain CDRs (e.g., L1 = LC CDR1, etc.). The ends of the Chothia CDR-H1 loop differ between H32 and H34 depending on the length of the loop when numbered using the Kabat numbering rules (this is because the Kabat numbering scheme places insertions at H35A and H35B, and if neither 35A nor 35B is present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions are the product of a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. [Table 1]

[0043] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. An epitope usually consists of a chemically active surface population of molecules such as amino acids or sugar side chains and typically has specific three-dimensional structural characteristics as well as specific charge characteristics. When the antigen is a polypeptide, the epitope can be formed from both contiguous and non-contiguous amino acids juxtaposed by the protein's tertiary folding. Epitopes formed from contiguous amino acids are usually retained upon protein denaturation, whereas epitopes formed by tertiary folding are usually lost upon protein denaturation. An epitope usually contains at least 3 amino acids, more generally at least 5 or 8 - 10 amino acids, in a unique spatial higher-order structure.

[0044] A "blocking" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds, such as EpCAM. Certain 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 EpCAM-related tyrosine kinase activity by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

[0045] An "isolated" antibody is one that has been separated and / or recovered from its natural environment. The contaminating components of its natural environment are materials that would interfere with the diagnostic or therapeutic use of the antibody, and these can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is purified (1) to greater than 95% by weight, and in some cases greater than 99% by weight, as measured, for example, by the Lowry method, (2) to at least 15 residues of the N-terminus or to a degree sufficient to obtain the internal amino acid sequence using a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) using Coomassie blue or, in some cases, silver staining, under reducing or non-reducing conditions. An isolated antibody lacks at least one component of its natural environment and thus includes an EpCAM antibody that is present in situ within a recombinant cell. However, usually, an isolated antibody will have been prepared by at least one purification step.

[0046] 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 intact or full-length antibodies, activatable antibodies, antigen (e.g., human and / or cynomolgus EpCAM) binding antibody fragments, and / or antibodies that include at least one human heavy chain and / or light chain polypeptide, such as an antibody that includes a mouse light chain and a human heavy chain polypeptide.

[0047] As used herein, the term "chimeric antibody" means an antibody in which the sequences of the immunoglobulin molecule are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of an antibody derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and function, while the constant regions are homologous to the sequences of an antibody derived from another species, usually human, to avoid eliciting an immune response in that species.

[0048] As used herein, the term "humanized antibody" refers to a form of a non-human (e.g., murine) antibody that is a specific immunoglobulin chain, chimeric immunoglobulin, or antigen-binding antibody fragment that contains a minimal amount of non-human (e.g., murine) sequence. Typically, a humanized antibody is a human immunoglobulin in which the residues derived from the complementarity determining regions (CDRs) have been replaced with residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity (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 cases, the Fv framework region (FR) residues of the human immunoglobulin have been replaced with the corresponding residues in an antibody from a non-human species having the desired specificity, affinity, and capacity. A humanized antibody can be further modified by substitution of additional residues either within the Fv framework region and / or within the substituted non-human residues to refine and optimize the specificity, affinity, and / or capacity of the antibody. Generally, an antibody will comprise substantially all of at least one, typically two or three variable regions that contain all or substantially all of the CDR regions corresponding to the non-human immunoglobulin, and all or substantially all of the FR regions will be those of a human immunoglobulin consensus sequence. The antibody may also comprise an immunoglobulin constant region or domain (Fc), typically at least a portion of that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent No. 5,225,539.

[0049] The "effector function" of an antibody refers to biological activities attributable to the Fc region of the antibody (either the native sequence Fc region or amino acid sequence variant Fc region), and which vary depending on the isotype of the antibody. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP).

[0050] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, the cells express at least FcγRIII and perform ADCC or ADCP effector function(s). Examples of human leukocytes that mediate ADCC or ADCP include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as blood.

[0051] As used herein, the term "Fc region" includes a polypeptide 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, as well as the last three constant region immunoglobulin domains of IgE and IgM, and the N-terminal flexible hinge to these domains. In the case of IgA and IgM, Fc may include a J chain. In the case of IgG, Fc includes the immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3), as well as the hinge between Cγ1 (Cγ1) and Cγ2 (Cγ2). The boundaries of the Fc region may vary, but the human IgG heavy chain Fc region is typically defined to include residue C226 or P230 at its carboxyl terminus, where the numbering follows the EU index shown by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va). The "EU index described in Kabat" refers to the residue numbering of the human IgG1 EU antibody described by Kabat et al. supra. Fc may refer to this region alone or 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 includes an Fc region. Certain proteins include a variant Fc region that is a non-natural variant of Fc. Polymorphisms have been observed at many Fc positions including, but not limited to, Kabat positions 270, 272, 312, 315, 356, and 358, and thus there may be minor differences between the presented sequences and those of the prior art, which will be understood by those skilled in the art based on the teachings herein.

[0052] As used herein, the terms "conjugate", "immunoconjugate", "ADC", or "AADC" refer to a compound or its derivative conjugated to a cell binding agent (i.e., an EpCAM antibody, its EpCAM-binding antibody fragment, or an EpCAM-activatable antibody), which is generally defined by the general formula C-L-A, wherein C = compound, L = linker, and A = an EpCAM binding agent (EpBA) (e.g., an EpCAM antibody, its EpCAM-binding antibody fragment, or an EpCAM-activatable antibody disclosed herein). In some embodiments, the general formula: D-L-A (wherein D = drug, L = linker, and A = cell binding agent) (e.g., an EpCAM antibody, its EpCAM-binding antibody fragment, or an EpCAM-activatable antibody) can be used as well. In some cases, the immunoconjugates described herein include a camptothecin derivative linker payload.

[0053] A "linker" is any chemical moiety capable of binding a compound, usually a drug such as a maytansinoid, a camptothecin derivative, or an indolinobenzodiazepine compound, to a cell binding agent, such as an anti-EpCAM antibody or its EpCAM-binding antibody fragment. The linker may be susceptible or substantially resistant to acid-induced cleavage, photo-induced cleavage, protease-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, protease-labile groups, and esterase-labile groups. Linkers include peptide linkers and charged linkers disclosed herein and known in the art, as well as their hydrophilic forms.

[0054] As used herein, "abnormal cell proliferation" refers to cell growth independent of normal regulatory mechanisms (e.g., loss of contact inhibition), unless otherwise indicated. Examples thereof include, for example, (1) tumor cells (tumors) that proliferate by expression of mutant tyrosine kinases or overexpression of receptor tyrosine kinases, (2) benign and malignant cells of other proliferative diseases in which abnormal tyrosine kinase activation occurs, (3) any tumor that proliferates by receptor tyrosine kinases, (4) any tumor that proliferates by abnormal serine / threonine kinase activation, (5) benign and malignant cells of other proliferative diseases in which abnormal serine / threonine kinase activation occurs, and (6) abnormal growth of benign and malignant cells of other proliferative diseases.

[0055] The terms "cancer" and "cancerous" generally refer to or describe a physiological state in mammals characterized by uncontrolled cell growth. A "tumor" contains one or more cancerous cells. The term "cancer" or "cancerous" as defined herein includes "precancerous" states that may become cancerous if left untreated. 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, cancers containing cancer stem cells, uterine cancer, gastric cancer, head and neck cancer, endometrial cancer, and pancreatic cancer.

[0056] The terms "cancer cell", "tumor cell", and grammatical equivalents refer to the entire cell population derived from a tumor or precancerous lesion, including non-tumorigenic cells that make up the majority of the tumor cell population and tumorigenic stem cells (cancer stem cells).

[0057] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents one or more cell functions and / or causes cell death.

[0058] As used herein, "treatment" refers to a clinical intervention to alter the natural course of an individual or cell being treated and can be implemented for prophylactic purposes or during the course of a clinical pathology. Desirable therapeutic effects include prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of any direct or indirect pathological consequence of the disease, prevention of metastasis, reduction in the rate of disease progression, remission or alleviation of the medical condition, and improvement of remission or prognosis. In some embodiments, the methods and compositions provided herein are useful for delaying the onset of a disease or disorder.

[0059] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both (1) therapeutic measures that cure, delay, weaken, and / or halt the progression of the symptoms of a diagnosed medical condition or disorder, and (2) prophylactic or preventative measures that prevent and / or delay the onset of the targeted medical condition or disorder. Accordingly, those in need of treatment include those already having a disorder, those prone to having a disorder, and those in whom a disorder should be prevented. In certain embodiments, a subject will have been successfully "treated" for cancer by the methods provided herein if the subject demonstrates one or more of a decrease in the number of cancer cells or complete disappearance thereof, a decrease in tumor size, inhibition or disappearance of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone of cancer, inhibition or disappearance of tumor metastasis, inhibition or disappearance of tumor growth, alleviation of one or more symptoms associated with a particular cancer, reduction of morbidity and mortality, improvement in quality of life, reduction of the tumorigenic, tumorigenic frequency, or tumorigenic capacity of the tumor, reduction in the number or frequency of cancer stem cells in the tumor, differentiation of tumorigenic cells into a non-tumorigenic state, or some combination of effects.

[0060] The "effective amount" of an antibody disclosed herein is an amount sufficient to effect the specific purpose shown. The "therapeutically effective amount" refers to an amount that is required to be administered for a required period of time and is effective to achieve the desired therapeutic result. The "therapeutically effective amount" of a therapeutic agent (e.g., conjugate or immunoconjugate) can vary depending on factors such as the individual's medical condition, age, gender, and weight, as well as the ability of the antibody to elicit the desired response in the individual. The therapeutically effective amount is also an amount such that any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.

[0061] "Therapeutic agent" includes biological agents such as antibodies, peptides, proteins, enzymes, chemotherapeutic agents, or both conjugates or immunoconjugates.

[0062] The terms "subject", "individual", "animal", "patient", and "mammal" refer to any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include, but are not limited to, humans, non-human primates, laboratory animals, domestic animals, rodents, etc., which are recipients of a particular treatment.

[0063] As used interchangeably herein, the terms "polynucleotide" or "nucleic acid" refer to polymers of nucleotides of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. Polynucleotides may include modified nucleotides such as methylated nucleotides and their analogs. Where present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component. Other types of modifications include, for example, "caps" which are substitutions of one or more natural nucleotides by analogs, internucleotide modifications such as those by uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), including those containing pendant moieties of proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those by intercalators (e.g., acridines, psoralens, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those by modified linkages (e.g., α-anomeric nucleic acids, etc.), as well as polynucleotides in unmodified form(s). Further, any of the hydroxyl groups that are normally present in the sugar can be substituted, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OHs can also be phosphorylated or substituted with an amine or an organic capping group moiety of 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized with standard protecting groups.Polynucleotides can generally also contain analogous forms of ribose or deoxyribose sugars known in the art, including, for example, 2′-O-methyl-2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester bonds may be replaced with alternative linking groups. These alternative linking groups include where the phosphate is replaced with P(O)S(“thioate”), P(S)S(“dithioate”), ”(O)NR2(“amidate”), P(O)R, P(O)OR’, CO, or CH2(“formacetal”), where each R or R’ is independently H or substituted or unsubstituted alkyl (1-20C) (optionally containing an ether (--O--) bond), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl, although embodiments are not limited thereto. All bonds of the polynucleotide need not be the same. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0064] The term “vector” means a construct capable of delivering and optionally expressing one or more genes or sequences of interest in a host cell. Examples of vectors include viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors complexed with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells, although embodiments are not limited thereto.

[0065] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also include amino acid polymers that are naturally or artificially modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids), and other modifications known in the art are included in this definition.

[0066] As is known in the art, the term "identical" and percent "identity" is a measure of correlation between two polynucleotides or two polypeptides, where the percent identity is determined by comparing their sequences. Identity or similarity to a sequence, as used herein, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, is defined as the percentage of amino acid residues in a candidate sequence that are identical (i.e., the same residue) or similar (i.e., amino acid residues derived from the same group based on common side chain characteristics, see below) to the EpCAM antibody residues. Neither N-terminal, C-terminal, or internal extensions, deletions, or insertions into antibody sequences outside the variable regions are to be construed as affecting sequence identity or similarity. Generally, the two sequences to be compared are aligned to give the maximum correlation between the sequences. The alignment of the two sequences is examined and the number of positions giving the exact amino acid or nucleotide correspondence between the two sequences determined is divided by the full length of the alignment and multiplied by 100 to obtain a number for the percent identity. This numerical value for percent identity can also be determined over the full length of the sequences being compared, which is particularly suitable for highly homologous sequences of the same or very similar length, or it can be determined over a shorter fixed length, which is more suitable for sequences of unequal length or lower levels of homology. Similarly, the percent similarity can be determined in a similar manner based on the presence of both identical and similar residues.

[0067] Percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. A variety of algorithms and software are known in the art and can be used to obtain an alignment 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), modified in Karlin et al., 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. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et al., Meth. Enzym. 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California), or Megalign (DNASTAR®) are further publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences can be determined using the GAP program of GCG software (e.g., using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 90 and length weights of 1, 2, 3, 4, 5, or 6).In certain alternative embodiments, the percent identity between two amino acid sequences can be determined using the GAP program of the GCG software package incorporating the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) (e.g., using the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences can be 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), using PAM120 with a residue table, a gap length penalty of 12, and a gap penalty of 4. Appropriate parameters for maximum alignment by a particular alignment software can be determined by those skilled in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percentage identity "X" of a first amino acid sequence to a second sequence amino acid is calculated as 100x(Y / Z), where Y is the number of amino acid residues scored as exact matches in the alignment of the first and second sequences (aligned by visual inspection or by a particular sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence.

[0068] As a non-limiting example, whether any particular polynucleotide has a particular percentage of sequence identity (e.g., 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 Applied 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 example, 95% identical to a reference sequence provided herein, the percentage of identity is calculated over the entire length of the reference nucleotide sequence, and the parameters are set such that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0069] "Conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include, for example, amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substituting tyrosine with phenylalanine is a conservative substitution. In some embodiments, conservative substitutions of the polypeptide and antibody sequences provided herein do not inhibit the binding of the polypeptide or antibody containing the amino acid sequence to the antigen(s) to which the polypeptide or antibody binds. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0070] As used herein, "alkyl" refers to a saturated straight-chain or branched-chain monovalent hydrocarbon radical having 1 to 20 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, -CH2CH(CH3)2), 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl), 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, etc. In some embodiments, alkyl has 1 to 10 carbon atoms. In some embodiments, alkyl has 1 to 4 carbon atoms.

[0071] The number of carbon atoms in a group can be specified herein by the prefix "Cx-xx", where x and xx are integers. For example, "C1-4 alkyl" is an alkyl group having 1 to 4 carbon atoms.

[0072] The terms "compound", "cytotoxic compound", or "cytotoxic agent" are used interchangeably. They are intended to include compounds whose structure or formula or any derivatives thereof are disclosed herein, or whose structure or formula or any derivatives thereof are incorporated by reference. The term also includes stereoisomers, geometric isomers, tautomers, solvates, metabolites, and salts (e.g., pharmaceutically acceptable salts) of all compounds of the formulas disclosed herein. The term includes solvates, hydrates, and polymorphs of any of the foregoing. Specific listings of "stereoisomers", "geometric isomers", "tautomers", "solvates", "metabolites", "salts", "conjugates", "conjugate salts", "solvates", "hydrates", or "polymorphs" in particular embodiments provided herein are not to be construed as intended omissions of these forms in other disclosed embodiments when the term "compound" is used without enumerating these other forms. In some cases, the compound includes a camptothecin derivative linker payload.

[0073] The term "imine-reactive reagent" refers to a reagent that can react with an imine group. Examples of imine-reactive reagents include, but are not limited to, sulfites (H2SO3, H2SO2, or salts of HSO3 - , SO3 2 , or HSO2 - ), metabisulfites (H2S2O5 or salts of S2O5 2- ), mono-, di-, tri-, and tetra-thiophosphates (PO3SH3, PO2S2H3, POS3H3, PS4H3, or salts of PO3S3 - , PO2S2 3- , POS3 3- , or PS4 3-salts), thiophosphates ((RiO)2PS(ORi), RiSH, RiSOH, RiSO2H, RiSO3H), various amines (hydroxylamines (e.g., NH2OH), hydrazines (e.g., NH2NH2), NH2O-Ri, Ri’NH-Ri, NH2-Ri), NH2-CO-NH2, NH2-C(=S)-NH2, thiosulfates (H2S2O3 or salts formed with cations of S2O3 2- salts), dithionites (H2S2O4 or salts formed with cations of S2O4 2- salts), phosphorodithioates (P(=S)(ORk)(SH)(OH) or salts thereof formed with cations), hydroxamic acids (RkC(=O)NHOH or salts formed with cations), hydrazides (RkCONHNH2), formaldehyde sulfoxylate (HOCH2SO2H or HOCH2SO2 formed with cations - salts, e.g., HOCH2SO2-Na + ), glycated nucleotides (such as GDP-mannose), fludarabine or mixtures thereof, wherein Ri and Ri’ are each independently a straight or branched alkyl having 1 to 10 carbon atoms, substituted with at least one substituent selected from N(Rj)2, -CO2H, SO3H, and PO3H, and Ri and Ri’ may be further optionally substituted with substituents of the alkyls disclosed herein, Rj is a straight or branched alkyl having 1 to 6 carbon atoms, and Rk is a straight, branched, or cyclic alkyl, alkenyl or alkynyl, aryl, heterocyclyl or heteroaryl having 1 to 10 carbon atoms (in some embodiments, Rk is a straight or branched alkyl having 1 to 4 carbon atoms, and in some embodiments, Rk is methyl, ethyl, or propyl). In some embodiments, the cation is Na + or K + and other monovalent cations. In some embodiments, the imine-reactive reagent is selected from sulfites, hydroxylamines, urea, and hydrazines. In some embodiments, the imine-reactive reagent is NaHSO3 or KHSO3.

[0074] The term "cation" refers to an ion having a positive charge. Cations can be monovalent (e.g., Na + , K + , NH4 + , etc.), divalent (e.g., Ca2 + , Mg2 + , etc.), or polyvalent (e.g., Al 3+ , etc.). In some embodiments, the cation is monovalent.

[0075] As used herein, the phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt 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, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharinate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salt, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (such as magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts may involve the inclusion of another molecule such as an acetate ion, succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Further, a pharmaceutically acceptable salt can have more than one charged atom within its structure. Instances where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.

[0076] When the compounds provided herein are bases, the desired pharmaceutically acceptable salts can be prepared by treating the free base with any suitable method available in the art, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, or organic acids such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, glucuronic acid or galacturonic acid, alphahydroxy acids such as citric acid or tartaric acid, amino acids such as aspartic acid or glutamic acid, aromatic acids such as benzoic acid or cinnamic acid, sulfonic acids such as p-toluenesulfonic acid or ethanesulfonic acid.

[0077] When the compounds provided herein are acids, the desired pharmaceutically acceptable salts can be prepared by any suitable method, for example, by treating the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or an alkaline earth metal hydroxide. Specific examples of suitable salts include 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, but are not limited thereto.

[0078] As used herein, the term "solvate" means a compound further comprising a stoichiometric or non-stoichiometric amount of a solvent, such as water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine dichloromethane, 2-propanol, etc., bound by non-covalent intermolecular forces. Solvates or hydrates of the compounds are readily prepared by adding at least one molar equivalent of a hydroxyl-containing solvent such as methanol, ethanol, 1-propanol, 2-propanol, or water to the compound to solvate or hydrate the imine moiety.

[0079] "Metabolite" or "catabolite" refers to the product of a specific compound, its derivatives, or its conjugates, or salts thereof, produced by metabolism or catabolism in the body. Metabolites of a compound, its derivatives, or its conjugates can be identified using conventional techniques known in the art, and their activity can be 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, etc. of the administered compound. Accordingly, the present disclosure includes metabolites, derivatives, or conjugates of the compounds of the EpCAM compositions of the present disclosure disclosed herein, which are produced by a process comprising contacting a mammalian subject with an EpCAM compound, its derivative, or its conjugate of the present disclosure for a period of time sufficient to obtain its metabolites, and which includes a compound, its derivative, or its conjugate.

[0080] The term "pharmaceutically acceptable" indicates that a substance or composition must be chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or the mammalian subject being treated therewith.

[0081] The term "protecting group" or "protecting moiety" means a substituent commonly used to block or protect a particular function while allowing other functional groups of a compound, its derivatives, or its conjugates to react. For example, an "amine protecting group" or "amino protecting moiety" is a substituent that binds to an amino group and blocks or protects the amino functional group of a compound. Such groups are well known in the art (see, e.g., P. Wuts and T. Greene, 2007, Protective Groups in Organic Synthesis, Chapter 7, J. Wiley & Sons, NJ), which are exemplified by carbamates such as methyl carbamate and ethyl carbamate, FMOC, substituted ethyl carbamate, carbamate salts cleaved by 1,6-β elimination (also called "self-destructive type"), ureas, amides, peptides, alkyl and aryl derivatives. Suitable amino protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyleneoxycarbonyl (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.

[0082] The term "amino acid" refers to either a naturally occurring amino acid or a non-naturally occurring amino acid. In one embodiment, the amino acid is represented by NH2-C(Raa’Raa)-C(=O)OH, where Raa and Raa’ are each independently H, a straight-chain, branched-chain, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms optionally substituted, aryl, heteroaryl or heterocyclyl, or Raa and the N-terminal nitrogen atom may together form a heterocyclic ring (such as in proline, etc.). The term "amino acid residue" refers to the residue corresponding to the case where one hydrogen atom is removed from the amine terminus and / or carboxy terminus of an amino acid, e.g., -NH-C(Raa’Raa)-C(=O)O-.

[0083] The term "peptide" refers to a short chain of amino acid monomers linked by peptide (amide) bonds. In some embodiments, the peptide contains 2 to 20 amino acid residues. In other embodiments, the peptide contains 2 to 10 amino acid residues. In still other embodiments, the peptide contains 2 to 5 amino acid residues. As used herein, when a peptide is part of a cytotoxic agent or linker disclosed herein represented by a specific sequence of amino acids, the peptide can bind to the remaining part of the cytotoxic agent or linker in both directions. For example, the dipeptide X1-X2 includes X1-X2 and X2-X1. Similarly, the tripeptide X1-X2-X3 includes X1-X2-X3 and X3-X2-X1, and the tetrapeptide X1-X2-X3-X4 includes X1-X2-X3-X4 and X4-X2-X3-X1. X1, X2, X3, and X4 represent amino acid residues.

[0084] The term "reactive ester group" refers to an ester group that can readily react with an amine group to form an amide bond. Exemplary reactive ester groups include, but are not limited to, N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, N-hydroxysulfo-succinimide ester, para-nitrophenyl ester, dinitrophenyl ester, pentafluorophenyl ester, and derivatives thereof, and these derivatives promote the formation of amide bonds. In certain embodiments, the reactive ester group is an N-hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester.

[0085] 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 halides, imide esters, or reactive thioester groups. In certain embodiments, the amine-reactive group is a reactive ester group. In one embodiment, the amine-reactive group is an N-hydroxysuccinimide ester or an N-hydroxysulfo-succinimide ester.

[0086] 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 thiol-reactive group is maleimide.

[0087] As used in this disclosure and the claims, the singular forms "a", "an", and "the" include the plural unless the context clearly dictates otherwise.

[0088] As used herein, the term "and / or" shall be construed as each specific disclosure of two or more specified features or components (regardless of the presence or absence of other features or components). Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0089] When embodiments are disclosed herein with the word "comprising", it shall always be assumed that other similar embodiments defined with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0090] 6.2 Immunoconjugates comprising EPCAM antibodies and EPCAM-binding antibody fragments thereof Immunoconjugates comprising EpCAM antibodies and EpCAM-binding antibody fragments thereof are disclosed herein. These proteins specifically bind to human EpCAM and are also provided herein. In some embodiments, the proteins are referred to herein as "EpCAM binding agents" or EpBAs.

[0091] In additional embodiments, the immunoconjugates disclosed herein include an EpCAM binder that is an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM activatable antibody. In some embodiments, the EpBA is a full-length EpCAM antibody (i.e., a full-length antibody that specifically binds to 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 multispecific antibody (e.g., a bispecific antibody), or an EpCAM-binding antibody fragment thereof. In some embodiments, the EpCAM antibody specifically binds to human EpCAM. In further embodiments, the EpCAM antibody specifically binds to human EpCAM and cynomolgus EpCAM. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof is a mouse, other rodent, chimeric, humanized, or fully human monoclonal antibody.

[0092] In some embodiments, the immunoconjugates disclosed herein include an EpCAM antibody that is an EpCAM-binding antibody fragment. In some embodiments, the immunoconjugates disclosed herein include a non-human mammalian antibody, a mouse antibody, a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the immunoconjugates disclosed herein include a full-length antibody. In some embodiments, the immunoconjugates disclosed herein include a human IgG1 antibody.

[0093] In some embodiments, the immunoconjugates disclosed herein include a Fab, Fab’, F(ab’)2, scFv, or disulfide-bonded Fv (dsFv). In additional aspects, the EpCAM-binding antibody fragment is a single-chain Fv (scFv), a disulfide-bonded Fv, IgGδCH2, a minibody, F(ab’)3, scAb, a tetrabody, a tribody, a diabody, a DVD-Ig, Fcab, mAb 2 , (scFv)2, scFv-Fc, or bis-scFv.

[0094] In some cases, the immunoconjugates disclosed herein optionally bind to EpCAM (e.g., human EpCAM and / or mouse EpCAM) with a broad affinity (K D ) and include the EpCAM antibodies provided herein and their EpCAM-binding antibody fragments. In certain embodiments, the antibody binds to human EpCAM with high affinity. For example, a human or human-modified or humanized or resurfaced mAb, measured using standard procedures by flow cytometry-based assay, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), or KinExA® method, has a K -7 of 10 -7 M or less, e.g., 0.1 - 9.9 (or any range or value therebetween) × 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M, or any range or value therein, and can bind to the human antigen. In some embodiments, the EpCAM antibody binds with a Kd of 10 D M or less, more specifically about 10 -9 M to 10 -9 M to 10 -10 M.

[0095] The affinity or binding activity of the antibody or its antibody fragment in the immunoconjugate described in this specification for EpCAM can be experimentally measured using any suitable method known in the art, such as flow cytometry, enzyme-linked immunosorbent 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, for example, Berzofsky, et al., “Antibody-Antigen 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 the methods disclosed herein. The measured affinity of a particular antibody-EpCAM interaction may vary when measured under different conditions (e.g., salt concentration, pH, temperature). Thus, the measurement of affinity and other EpCAM binding parameters (e.g., KD or Kd, Kon, Koff) is performed using standardized solutions of the antibody and EpCAM, and standardized buffers such as those known in the art and disclosed herein.

[0096] In one embodiment, the binding assay is performed using flow cytometry on cells expressing the EpCAM antigen on their surface. For example, such EpCAM-positive cells are resuspended at 1 × 10 per sample in 100 μL of FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum) 5Incubate with various concentrations of EpCAM antibody using individual cells. Subsequently, pellet the cells, wash them, and incubate for 1 hour with 100 μL of FITC-conjugated goat anti-mouse IgG antibody (such as those available from Jackson ImmunoResearch) in FACS buffer. Pellet the cells again, wash them with FACS buffer, and resuspend them in 200 μL of PBS containing 1% formaldehyde. For example, acquire samples using a FACSCalibur (trademark) flow cytometer equipped with an HTS multiwell sampler and analyze using CellQuest (registered trademark) Pro (all manufactured by BD Biosciences, San Diego, US). Export the mean fluorescence intensity (MFI) of FL1 for each sample and plot it on a semi-logarithmic plot against the antibody concentration to generate a binding curve. Fit a sigmoidal dose-response curve to the binding curve and use a program such as GraphPad Prism v4 (GraphPad software, San Diego, CA) with default parameters to calculate the EC 50 value. For each antibody, the EC 50 value can be used as a measure of the apparent dissociation constant "K d " or "K D ".

[0097] In certain embodiments, the EpCAM antibody or fragment thereof in any of the immunoconjugates described herein is modified to alter its binding affinity for EpCAM and / or its EpCAM antigen fragment. Binding characteristics can be determined using standard procedures known in the art by various in vitro assay methods, including, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or kinetics (e.g., BIACORE (trademark) analysis).

[0098] In one embodiment, the EpCAM antibody or its EpCAM-binding antibody fragment in any of the immunoconjugates described herein is less than 10 -5 M, or less than 10 -6M or less than 10-7 less than M, or 10 -8 less than M, or 10 -9 less than M, or 10 -10 less than M, or 10 -11 less than M, or 10 -12 less than M, or 10 -13M dissociation constant or KD or Kd (koff / kon) less than, and specifically binds to human and / or cynomolgus monkey EpCAM and / or its EpCAM antigen fragments. In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment is 1.0×10 -9 M or less, 2.0×10 -9 M or less, 3.0×10 -9 M or less, 4.0×10 -9 M or less, 5.0×10 -9 M or less, 6.0×10 -9 M or less, 7.0×10 -9 M or less, 8.0×10 -9 M or less, or 9.0×10 -9 KD of M or less and specifically binds to human and / or cynomolgus monkey EpCAM and / or its EpCAM antigen fragments. In certain embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment binds to EpCAM of both human and cynomolgus monkey and / or its EpCAM antigen fragments with a KD of 3.0×10 -9 M or less. In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment binds to human EpCAM with a KD of about 0.4×10 -9 In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment binds to human EpCAM with a KD of about 0.8×10 -9 In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment binds to cynomolgus monkey EpCAM with a KD of about 0.8×10 -9 In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment binds to cynomolgus monkey EpCAM with a KD of about 2.2×10 -9binds with a KD of. In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment binds with a KD of about 2.8×10 -9 binds with a KD of.

[0099] In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment in any of the immunoconjugates described herein specifically binds to an epitope within the extracellular region of human EpCAM (SEQ ID NO: 1). The extracellular region of human EpCAM can be further divided into three distinct domains, namely, D1 (SEQ ID NO: 2), D2 (SEQ ID NO: 3), and D3 (SEQ ID NO: 4). In certain embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment specifically binds to an epitope within the first extracellular domain (D1) of human EpCAM.

[0100] In one embodiment, the EpCAM antibody or its EpCAM-binding antibody fragment in any of the immunoconjugates described herein comprises X1YX3X4H, wherein X1 is selected from N and S, X3 is selected from Y, N, F, S, H, D, L, I, and W, X4 is selected from I and M, the VH-CDR1, and the sequence WX2X3PGX6VYIQYX 12 X 13 KFX 17 comprises G, wherein X2 is selected from I and F, X3 is selected from Y and N, X6 is selected from N and D, X 12 is selected from N and S, X 13 is selected from E and Q, X 17 is selected from K and Q, the VH-CDR2 (SEQ ID NO: 7), and X1GX3X4FAY, wherein X1 is selected from D and E, X3 is selected from P, A, S, Y, F, G, T, and V, X4 is selected from Y and W, the VH-CDR3 (SEQ ID NO: 8), and comprises.

[0101] In a further embodiment, the disclosure provides RSSX4SLLHSX 10 GX 12 TYLX 16 comprises, wherein X4 is selected from R and K, X10 is selected from N and D, X 12 is selected from F and I, X 16 is selected from Y and S, a light chain CDR1 (VL-CDR1) (SEQ ID NO: 10), a light chain VL-CDR2 (SEQ ID NO: 40) containing QTSNLAS, and X1QX3LELPX8T, wherein X1 is selected from A, L, and Q, X3 is selected from S, G, Y, and N, and X8 is selected from N and W, a VL-CDR3 (SEQ ID NO: 11), and provides an EpCAM antibody or an EpCAM-binding antibody fragment thereof in any of the immunoconjugates described herein. In some embodiments, the present disclosure provides a heavy chain CDR1 (VH-CDR1) containing the sequence of SEQ ID NO: 13, a heavy chain CDR2 (VH-CDR2) containing the sequence of SEQ ID NO: 14, a heavy chain CDR3 (VH-CDR3) containing the sequence of SEQ ID NO: 15, a light chain CDR1 (VL-CDR1) containing the sequence of SEQ ID NO: 42, a light chain CDR2 (VL-CDR2) containing the sequence of SEQ ID NO: 40, and a light chain CDR3 (VL-CDR3) containing the sequence of SEQ ID NO: 41, and provides an EpCAM antibody or an EpCAM-binding antibody fragment thereof.

[0102] In some embodiments, VH-CDR1 contains 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, VH-CDR3 contains the sequence DGPX4FAY, wherein X4 is selected from Y and W (SEQ ID NO: 9). In some embodiments, VL-CDR3 contains the sequence AQX3LELPNT, wherein X3 is selected from S, G, Y, and N (SEQ ID NO: 12). In some embodiments, the present disclosure provides a heavy chain CDR1 (VH-CDR1) containing the sequence of SEQ ID NO: 13, a heavy chain CDR2 (VH-CDR2) containing the sequence of SEQ ID NO: 14, a heavy chain CDR3 (VH-CDR3) containing the sequence of SEQ ID NO: 15, a light chain CDR1 (VL-CDR1) containing the sequence of SEQ ID NO: 42, a light chain CDR2 (VL-CDR2) containing the sequence of SEQ ID NO: 40, and a light chain CDR3 (VL-CDR3) containing the sequence of SEQ ID NO: 41, and provides an EpCAM antibody or an EpCAM-binding antibody fragment thereof.

[0103] In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment in any of the immunoconjugates described herein comprises a series of complementarity-determining regions (CDRs): heavy-chain variable region (VH)-CDR1, VH-CDR2, VH-CDR3, light-chain variable region (VL) CDR1, VL-CDR2, and VL-CDR3, where the heavy-chain CDRs are disclosed in Table 2.

Table 2

[0104] In some embodiments, the present disclosure provides an EpCAM antibody or its EpCAM-binding antibody fragment in any of the immunoconjugates described herein that comprises the heavy-chain CDRs of a single row in Table 2. In some embodiments, the present disclosure provides an EpCAM antibody or its EpCAM-binding antibody fragment that comprises VH-CDR1 selected from SEQ ID NOs: 13 and 16-25, VH-CDR2 selected from SEQ ID NOs: 14 and 26-29, and VH-CDR3 selected from SEQ ID NOs: 15 and 30-38. In some embodiments, the present disclosure provides an EpCAM antibody or its EpCAM-binding antibody fragment that comprises VH-CDR1 of SEQ ID NO: 13, VH-CDR2 of SEQ ID NO: 14, and VH-CDR3 of SEQ ID NO: 15. In some embodiments, the present disclosure provides an EpCAM antibody or its EpCAM-binding antibody fragment that comprises VH-CDR1 of SEQ ID NO: 13, VH-CDR2 of SEQ ID NO: 26, and VH-CDR3 of SEQ ID NO: 15.

[0105] In one embodiment, the present disclosure provides an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an immunoconjugate having an EpCAM-activatable antibody, comprising VH-CDR1 of NYYIH (SEQ ID NO: 13), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, WIYPGNVYIQYNEKFKG (SEQ ID NO: 14), or a variant thereof comprising 1, 2, 3, or 4 amino conservative acid substitutions, and heavy chain CDR3 of DGPWFAY (SEQ ID NO: 15), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

[0106] In some embodiments, the present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising VH-CDR1 of SEQ ID NO: 22, VH-CDR2 of SEQ ID NO: 14; and VH-CDR3 of SEQ ID NO: 15. In some embodiments, the present disclosure provides an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising VH-CDR1 of SEQ ID NO: 13, VH-CDR2 of SEQ ID NO: 14, and VH-CDR3 of SEQ ID NO: 33. In some embodiments, the present disclosure provides an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising VH-CDR1 of SEQ ID NO: 23, VH-CDR2 of SEQ ID NO: 14, and VH-CDR3 of SEQ ID NO: 15. In some embodiments, the present disclosure provides an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising VH-CDR1 of SEQ ID NO: 25; VH-CDR2 of SEQ ID NO: 14; and VH-CDR3 of SEQ ID NO: 15.

[0107] In one embodiment, the present disclosure provides an anti-EpCAM antibody, an anti-EpCAM antibody fragment thereof, or an immunoconjugate having an anti-EpCAM activatable antibody, comprising VH-CDR1 comprising NYHIH (SEQ ID NO: 22), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, WIYPGNVYIQYNEKFKG (SEQ ID NO: 14), or a variant thereof comprising 1, 2, 3, or 4 amino acid conservative substitutions, and 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 present disclosure provides an anti-EpCAM antibody, an anti-EpCAM antibody fragment thereof, or an immunoconjugate having an anti-EpCAM activatable antibody, comprising VH-CDR1 comprising NYYIH (SEQ ID NO: 13), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions, WIYPGNVYIQYNEKFKG (SEQ ID NO: 14), or a variant thereof comprising 1, 2, 3, or 4 amino acid conservative substitutions, and heavy chain CDR3 comprising DGYWFAY (SEQ ID NO: 33), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

[0108] In some embodiments, the anti-EpCAM antibody or the anti-EpCAM antibody fragment thereof in any of the immunoconjugates described herein comprises a set of complementarity determining regions (CDRs): heavy chain variable region (VH)-CDR1, 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. [Table 3]

[0109] In some embodiments, the present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof that includes a single row of light chain CDRs in Table 3. In some embodiments, the present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof that includes 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 present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof that includes 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 present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof that includes 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.

[0110] In one embodiment, the present disclosure provides an immunoconjugate having an EpCAM antibody, an EpCAM-binding antibody fragment, or an EpCAM-activatable antibody, which comprises VL-CDR1 comprising RSSRSLLHSDGFTYLY (SEQ ID NO: 42), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; VL-CDR2 comprising QTSNLAS (SEQ ID NO: 40), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and 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 present disclosure provides an immunoconjugate having an EpCAM antibody, an EpCAM-binding antibody fragment, or an EpCAM-activatable antibody, which comprises VL-CDR1 comprising RSSKSLLHSDGFTYLY (SEQ ID NO: 39), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; VL-CDR2 comprising QTSNLAS (SEQ ID NO: 40), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and VL-CDR3 comprising AQNLELPNT (SEQ ID NO: 41), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

[0111] In some embodiments, the present disclosure provides an immunoconjugate having an EpCAM antibody or an 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 present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising 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 present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising 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 present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having the sequences of SEQ ID NOs: 13, 26, 15, and 39-41, respectively. In some embodiments, the present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising VH-CDR1, 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.

[0112] In some embodiments, the present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising VH-CDR1, 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 present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising 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 present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, having the sequences of SEQ ID NOs: 23, 14, 15, 42, 40, and 41, respectively. In some embodiments, the present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising 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.

[0113] In some embodiments, the immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a heavy chain variable region (VH) sequence disclosed in Table 4. In some embodiments, the EpCAM antibody or an 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 the VH sequence disclosed in Table 4. In some embodiments, the percent identity in each case is 95%. In some embodiments, the percent identity in each case is 98%. [Table 4] TIFF2025524628000017.tif236159TIFF2025524628000018.tif155159

[0114] In some embodiments, the immunoconjugates described herein comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof that comprises a VH sequence having a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, less than 15, or 0 amino acid 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 the framework regions of the reference sequence. In some embodiments, the substitutions are conservative. In other embodiments, the substitutions are non-conservative.

[0115] In some embodiments, the immunoconjugates described herein comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof that comprises a VH having a sequence selected from SEQ ID NOs: 53-84. In some embodiments, the EpCAM antibody or an 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 an EpCAM-binding antibody fragment thereof comprises a VH having the sequence of SEQ ID NO: 54. In some embodiments, the EpCAM antibody or an 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: 53-56. In some embodiments, the EpCAM antibody or an 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%.

[0116] In some embodiments, the immunoconjugates disclosed herein include an EpCAM antibody or an EpCAM-binding antibody fragment thereof that includes a VH having a sequence selected from SEQ ID NOs: 75-77, and 84. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof includes a VH having the sequence of SEQ ID NO: 75. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof includes a VH having the sequence of SEQ ID NO: 77. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof includes 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 an EpCAM-binding antibody fragment thereof includes 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 an EpCAM-binding antibody fragment thereof includes 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%.

[0117] In some embodiments, the immunoconjugates disclosed herein include an EpCAM antibody or an EpCAM-binding antibody fragment thereof that includes a light chain variable region (VL) sequence disclosed in Table 5. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof includes a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the 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] TIFF2025524628000020.tif94159

[0118] In some embodiments, the immunoconjugates disclosed herein include an anti-EpCAM antibody or an anti-EpCAM binding antibody fragment thereof that includes a VL sequence having a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, less than 15, or 0 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 the framework region of the reference sequence. In some embodiments, the substitutions are conservative. In other embodiments, the substitutions are non-conservative.

[0119] In some embodiments, the immunoconjugates disclosed herein comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof that comprises a VL having a sequence selected from SEQ ID NOs: 86-99. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a VL having the sequence of SEQ ID NO: 89. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a VL having the sequence of SEQ ID NO: 87. In some embodiments, the EpCAM antibody or an 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 an 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: 89. In some embodiments, the EpCAM antibody or an 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%.

[0120] In some embodiments, the immunoconjugates disclosed herein comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof that comprises 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 an 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 an 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 an 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 an 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 an 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 an 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.

[0121] In some embodiments, the immunoconjugates disclosed herein comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof that comprises a VH comprising the sequence of SEQ ID NO: 75 and a VL comprising the sequence of SEQ ID NO: 89. In some embodiments, the EpCAM antibody or an 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 an 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 an 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.

[0122] In some embodiments, the immunoconjugates disclosed herein include an EpCAM antibody or an EpCAM-binding antibody fragment thereof that competes with an antibody comprising the VH and VL sequences disclosed in Tables 4 and 5, respectively, for binding to human EpCAM.

[0123] In one embodiment, the present disclosure provides an immunoconjugate comprising an EpCAM antibody or an EpCAM-binding antibody fragment thereof that competes with an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL) selected from: (a) the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 89, (b) the VH of SEQ ID NO: 54 and the VL of SEQ ID NO: 87, (c) the VH of SEQ ID NO: 75 and the VL of SEQ ID NO: 89, and (d) the VH of SEQ ID NO: 77 and the VL of SEQ ID NO: 89, for binding to human EpCAM.

[0124] An EpCAM antibody or an EpCAM-binding antibody fragment thereof (or an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof) is said to "compete" for binding to EpCAM if it binds to human EpCAM to such an extent that it blocks to some degree the binding of a reference molecule to human EpCAM. The ability of a protein to compete for binding to EpCAM and thus to interfere with, block, or "cross-block" each other's binding to EpCAM can be determined, for example, by any standard competitive binding assay known in the art, including competitive ELISA assays, surface plasmon resonance (SPR; BIACORE®, Biosensor, Piscataway, N.J.), or according to the method described by Scatchard et al. (Ann. N.Y. Acad. Sci. 51:660 - 672 (1949)). An antibody can be said to competitively inhibit the binding of a reference EpCAM antibody to human EpCAM by, for example, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0125] In some embodiments, the immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof further comprises a heavy chain constant region or a 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 a fragment thereof, (b) a human IgD constant region, or a fragment thereof, (c) a human IgE constant domain, or a fragment thereof, (d) a human IgG1 constant region, or a fragment thereof, (e) a human IgG2 constant region, or a fragment thereof, (f) a human IgG3 constant region, or a fragment thereof, (g) a human IgG4 constant region, or a fragment thereof, and (h) a human IgM constant region, or a fragment thereof. In certain embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a heavy chain constant region or a fragment thereof, such as a human IgG constant region or a fragment thereof. In further embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a heavy chain immunoglobulin constant domain having altered effector function and / or half-life, or mutated to have such.

[0126] In some embodiments, the immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a heavy chain sequence disclosed in Table 6. [Table 6] TIFF2025524628000022.tif236159TIFF2025524628000023.tif235159TIFF2025524628000024.tif236159TIFF2025524628000025.tif233159TIFF2025524628000026.tif233159TIFF2025524628000027.tif227159TIFF2025524628000028.tif21159

[0127] In some embodiments, the immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a heavy chain (HC) sequence selected from SEQ ID NOs: 102-134. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof of the immunoconjugate described herein comprises a heavy chain (HC) sequence selected from SEQ ID NOs: 102-106. In certain embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof of the immunoconjugate described herein comprises the HC sequence of SEQ ID NO: 103. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises an 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, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises an 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: 102-106. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises an 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%.

[0128] In alternative embodiments, the EpCAM antibody of the immunoconjugate described herein or its EpCAM-binding antibody fragment comprises an HC sequence selected from SEQ ID NOs: 125-127 and 134. In certain embodiments, the EpCAM antibody of the immunoconjugate described herein or its EpCAM-binding antibody fragment comprises the HC sequence of SEQ ID NO: 125. In certain embodiments, the EpCAM antibody of the immunoconjugate described herein or its EpCAM-binding antibody fragment comprises the HC sequence of SEQ ID NO: 127. In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment comprises an 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 its EpCAM-binding antibody fragment comprises an 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: 125. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0129] In additional aspects, the EpCAM antibody of the immunoconjugate described herein or its EpCAM-binding antibody fragment comprises a light chain immunoglobulin constant region. In further aspects, the antibody comprises a human Ig kappa constant region or a human Ig lambda constant region.

[0130] In some embodiments, the EpCAM antibody of the immunoconjugate described herein or its EpCAM-binding antibody fragment comprises the light chain sequences disclosed in Table 7.

Table 7

[0131] In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment of the immunoconjugate described herein comprises a light chain (LC) sequence selected from SEQ ID NOs: 137-150. In certain embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment of the immunoconjugate described herein comprises the LC sequence of SEQ ID NO: 140. In another embodiment, the EpCAM antibody or its EpCAM-binding antibody fragment of the immunoconjugate described herein comprises the LC sequence of SEQ ID NO: 138. In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment comprises an 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 its EpCAM-binding antibody fragment comprises an 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 its EpCAM-binding antibody fragment comprises an 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%.

[0132] In additional embodiments, the anti-EpCAM antibody or EpCAM-binding antibody fragment of the immunoconjugate described herein comprises an HC 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 anti-EpCAM antibody or EpCAM-binding antibody fragment of the immunoconjugate described herein comprises an HC having the sequence of SEQ ID NO: 103 and an LC having the sequence of SEQ ID NO: 140. In additional embodiments, the anti-EpCAM antibody or EpCAM-binding antibody fragment of the immunoconjugate described herein comprises an HC having the sequence of SEQ ID NO: 103 and an LC having the sequence of SEQ ID NO: 138. In additional embodiments, the anti-EpCAM antibody or EpCAM-binding antibody fragment of the immunoconjugate described herein comprises an HC having the sequence of SEQ ID NO: 105 and an LC having the sequence of SEQ ID NO: 138. In additional embodiments, the anti-EpCAM antibody or EpCAM-binding antibody fragment of the immunoconjugate described herein comprises an HC having the sequence of SEQ ID NO: 106 and an LC having the sequence of SEQ ID NO: 139. In additional embodiments, the anti-EpCAM antibody or EpCAM-binding antibody fragment of the immunoconjugate described herein comprises an HC having the sequence of SEQ ID NO: 105 and an LC having the sequence of SEQ ID NO: 140. In additional embodiments, the anti-EpCAM antibody or EpCAM-binding antibody fragment of the immunoconjugate described herein comprises an HC having the sequence of SEQ ID NO: 106 and an LC having the sequence of SEQ ID NO: 140. In some embodiments, the anti-EpCAM antibody or EpCAM-binding antibody fragment of the immunoconjugate described herein comprises an HC having the sequence of SEQ ID NO: 125 and an LC having the sequence of SEQ ID NO: 140. In additional embodiments, the anti-EpCAM antibody or EpCAM-binding antibody fragment of the immunoconjugate described herein comprises an HC having the sequence of SEQ ID NO: 127 and an LC having the sequence of SEQ ID NO: 140. In additional embodiments, the anti-EpCAM antibody or EpCAM-binding antibody fragment of the immunoconjugate described herein comprises an 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 of the immunoconjugate described herein, or an EpCAM-binding antibody fragment thereof, comprises an HC having the sequence of SEQ ID NO: 134 and an LC having the sequence of SEQ ID NO: 140.

[0133] In some embodiments, the EpCAM antibody of the immunoconjugate described herein comprises an altered (e.g., mutated or modified) Fc region. For example, in some aspects, the Fc region has been altered to reduce or enhance the effector function of the antibody and to modify the serum half-life or other functional characteristics of the antibody. In certain cases, for example, in the case of an antibody having a mechanism of action that involves blocking or antagonizing, but not killing, cells having a target antigen, reduction or elimination of effector function is desirable. An increase in effector function is generally desirable when directed against undesirable cells such as tumors and foreign cells that express low levels of FcγR, e.g., tumor-specific B cells having low levels of FcγRIIB (e.g., non-Hodgkin lymphoma, CLL, and Burkitt lymphoma). The immunoconjugates of the invention having such imparted or altered effector function activity are useful for the treatment and / or prevention of diseases, disorders, or infections where improvement in the effectiveness of effector function activity is desired. In some aspects, the Fc region is an isotype selected from IgM, IgA, IgG, IgE, or other isotypes.

[0134] The Fc region of an EpCAM antibody and its EpCAM-binding antibody fragment may have the ability to bind to one or more Fc receptors (e.g., FcγR(s)), but in certain embodiments, the antibody or antibody fragment has a variant Fc region with altered binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b) compared to the binding exhibited by the wild-type Fc region. For example, the binding to activating receptors is enhanced, and / or the ability to bind to inhibitory receptor(s) is substantially reduced or eliminated. Thus, the Fc region of an EpCAM antibody or its EpCAM-binding antibody fragment may comprise part or all of the CH2 domain of the full Fc region and / or part or all of the CH3 domain, or may comprise variant CH2 and / or variant CH3 sequences (e.g., may contain one or more insertions and / or one or more deletions with respect to the CH2 or CH3 domain of the full Fc region). Such Fc regions may contain a non-Fc polypeptide portion, may contain part of a non-native full Fc region, or may contain a non-native orientation of the CH2 and / or CH3 domains (e.g., two CH2 domains or two CH3 domains, or a CH3 domain linked to a CH2 domain in the N-terminal to C-terminal direction, etc.).

[0135] Modifications of the Fc region identified as altering effector function are known in the art and include, for example, modifications that increase binding to activating receptors (e.g., FcγRIIA (CD16A)) and reduce binding to inhibitory receptors (e.g., FcγRIIB (CD32B)) (see, e.g., Stavenhagen, et al., Cancer Res. 57(18):8882-8890 (2007)). Table 8 lists exemplary single, double, triple, quadruple, and quintuple substitutions (numbering is according to the Kabat EU index, and the substitutions are with respect to the amino acid sequence of SEQ ID NO: 304) of exemplary modifications that increase binding to activating receptors and / or reduce binding to inhibitory receptors.

Table 8

[0136] Exemplary variants of the human IgG1 Fc region with reduced binding to CD32B and / or increased binding to CD16A include the F243L, R292P, Y300L, V305I, or P396L substitutions, where the numbering is according to the Kabat EU index. These amino acid substitutions can be present in the human IgG1 Fc region in any combination. In one embodiment, the variant human IgG1 Fc region contains the substitutions F243L, R292P, and Y300L. In another embodiment, the variant human IgG1 Fc region contains the substitutions F243L, R292P, Y300L, V305I, and P396L.

[0137] In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment of the immunoconjugates described herein comprises an immunoglobulin heavy chain constant region containing a modification that reduces effector function (see, e.g., Idusogie et al., 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 (1994); Xu et al., Cell Immunol. 200:16-26 (2000); Cole et al., Transplantation 68:563-571 (1999); Hutchins et al., PNAS USA 92:11980-11984 (1995); Reddy et al., J. Immunol. 164:1925-1933 (2000); WO 97 / 11971 and WO 07 / 106585; US Patent Application Publication No. 2007 / 0148167A1; McEarchern 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), each of which is incorporated herein by reference in its entirety).

[0138] In some embodiments, the Fc region of the anti-EpCAM antibody of the immunoconjugates described herein or its EpCAM-binding antibody fragment exhibits (or substantially does not exhibit) a reduction in binding to effector receptors selected from the group consisting of FcγRIA (CD64), FcγRIIA (CD32A) (allotypes R131 and H131), FcγRIIB (CD32B), FcγRIIIA (CD16a) (allotypes V158 and F158), and FcγRIIIB (CD16b) (allotypes FcγIIIb-NA1 and FcγIIIb-NA2) as compared to the binding exhibited by the wild-type IgG Fc region (SEQ ID NO: 304). In some embodiments, the binding affinity of the Fc region variant of the anti-EpCAM antibody or its EpCAM-binding antibody fragment to effector receptors is reduced to 1 / 10 or less, 1 / 50 or less, or 1 / 100 or less compared to the binding affinity of the corresponding antibody or antibody-binding fragment comprising the wild-type Fc region of the corresponding immunoglobulin.

[0139] In certain embodiments, the EpCAM antibody of the immunoconjugates described herein, or an EpCAM-binding antibody fragment thereof, exhibits reduced effector function (e.g., reduced ADCC), and comprises an IgG Fc region comprising 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 follows the EU index described in Kabat. In one embodiment, the CH2-CH3 domain of the EpCAM antibody comprises any 1, 2, 3, or 4 of the substitutions: L234A, L235A, D265A, N297Q, N297A, and N297G, and this numbering is that of the Kabat EU index. In another embodiment, the CH2-CH3 domain comprises an N297Q substitution, an N297A substitution, or an L234A and L235A substitution, because these mutations abrogate FcR binding. Alternatively, the EpCAM antibody of the immunoconjugates described herein, or an EpCAM-binding antibody fragment thereof, comprises a CH2-CH3 domain of a native Fc region that exhibits reduced (or substantially absent) binding to FcγRIIIA (CD16a) and / or reduced effector function (compared to the binding and effector functions exhibited by the wild-type IgG1 Fc region (SEQ ID NO: 304)). In certain embodiments, 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). The N297A, N297G, N297Q, L234A, L235A, and D265A substitutions abrogate effector function and thus, in situations where effector function is desired, these substitutions may not be employed in some cases.

[0140] The specific IgG1 sequences of the CH2 and CH3 domains of the Fc region-containing EpCAM antibody of the immunoconjugate described herein or its EpCAM-binding antibody fragment having a reduced or abrogated effector function contain the substitutions L234A / L235A (underlined) (SEQ ID NO: 307):

Table 9

[0141] The specific IgG1 sequences of the CH2 and CH3 domains of the Fc region-containing EpCAM antibody of the immunoconjugate described herein or its EpCAM-binding antibody fragment having a reduced or abrogated effector function contain the substitution N297A (underlined) (SEQ ID NO: 308):

Table 10

[0142] The specific IgG1 sequences of the CH2 and CH3 domains of the Fc region-containing EpCAM antibody of the immunoconjugate described herein or its EpCAM-binding antibody fragment having a reduced or abrogated effector function contain the substitution N297Q (underlined) (SEQ ID NO: 309):

Table 11

[0143] In some embodiments, the EpCAM antibody of the immunoconjugates described herein or its EpCAM-binding antibody fragment comprises one or more modifications corresponding to IgG1-C220S, C226S, C229S, P238S; IgG1-C226S, C229S; IgG1-C226S, C229S, E233P, L234V, L235A; IgG1-L234A, L235A; IgG1-L234F, L235E, P331S; IgG1-L234F, L235E, P331S; IgG1-H268Q, A330S, P331S; IgG1-G236R, L328R; IgG1-L235G, G236R, IgG1-N297A; IgG1-N325A, L328R; IgG1-N325L, L328R; IgG1-K326W, E333S; IgG2-V234A, 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, where the position numbering follows the Kabat EU index.

[0144] In some embodiments, the EpCAM antibody of the immunoconjugates described herein or its EpCAM-binding antibody fragment comprises a heavy chain immunoglobulin constant domain with reduced CDC activity. In certain aspects, the EpCAM antibody of the immunoconjugates described herein or its EpCAM-binding antibody fragment comprises an IgG1 heavy chain constant region that contains mutations that reduce CDC activity (see, e.g., WO 1997 / 11971 and WO 2007 / 106585, US Patent Application Publication No. 2007 / 0148167A1; McEarchern et al., Blood 109:1185-1192 (2007); Hayden-Ledbetter et al., Clin. Cancer 15:2739-2746 (2009); Lazar et al., PNAS USA 103:4005-4010 (2006); Bruckheimer et al., Neoplasia 11:509-517 (2009); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Sazinsky et al., PNAS USA 105:20167-20172 (2008), each of which is incorporated herein by reference in its entirety). Examples of heavy chain constant domain sequence modifications that reduce CDC include IgG1-C226S, C229S, E233P, L234V, L235A; IgG1-C226S, P230S; IgG1-L234F, L235E, P331S; IgG1-S239D, A330L, I332E; IgG2 EU sequence 118-260; IgG4-EU sequence 261-447; and one or more modifications corresponding to IgG2-H268Q, V309L, A330S, A331S (according to EU index).

[0145] In some embodiments, the provided anti-EpCAM antibody of the immunoconjugates described herein or its anti-EpCAM binding antibody fragment comprises a heavy chain immunoglobulin constant domain that contains one or more half-life extending amino acid modifications (e.g., substitutions). A number of mutations capable of extending the half-life of an Fc region-containing molecule are known in the art and are included as components of the anti-EpCAM antibodies and their anti-EpCAM binding antibody fragments provided herein. See, for example, U.S. Patent Nos. 6,277,375; 7,083,784; 7,217,797; and 8,088,376; U.S. Patent Application Publication Nos. 2002 / 0147311; and 2007 / 0148164; and International Publication Pamphlets Nos. WO 1998 / 23289; WO 2009 / 058492; and WO 2010 / 033279 (each in its entirety incorporated herein by reference).

[0146] The serum half-life of a protein containing an Fc region can be increased by increasing the binding affinity of the Fc region for FcRn. As used herein, the term "half-life" refers to the pharmacokinetic property of a molecule that is a measure of the average survival time of the molecule after administration. Half-life can be expressed, for example, as the time required to remove 50 percent (50%) of a known amount of a molecule from the body or a particular compartment thereof of a subject (e.g., a human patient or other mammal), measured in serum (i.e., circulating half-life) or in other tissues. Generally, an increase in half-life results in an increase in the mean residence time (MRT) of the administered molecule in circulation.

[0147] In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment of the 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, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428, 433, 434, 435, and 436, wherein the amino acid position numbering follows the EU index. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises 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 follows the EU index. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a substitution of the amino acid by Tyr, Phe, Trp, or Thr at Kabat position 252; a substitution of the amino acid by Thr at Kabat position 254; a substitution of the amino acid by Ser, Arg, Gln, Glu, Asp, or Thr at Kabat position 256; a substitution of the amino acid by Leu at Kabat position 257; a substitution of the amino acid by Pro at Kabat position 309; a substitution of the amino acid by Ser at Kabat position 311; a substitution of the amino acid by Thr, Leu, Phe, or Ser at Kabat position 428; a substitution of the amino acid by Arg, Ser, Iso, Pro, or Gln at Kabat position 433; or a substitution of the amino acid by Trp, Met, Ser, His, Phe, or Tyr at Kabat position 434, or one or more of the above. More specifically, the domain of the EpCAM antibody or an EpCAM-binding antibody fragment thereof can comprise amino acid substitutions such as a substitution of the amino acid by Tyr at Kabat position 252, a substitution of the amino acid by Thr at Kabat position 254, and a substitution of the amino acid by Glu at Kabat position 256, relative to the wild-type human IgG constant domain.

[0148] In some embodiments, the anti-EpCAM antibody or an EpCAM-binding antibody fragment of the immunoconjugates described herein comprises at least one substitution selected from T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, N434S, N434H, N434Y, H435K, and Y436I, where the numbering is that of the Kabat EU index. In further embodiments, the anti-EpCAM antibody or an 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) A378V and N434A; (g) N434A and Y436I; (h) V308P and N434A; and (i) K288D and H435K.

[0149] In certain embodiments, the anti-EpCAM antibody or an EpCAM-binding antibody fragment of the immunoconjugates described herein comprises a variant IgG Fc region comprising any one, two, or three of the substitutions: M252Y, S254T, and T256E. The disclosure further provides an anti-EpCAM antibody or an EpCAM-binding antibody fragment thereof having a variant Fc region comprising (a) one or more mutations that alter effector function and / or FcγR; and (b) one or more mutations that extend serum half-life.

[0150] 6.3 Immunoconjugates Comprising EPCAM-Activatable Antibodies In additional embodiments, the present disclosure provides immunoconjugates comprising EpCAM activatable antibodies (e.g., activatable EpCAM antibodies and activatable EpCAM-binding antibody fragments thereof). In some embodiments, the EpCAM activatable antibody comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof that specifically binds to 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 its EpCAM-binding antibody fragment to bind to EpCAM. In some embodiments, the MM is coupled via a sequence comprising a substrate for a protease, e.g., a protease that is active in diseased tissue and / or co-localizes with EpCAM at the treatment site of the subject. In some embodiments, the immunoconjugate comprising an EpCAM activatable antibody comprises an EpCAM antibody described in Section 6.2 coupled to the MM via a protease substrate.

[0151] The EpCAM activatable antibody is, in some cases, stable in circulation and is activated at the intended treatment and / or diagnostic site, but not in normal, e.g., healthy tissue or other tissues that are not targets of the treatment and / or diagnosis, and upon activation, exhibits binding to EpCAM that is at least equivalent to that of the corresponding unmodified antibody. Also provided are immunoconjugates comprising an EpCAM activatable antibody, as well as nucleic acids or a series of nucleic acids encoding the EpCAM activatable antibody, and vectors and host cells comprising the nucleic acids. Pharmaceutical compositions comprising the activatable antibody, immunoconjugate, nucleic acid, vector, and host cell are also provided.

[0152] In some embodiments, an immunoconjugate having an EpCAM activatable antibody or antibody fragment (a) a cleavable moiety coupled to an antibody or antibody fragment that functions as a substrate for a protease; and (b) When the activatable antibody is in the uncleaved state, it contains a masking moiety coupled to an antibody or antibody fragment that inhibits the binding of the masking moiety to an antibody or antibody fragment against EpCAM. The uncleaved activatable antibody has a structural sequence from the N-terminus to the C-terminus: (masking moiety)-(cleavable moiety)-(antibody or antibody fragment) or (antibody or antibody fragment)-(cleavable moiety)-(masking moiety).

[0153] In some cases, the immunoconjugates disclosed herein include an EpCAM-activatable antibody, and the EpCAM-activatable antibody (a) (i) having VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 with sequences of members selected from the group consisting of SEQ ID NOs: 13-15, 42, 40, and 41, respectively; (ii) having VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 with sequences of members selected from the group consisting of SEQ ID NOs: 13-15 and 39-41, respectively; (iii) having VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 with sequences of members selected from the group consisting of SEQ ID NOs: 13, 26, 15, and 39-41, respectively; and (iv) an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having sequences of members selected from the group consisting of SEQ ID NOs: 13, 24, 15, 42, 40, and 41, respectively; (b) When the activatable antibody is in the uncleaved state, a masking moiety coupled to the EpCAM antibody or an EpCAM-binding antibody fragment thereof that inhibits the binding of the masking moiety to an antibody or antibody fragment against EpCAM, and (c) a cleavable moiety coupled to the EpCAM antibody or an EpCAM-binding antibody fragment thereof, wherein the cleavable moiety is a polypeptide that functions as a substrate for a protease. The uncleaved activatable antibody has a structural sequence from the N-terminus to the C-terminus: (masking moiety)-(cleavable moiety)-(antibody or antibody fragment) or (antibody or antibody fragment)-(cleavable moiety)-(masking moiety).

[0154] The EpCAM-activatable antibody of the immunoconjugate described herein binds to human EpCAM when in the activated state and comprises: (i) an EpCAM antibody of the immunoconjugate described herein or an EpCAM-binding antibody fragment thereof (Ab) that specifically binds to human EpCAM (e.g., as disclosed in Section 6.2 herein); (ii) a masking moiety (MM) that inhibits the binding of the EpCAM-activatable antibody to EpCAM when the EpCAM-activatable antibody is in the uncleaved state; and (c) a cleavable moiety (CM) coupled to the EpCAM antibody or an EpCAM-binding antibody fragment thereof, which is a polypeptide that functions as a substrate for a protease. In some embodiments, the uncleaved EpCAM-activatable antibody has a structural sequence from the N-terminus to the C-terminus of MM-CM-Ab or Ab-CM-MM. In some embodiments, the EpCAM-activatable antibody comprises a linker peptide between MM and CM. In some embodiments, the EpCAM-activatable antibody comprises a linker peptide between CM and Ab.

[0155] In some embodiments, the uncleaved EpCAM-activatable antibody of the immunoconjugate described herein specifically binds to mammalian EpCAM with a dissociation constant of 1 nM or less, 5 nM or less, 10 nM or less, 15 nM or less, 20 nM or less, 25 nM or less, 50 nM or less, 100 nM or less, 150 nM or less, 250 nM or less, 500 nM or less, 750 nM or less, 1000 nM or less, and 122 / , or 2000 nM or less.

[0156] In some embodiments, the uncleaved EpCAM-activatable antibody of the immunoconjugate described herein specifically binds to mammalian EpCAM (e.g., human EpCAM or cynomolgus monkey EpCAM) with a dissociation constant of 1 nM or more, 5 nM or more, 10 nM or more, 15 nM or more, 20 nM or more, 25 nM or more, 50 nM or more, 100 nM or more, 150 nM or more, 250 nM or more, 500 nM or more, 750 nM or more, 1000 nM or more, and 122 / , or 2000 nM or more.

[0157] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein in the non-cleaved state has 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, 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, 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,Specifically binds to mammalian EpCAM (e.g., human EpCAM or cynomolgus monkey EpCAM).

[0158] In some embodiments, the EpCAM-activatable antibody in the activated state specifically binds to mammalian EpCAM (e.g., human EpCAM or cynomolgus monkey EpCAM) with a dissociation constant of 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, or 10 nM or less. In some embodiments, the EpCAM-activatable antibody in the activated state specifically binds to mammalian EpCAM with a dissociation constant of 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, or 10 nM or more.

[0159] In some embodiments, the EpCAM-activatable antibody in the activated state specifically binds to mammalian EpCAM (e.g., human EpCAM or cynomolgus monkey 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.

[0160] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein specifically binds to human EpCAM with a dissociation constant of less than 1 nM. In some embodiments, the EpCAM-activatable antibody specifically binds to cynomolgus monkey EpCAM with a dissociation constant of less than 1 nM. In some embodiments, the EpCAM-activatable antibody specifically binds to both human EpCAM and cynomolgus monkey EpCAM with a dissociation constant of less than 1 nM.

[0161] In some embodiments, the serum half-life of the EpCAM-activatable antibody of the immunoconjugates described herein is longer than that of the corresponding antibody, e.g., the pK of the EpCAM-activatable antibody is longer than that of the corresponding antibody. In some embodiments, the serum half-life of the EpCAM-activatable antibody is the same as that of the corresponding antibody.

[0162] In some embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof that comprises VH-CDR1, VH-CDR2, and VH-CDR3 having the sequences set forth in one row of Table 2. In some embodiments, the EpCAM-activatable antibody comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof that comprises VL-CDR1, VL-CDR2, and VL-CDR3 having the sequences set forth in one row of Table 3.

[0163] In some embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof that comprises VH-CDR1 selected from SEQ ID NOs: 13 and 16-25, VH-CDR2 selected from SEQ ID NOs: 14 and 26-29, VH-CDR3 selected from SEQ ID NOs: 15 and 30-38, VL-CDR1 selected from SEQ ID NOs: 39 and 42-45, VL-CDR2 of SEQ ID NO: 40, and VL-CDR3 selected from SEQ ID NOs: 41 and 46-51.

[0164] In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein include an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having sequences selected from the group consisting of (i) SEQ ID NOs: 13-15, 42, 40, and 41; (ii) SEQ ID NOs: 13-15, and 39-41; (iii) SEQ ID NOs: 13, 26, 15, and 39-41; and (iv) SEQ ID NOs: 13, 26, 15, 42, 40, and 41. In some embodiments, the EpCAM-activatable antibody includes an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising 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.

[0165] In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having sequences selected from the group consisting of (i) SEQ ID NOs: 22, 14, 15, 42, 40, and 41; (ii) SEQ ID NOs: 13, 14, 33, 42, 40, and 41; (iii) SEQ ID NOs: 23, 14, 15, 42, 40, and 41; and (iv) SEQ ID NOs: 25, 14, 15, 42, 40, and 41. In some embodiments, the EpCAM-activatable antibody comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising VH-CDR1, 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-activatable antibody comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising 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.

[0166] In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise a VH disclosed in Table 4. In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise a VL disclosed in Table 5. In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise 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-activatable antibodies of the immunoconjugates described herein comprise a VH having the sequence of SEQ ID NO: 75 and a VL having the sequence of SEQ ID NO: 89, and comprise the EpCAM antibodies or EpCAM-binding antibody fragments thereof described herein. In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise a VH having the sequence of SEQ ID NO: 77 and a VL having the sequence of SEQ ID NO: 89, and comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof.

[0167] In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise VH sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 54, 75, and 77. In some embodiments, the EpCAM-activatable antibodies comprise VL sequences that are 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%.

[0168] In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise the HC disclosed in Table 6. In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise the LC disclosed in Table 7. In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise an HC having the sequence of SEQ ID NO: 103 and an LC having the sequence of SEQ ID NO: 140. In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise an HC having the sequence of SEQ ID NO: 125 and an LC having the sequence of SEQ ID NO: 140. In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise an HC having the sequence of SEQ ID NO: 127 and a light chain having the sequence of SEQ ID NO: 140.

[0169] In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise an EpCAM antibody or an 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-activatable antibodies of the immunoconjugates described herein comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof that specifically binds to an epitope within the first extracellular domain (D1) of human EpCAM (SEQ ID NO: 2).

[0170] In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein comprise X1YX3X4H, wherein X1 is selected from N and S, X3 is selected from Y, N, F, S, H, D, L, I, and W, X4 is selected from I and M, a VH-CDR1, and the sequence WX2X3PGX6VYIQYX 12 X 13 KFX 17 comprising G, wherein X2 is selected from I and F, X3 is selected from Y and N, X6 is selected from N and D, X 12 is selected from N and S, X 13 is selected from E and Q, X 17It comprises a VH-CDR2 (SEQ ID NO: 7) selected from K and Q, and a VH-CDR3 (SEQ ID NO: 8) comprising X1GX3X4FAY, wherein X1 is selected from D and E, X3 is selected from P, A, S, Y, F, G, T, and V, and X4 is selected from Y and W. In some embodiments, the EpCAM-activatable antibody is RSSX4SLLHSX 10 GX 12 TYLX 16 It comprises, wherein X4 is selected from R and K, X 10 is selected from N and D, X 12 is selected from F and I, X 16 is selected from Y and S, a VL-CDR1 (SEQ ID NO: 10), a light chain VL-CDR2 (SEQ ID NO: 40) comprising QTSNLAS, and a VL-CDR3 (SEQ ID NO: 11) comprising X1QX3LELPX8T, wherein X1 is selected from A, L, and Q, X3 is selected from S, G, Y, and N, and X8 is selected from N and W. In some embodiments, the EpCAM-activatable 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-CDR2 comprising the sequence of SEQ ID NO: 40, and a VL-CDR3 comprising the sequence of SEQ ID NO: 41.

[0171] In some embodiments, the VH-CDR1 of the EpCAM-activatable antibody of the immunoconjugates described herein comprises the sequence NYX3IH, where X3 is 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-activatable antibody comprises the sequence DGPX4FAY, where X4 is selected from Y and W (SEQ ID NO: 9). In some embodiments, the VL-CDR3 of the EpCAM-activatable antibody comprises the sequence AQX3LELPNT, where X3 is selected from S, G, Y, and N (SEQ ID NO: 12). In some embodiments, the EpCAM-activatable 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-CDR2 comprising the sequence of SEQ ID NO: 40, and a VL-CDR3 comprising the sequence of SEQ ID NO: 41.

[0172] Suitable components of the EpCAM-activatable antibodies of the immunoconjugates described herein disclosed also include EpCAM antibodies or EpCAM-binding antibody fragments thereof that cross-compete 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 with respect to binding to human EpCAM and / or cynomolgus EpCAM. Additional suitable EpCAM-activatable antibodies cross-compete 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 with respect to binding to human EpCAM and / or cynomolgus EpCAM. Additional suitable EpCAM-activatable antibodies cross-compete 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 with respect to binding to human EpCAM and / or cynomolgus EpCAM.

[0173] The EpCAM-activatable antibodies of the immunoconjugates described herein provided in this specification include a masking moiety (MM). In some embodiments, the masking moiety (or "mask") is coupled or otherwise attached to the EpCAM antibody and is an amino acid sequence disposed within the EpCAM-activatable antibody construct such that the masking moiety reduces the ability of the EpCAM antibody to specifically bind to 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 pamphlet, the entire content of which is incorporated herein by reference.

[0174] In some embodiments, the MM of the activatable antibody has a dissociation constant for binding to the Ab that is greater than the dissociation constant of the Ab for EpCAM. In some embodiments, the MM has a dissociation constant for binding to the Ab that is less than or equal to the dissociation constant of the Ab for EpCAM.

[0175] In some embodiments, the MM has a dissociation constant for binding to the Ab that is less than the dissociation constant of the Ab for EpCAM.

[0176] In some embodiments, the dissociation constant (K d) is 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 times or more, or does not exceed 1 to 5, 5 to 10, 10 to 100, 10 to 1,000, 10 to 10,000, 10 to 100,000, 10 to 1,000,000, 10 to 10,000,000, 100 to 1,000, 100 to 10,000, 100 to 100,000, 100 to 1,000,000, 100 to 10,000,000, 1,000 to 10,000, 1,000 to 100,000, 1,000 to 1,000,000, 1000 to 10,000,000, 10,000 to 100,000, 10,000 to 1,000,000, 10,000 to 10,000,000, 100,000 to 1,000,000, or 100,000 to 10,000,000 times or more of the dissociation constant for the target of Ab.

[0177] In some embodiments, when the EpCAM-activatable antibody of the immunoconjugate described herein is in a cleaved state, MM does not interfere with or compete with Ab with respect to binding to EpCAM. In some embodiments, MM is a polypeptide about 2 to 40 amino acids in length. In some embodiments, MM is a polypeptide up to about 40 amino acids in length.

[0178] In some embodiments, the polypeptide sequence of MM is different from the polypeptide sequence of EpCAM. In some embodiments, the MM polypeptide sequence has 50% or less identity with any natural binding partner of Ab. In some embodiments, the MM polypeptide sequence is different from the sequence of EpCAM and has 40%, 30%, 25%, 20%, 15%, or 10% or less identity with any natural binding partner of Ab.

[0179] In some embodiments, the coupling of MM to Ab reduces the ability of the Ab to bind to EpCAM such that the dissociation constant (Kd) of the Ab for EpCAM when coupled to MM is at least 2, 5, 10, 20, 40, 100, 1000, 10,000 times greater than the Kd of the Ab for EpCAM when not coupled to MM.

[0180] In some embodiments, in the presence of EpCAM, for example, when assayed in vitro using a target displacement assay such as the assay described in WO 2010 / 081173, the entire contents of which are incorporated herein by reference, when CM is uncleaved, MM reduces the ability of the Ab to bind to EpCAM by at least 90% compared to when CM is cleaved.

[0181] When the Ab is modified with MM and is in the presence of human EpCAM, the specific binding of the Ab to human EpCAM is reduced or inhibited compared to the specific binding of the unmodified Ab or the parental Ab to human EpCAM.

[0182] The Kd of the MM-modified Ab for 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 more, or 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 greater than the Kd of the unmodified Ab or the parental Ab for human EpCAM. Conversely, the binding affinity of the MM-modified Ab for 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 more, or 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 smaller than the binding affinity of the unmodified Ab or the parental Ab for human EpCAM.

[0183] In some embodiments, the dissociation constant (Kd) of MM for Ab is approximately equal to the Kd of the Ab for human EpCAM. In some embodiments, the dissociation constant (Kd) of MM for Ab is less than or equal to the dissociation constant of the Ab for human EpCAM. In some embodiments, the dissociation constant (Kd) of MM for Ab is less than the dissociation constant of the Ab for human EpCAM. In some embodiments, the dissociation constant (Kd) of MM for Ab is greater than the dissociation constant of the Ab for human EpCAM.

[0184] In some embodiments, MM has a Kd for binding to Ab that is less than or equal to the Kd for binding of the Ab to human EpCAM.

[0185] In some embodiments, MM has a Kd for binding to Ab that is greater than or equal to the Kd for binding of the Ab to human EpCAM. In some embodiments, MM has a Kd for binding to Ab that is approximately equal to the Kd for binding of the Ab to human EpCAM. In some embodiments, MM has a Kd for binding to Ab that is less than the Kd for binding of the Ab to human EpCAM. In some embodiments, MM has a Kd for binding to Ab that is greater than the Kd for binding of the Ab to human EpCAM. In some embodiments, MM has a Kd for binding to Ab that is no more than 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times greater than the Kd for binding of the Ab to human EpCAM. In some embodiments, MM has a Kd for binding to Ab that is 1 - 5, 2 - 5, 2 - 10, 5 - 10, 5 - 20, 5 - 50, 5 - 100, 10 - 100, 10 - 1,000, 20 - 100, 20 - 1,000, or 100 - 1,000 times greater than the Kd for binding of the Ab to human EpCAM.

[0186] In some embodiments, the MM has an affinity for the Ab that is less than the affinity of the Ab for binding to human EpCAM. In some embodiments, the MM has an affinity for the Ab that is less than or equal to the affinity of the Ab for binding to human EpCAM. In some embodiments, the MM has an affinity for the Ab that is approximately equal to the affinity of the Ab for binding to human EpCAM. In some embodiments, the MM has an affinity for the Ab that is greater than or equal to the affinity of the Ab for binding to human EpCAM. In some embodiments, the MM has an affinity for the Ab that is greater than the affinity of the Ab for binding to human EpCAM.

[0187] In some embodiments, the MM has an affinity for the Ab that is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times less than the affinity of the Ab for binding to human EpCAM. In some embodiments, the MM has an affinity for the Ab that is 1 - 5, 2 - 5, 2 - 10, 5 - 10, 5 - 20, 5 - 50, 5 - 100, 10 - 100, 10 - 1,000, 20 - 100, 20 - 1,000, or 100 - 1,000 times less than the affinity of the Ab for binding to human EpCAM. In some embodiments, the MM has an affinity for the Ab that is 2 - 20 times less than the affinity of the Ab for binding to human EpCAM. In some embodiments, the MM that is not covalently bound to the Ab and is present at an equimolar concentration with respect to the EpCAM-activatable antibody does not inhibit the binding of the Ab to human EpCAM.

[0188] When Ab is modified with MM and in the presence of human EpCAM, the specific binding of the Ab to human EpCAM is reduced or inhibited compared to the specific binding of the Ab not modified with MM or the parental Ab to human EpCAM. When compared to the binding of the Ab not modified with MM or the parental Ab to human EpCAM, the ability of the Ab modified with MM to bind to human EpCAM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and even 100% over a period of 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, as measured in an in vivo or in vitro assay.

[0189] MM inhibits the binding of the Ab to human EpCAM. MM binds to the antigen-binding domain of the Ab and inhibits the binding of the Ab to human EpCAM. MM can sterically inhibit the binding of the Ab to human EpCAM. MM can allosterically inhibit the binding of the Ab to its target. In these embodiments, when the Ab is modified with or coupled to MM and in the presence of the target, as measured in an in vivo or in vitro assay, 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, there is no or substantially no binding of the Ab to human EpCAM, or the binding of the Ab not modified with MM, the parental Ab, or the Ab not coupled to MM to human EpCAM is 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% or less compared to the binding of the Ab to human EpCAM.

[0190] When the Ab is coupled to MM or modified by MM, MM "masks", or reduces, or otherwise inhibits the specific binding of the Ab to human EpCAM. When the Ab is coupled to MM or modified by MM, such coupling or modification can result in a structural change that reduces or inhibits the ability of the Ab to specifically bind to its target.

[0191] An Ab coupled to or modified with MM can be represented by the following formula (in order from the amino (N)-terminal region to the carboxyl (C)-terminal region): (MM)-(Ab) (Ab)-(MM) (MM)-L-(Ab) (Ab)-L-(MM) In the formula, MM is a masking portion, Ab is an EpCAM antibody, an EpCAM-binding antigen fragment, and L is a linker. In many embodiments, it may be desirable to insert one or more linkers, such as flexible linkers, into the composition to impart flexibility.

[0192] In certain embodiments, MM is not the natural binding partner of Ab. In some embodiments, MM has no homology or substantially no homology to any natural binding partner of Ab. In some embodiments, MM is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% or less similar to any natural binding partner of Ab. In some embodiments, MM has 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% or less identity to any natural binding partner of Ab. In some embodiments, MM has 25% or less identity to any natural binding partner of Ab. In some embodiments, MM has 50% or less identity to any natural binding partner of Ab. In some embodiments, MM has 20% or less identity to any natural binding partner of Ab. In some embodiments, MM has 10% or less identity to any natural binding partner of Ab.

[0193] In some embodiments, MM comprises the sequences disclosed in Table 9. In some embodiments, MM comprises a sequence selected from SEQ ID NOs: 151-157. In some embodiments, MM comprises a sequence selected from SEQ ID NOs: 158-161. In some embodiments, MM comprises a sequence selected from SEQ ID NOs: 162-167. In some embodiments, MM comprises the sequence of SEQ ID NO: 155. [Table 12]

[0194] The EpCAM-activatable antibodies of the immunoconjugates provided herein and described herein include a cleavable moiety. In some embodiments, the cleavable moiety (or "substrate") comprises an amino acid sequence that is a substrate for a protease, which is typically 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 U.S. Patent Nos. 7,666,817 and 8,563,269, and International Publication No. 2014 / 026136, the entire contents of each of which are incorporated herein by reference. (See also Boulware et al., Biotechnol Bioeng. 106(3):339-346(2010)).

[0195] In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein include an Ab that is modified by an MM and also includes one or more cleavable moieties (CM). Such EpCAM-activatable antibodies exhibit activatable / switchable binding to human EpCAM. EpCAM-activatable antibodies typically include an antibody or antigen-binding antibody fragment (Ab) that is modified or coupled to a masking moiety (MM), and a modifiable or cleavable moiety (CM). In some embodiments, the CM comprises an amino acid sequence that functions as a substrate for at least one protease. In some embodiments, the MM and the cleavable moiety are directly coupled to the EpCAM-activatable antibody. In other embodiments, the MM and the cleavable moiety are indirectly coupled to the EpCAM-activatable antibody (e.g., via one or more linkers).

[0196] The elements of the EpCAM-activatable antibody of the immunoconjugate described herein are in a cleaved (or relatively active) state, and in the presence of human EpCAM, the EpCAM-activatable antibody binds to human EpCAM. However, when the EpCAM-activatable antibody is in an uncleaved (or relatively inactive) state and in the presence of human EpCAM, the MM and CM are arranged such that specific binding of the EpCAM-activatable antibody to human EpCAM is reduced or inhibited. Specific binding of the EpCAM-activatable antibody to human EpCAM can be reduced by inhibiting or masking the ability of the EpCAM-activatable antibody that specifically binds to human EpCAM with MM.

[0197] The Kd of the EpCAM-activatable antibody of the immunoconjugate described herein modified with MM and CM against 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-fold, or more, or 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-fold greater than the Kd of the EpCAM-activatable antibody not modified with MM and CM or the parental Ab against human EpCAM.In contrast, the binding affinity of the MM- and CM-modified Abs for 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 more, or 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 unmodified EpCAM-activatable antibody or parental Ab for human EpCAM.

[0198] The EpCAM-activatable antibody of the immunoconjugate described herein is modified with MM and CM and in the presence of human EpCAM, but in the absence of a modifying agent (e.g., at least one protease), the specific binding of the EpCAM-activatable antibody to human EpCAM is reduced or inhibited compared to the specific binding of an unmodified EpCAM-activatable antibody or parental Ab to human EpCAM. When compared to the binding of the parental Ab to human EpCAM or the binding of an unmodified EpCAM-activatable antibody to human EpCAM, the ability of the EpCAM-activatable antibody to bind to human EpCAM when modified with MM and CM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and even 100% as measured in an in vivo or in vitro assay 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 for a longer period.

[0199] As used herein, the term "cleaved state" refers to the state of the EpCAM-activatable antibody of the immunoconjugate described herein after CM has been modified by at least one protease. As used herein, the term "non-cleaved or intact state" refers to the state of the EpCAM-activatable antibody in the absence of cleavage of CM by a protease. As described above, the term "activatable antibody" or "activatable antibody" refers herein to the EpCAM-activatable antibody both in its non-cleaved (native or intact) state and in its cleaved state. One of ordinary skill in the art will appreciate that in some embodiments, the cleaved EpCAM-activatable antibody does not have MM due to cleavage of CM by a protease and can thus release at least MM by covalent bonding (e.g., disulfide bonding between cysteine residues) (e.g., if MM is not bound to the EpCAM-activatable antibody).

[0200] "Activatable or switchable" means that the EpCAM-activatable antibody of the immunoconjugate described herein exhibits a first level of binding to the target when the EpCAM-activatable antibody is inhibited, masked, intact, or in an uncut state (i.e., the first higher-order structure), and exhibits a second level of binding to human EpCAM when not inhibited, unmasked, and / or in a cut state (i.e., the second higher-order structure), and the second level of target binding is greater than the first level of binding. Generally, the access of human EpCAM to the Ab of the EpCAM-activatable antibody is greater in the presence of a cleavage agent capable of cleaving the CM, i.e., a protease, than in the absence of such a cleavage agent. Thus, when the EpCAM-activatable antibody is in an uncut state, the Ab can be inhibited from binding to human EpCAM and masked from human EpCAM binding (i.e., the Ab cannot bind to human EpCAM due to the first higher-order structure), and in a cut state, the Ab is not inhibited from binding to the target or masked.

[0201] The CM and Ab of the EpCAM-activatable antibody of the immunoconjugate described herein are selected such that the Ab serves as the binding moiety for a given target and the CM serves as the substrate for a protease. In some embodiments, the protease co-localizes with human EpCAM at the treatment or diagnostic site of the subject. As used herein, co-localize refers to being at the same site or relatively close. In some embodiments, the protease cleaves the CM to yield an activated antibody that binds to a target located in the vicinity of the cleavage site. The EpCAM-activatable antibodies disclosed herein find particular use, for example, when the protease is capable of cleaving a site in the CM, i.e., when the protease is present at a relatively higher level in the target-containing tissue at the treatment or diagnostic site than in tissue at a non-treatment site (e.g., healthy tissue). In some embodiments, the CM of the present disclosure is also cleaved by one or more other proteases. In some embodiments, this is one or more other proteases that co-localize with human EpCAM and are involved in the cleavage of the CM in vivo.

[0202] In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein result in a reduction of the toxicity and / or adverse side effects that may occur upon binding of the EpCAM-activatable antibody at a non-treatment site in other cases where the EpCAM-activatable antibody was not masked or binding to human EpCAM was not inhibited.

[0203] Generally, the EpCAM-activatable antibodies of the immunoconjugates described herein can be designed by selecting the Ab of interest and constructing the remainder of the EpCAM-activatable antibody such that, when structurally constrained, the MM results in masking of the EpCAM-activatable antibody or reduction of the binding of the EpCAM-activatable antibody to human EpCAM. To provide this functional feature, structural design criteria can be taken into account.

[0204] Provided herein are EpCAM-activatable antibodies of the immunoconjugates described herein that exhibit a switchable phenotype for target binding in an inhibited higher-order structure relative to an uninhibited higher-order structure. The dynamic range generally refers to the ratio of (a) the maximum detectable level of a parameter under a first set of conditions to (b) the minimum detectable value of that parameter under a second set of conditions. For example, in the case of an EpCAM-activatable antibody, the dynamic range refers to the ratio of (a) the maximum detectable level of target protein binding to the EpCAM-activatable antibody in the presence of at least one protease capable of cleaving the CM of the EpCAM-activatable antibody to (b) the minimum detectable level of target protein binding to the EpCAM-activatable antibody in the absence of protease. The dynamic range of an EpCAM-activatable antibody can be calculated as the ratio of the dissociation constant of the EpCAM-activatable antibody in the presence of an EpCAM-activatable antibody cleaving agent (e.g., an enzyme) to the dissociation constant of the EpCAM-activatable antibody in the presence of the EpCAM-activatable antibody cleaving agent. The greater the dynamic range of the EpCAM-activatable antibody, the better the switchable phenotype of the EpCAM-activatable antibody.

[0205] EpCAM-activatable antibodies of the immunoconjugates described herein having a relatively high dynamic range value (e.g., greater than 1) exhibit a more desirable switching phenotype, such that in the presence of a cleaving agent (e.g., an enzyme) capable of cleaving the CM of the EpCAM-activatable antibody, target protein binding by the EpCAM-activatable antibody occurs to a greater extent (e.g., predominantly) compared to when the cleaving agent is absent.

[0206] EpCAM-activatable antibodies of the immunoconjugates described herein can be provided in a variety of structural arrangements. Exemplary formulas for EpCAM-activatable antibodies are shown below. It is specifically contemplated that the order from the N-terminus to the C-terminus of Ab, MM, and CM can be reversed within the EpCAM-activatable antibody. Also, for example, it is specifically contemplated that the amino acid sequences of CM and MM may overlap such that CM is contained within MM.

[0207] For example, the EpCAM-activatable antibody of the immunoconjugate described in this specification can be represented by the following formula (in order from the amino (N)-terminal region to the carboxyl (C)-terminal region): (MM)-(CM)-(Ab) (Ab)-(CM)-(MM) In the formula, MM is a masking moiety, CM is a cleavable moiety, and Ab is an EpCAM antibody or an EpCAM-binding antibody fragment thereof. In the above formula, MM and CM are shown as separate components, but in all exemplary embodiments (including the formula) disclosed herein, it should be noted that the amino acid sequences of MM and CM can overlap such that, for example, CM is wholly or partially contained within MM. Further, the above formula provides for additional amino acid sequences that can be located at the N-terminus or C-terminus of the EpCAM-activatable antibody element.

[0208] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described in this specification comprises a CM that is cleavable by a protease. In some embodiments, the protease that cleaves CM is active in diseased tissue, for example, is upregulated or otherwise unregulated, and the protease cleaves CM when the EpCAM-activatable antibody is exposed to the protease in the EpCAM-activatable antibody.

[0209] In some embodiments, the protease is co-localized with EpCAM in the tissue, and the protease cleaves CM when the EpCAM-activatable antibody is exposed to the protease in the EpCAM-activatable antibody.

[0210] In some embodiments, the CM is disposed within the EpCAM-activatable antibody of the immunoconjugates described herein, such that when the EpCAM-activatable antibody is in the un-cleaved state, the binding of the EpCAM-activatable antibody to EpCAM occurs with a dissociation constant that is at least 2, 5, 10, 20, 40, 50, 100 or 200 times greater than the dissociation constant of the unmodified Ab binding to EpCAM, while in the cleaved state (i.e., when the EpCAM-activatable antibody is in the cleaved state), the Ab binds to EpCAM.

[0211] In some embodiments, the CM is a polypeptide up to 15 amino acids in length.

[0212] In some embodiments, the CM is a polypeptide comprising 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 CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate is independently a polypeptide up to 15 amino acids in length.

[0213] In some embodiments, the CM is a substrate for at least one protease that is upregulated in cancer or otherwise unregulated or thought to be so. In some embodiments, the CM is a substrate for at least one protease that is upregulated during inflammation or thought to be so. In some embodiments, the CM is a substrate for at least one protease that is upregulated during autoimmunity or otherwise unregulated or thought to be so.

[0214] In some embodiments, the CM is a substrate for at least one protease selected from serine proteases such as matrix metalloprotease (MMP), thrombin, neutrophil elastase, cysteine protease, legumain, and matriptase (MT-SPl), and urokinase (uPA). Without being bound by theory, these proteases are thought to be upregulated or otherwise uncontrolled in at least one of cancer, inflammation, and / or autoimmunity.

[0215] Exemplary substrates include, but are not limited to, substrates cleavable by one or more of the following enzymes or proteases: ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5); aspartic proteases (e.g., BACE, renin); aspartic cathepsins (e.g., cathepsin D and cathepsin E); caspases (e.g., caspases 1-10, and caspase 14); cysteine cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, and cathepsin X / Z / P); cysteine proteases (e.g., cruzain, legumain, and Otubain-2); KLK (e.g., KLK4-8, KLK10, KLK11, KLK13, and KLK14); metalloproteases (e.g., meprin, neprilysin, PSMA, and BMP1); MMP (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27); serine proteases (e.g., activated protein C, cathepsin A, cathepsin C, chymase, and coagulation factor proteases such as FVIIa, FIXa, FXa, FXIa, and FXIIa), elastase (e.g., human neutrophil elastase); granzyme B; guanidino benzoatase; HtrA1; lactoferrin; Marapsin; NS3 / 4A; PACE4; plasmin; PSA, tPA; thrombin; tryptase; uPA; type II transmembrane serine protease (TTSP) (e.g., DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, and TMPRSS2-4).

[0216] In some embodiments, the CM is selected for use with a specific protease, e.g., a protease known to co-localize with the target of an EpCAM activating antibody.

[0217] In some embodiments, the CM is a substrate for at least one MMP. Examples of MMPs include MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27. In some embodiments, the CM is a substrate for a protease selected from MMP9, MMP14, MMP1, MMP3, MMP13, MMP17, MMP11, and MMP19. In some embodiments, the CM is a substrate for MMP9. In some embodiments, the CM is a substrate for MMP14.

[0218] Suitable CMs that can be incorporated in the provided activatable antibodies in a conventional manner are known in the art. See, for example, pages 40-47 of WO 2016 / 179285, the entire content of which is incorporated herein by reference.

[0219] In some embodiments, the CM is a substrate for 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 cathepsin. In other embodiments, the CM is a substrate for uPA.

[0220] In certain embodiments, the CM is a substrate for uPA. In some embodiments, the CM comprises the sequences disclosed in Table 10. [Table 13]

[0221] 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).

[0222] In some embodiments, the CM is a substrate for at least two proteases. In some embodiments, each protease is selected from ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5); aspartic proteases (e.g., BACE, renin); aspartic cathepsins (e.g., cathepsin D and cathepsin E); caspases (e.g., caspases 1-10, and caspase 14); cysteine cathepsins (e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, and cathepsin X / Z / P); cysteine proteases (e.g., cruzain, legumain, and Otubain-2); KLK (e.g., KLK4-8, KLK10, KLK11, KLK13, and KLK14); metalloproteases (e.g., meprin, neprilysin, PSMA, and BMP1); MMP (e.g., MMP1-3, MMP7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27); serine proteases (e.g., activated protein C, cathepsin A, cathepsin C, chymase, and coagulation factor proteases such as FVIIa, FIXa, FXa, FXIa, and FXIIa), elastase (e.g., human neutrophil elastase); granzyme B; guanidino benzoatase; HtrAl; lactoferrin; malapsin; NS3 / 4A; PACE4; plasmin; PSA, tPA; thrombin; tryptase; uPA; type II transmembrane serine protease (TTSP) (e.g., DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, and TMPRSS2-4). In some embodiments, the CM is a substrate for at least two proteases, one of the proteases is selected from MMP, thrombin, neutrophil elastase, 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 consisting of MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase.

[0223] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises at least a first CM and a second CM. In some embodiments, the first CM and the second CM are each a polypeptide of 15 amino acids in length or less. In some embodiments, the first CM and the second CM in the non-cleaved state of the EpCAM-activatable antibody have a structural sequence from the N-terminus to the C-terminus of 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 MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase. In some embodiments, the first CM is cleaved by a first cleaving agent selected from MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase in the target tissue, and the second CM is cleaved by a second cleaving agent in the target tissue. In some embodiments, the other protease is selected from the list shown in the previous paragraph. In some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from MMP, thrombin, neutrophil elastase, cysteine protease, uPA, legumain, and matriptase, and the first CM and the second CM are different substrates for the enzyme. In some embodiments, the first cleaving agent and the second cleaving agent are the same protease selected from the list in the previous 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.

[0224] In some embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein also comprises a signal peptide. In some embodiments, the signal peptide is conjugated to the EpCAM-activatable antibody via a spacer. In some embodiments, the spacer is conjugated to the EpCAM-activatable antibody in the absence of the signal peptide. In some embodiments, the spacer is directly bound to the MM of the EpCAM-activatable antibody. In some embodiments, the spacer is directly bound to the MM of the EpCAM-activatable antibody in the structural sequence from the N-terminus to the C-terminus of the spacer-MM-CM-Ab.

[0225] Suitable spacers and spacer technologies are known in the art and can be routinely used to incorporate a spacer in some embodiments of the activatable antibodies provided. See, for example, WO 2016 / 179285 pamphlet (pages 52-53, for example), the entire content of which is incorporated herein by reference.

[0226] In many embodiments, it may be desirable to insert one or more linkers, such as flexible linkers, into the EpCAM-activatable antibody construct to impart flexibility at one or more of the MM-CM linker, the CM-Ab linker, or both. For example, the Ab, MM, and / or CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, particularly Gly and Ser, particularly Gly) to provide the desired flexibility. Thus, the switchable phenotype of such EpCAM-activatable antibody constructs can benefit from the introduction of one or more amino acids to provide a flexible linker. Further, as described below, when the EpCAM-activatable antibody is provided as a higher-order structure-constrained construct, the flexible linker can be operably inserted to facilitate the formation and maintenance of a cyclic structure in the uncleaved EpCAM-activatable antibody.

[0227] For example, in certain embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein comprises one of the following formulas (the following formulas represent amino acid sequences in either the N-terminal to C-terminal direction or the C-terminal to N-terminal direction): (MM)-L1-(CM)-(Ab) (MM)-(CM)-L2-(Ab) (MM)-L1-(CM)-L2-(Ab) Wherein, MM, CM, and Ab are as defined above, and L1 and L2 are each independently the same or different flexible linkers that are optionally present or absent, and contain at least one flexible amino acid (e.g., Gly). Further, the above formulas provide additional amino acid sequences that can be located at the N-terminus or C-terminus of the EpCAM-activatable antibody element. Examples include a target site (e.g., a ligand of a receptor of a cell present in a target tissue), and a serum half-life extending moiety (e.g., a polypeptide that binds to a serum protein such as an immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HSA))), but are not limited thereto.

[0228] In some embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein, in the activated or cleaved state, is exposed to a protease and thereby cleaved such that after the protease cleaves CM, the activated antibody contains a light chain sequence that includes at least a portion of the LP2 and / or CM sequence.

[0229] CM is cleaved by at least one protease at a rate of about 0.001 - 1500x10 4 M -1 S -1 or 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 1500x10 4 M -1 S -1is specifically cleaved at a rate of. In some embodiments, CM is about 100,000 M -1 S -1 is specifically cleaved at a rate of. In some embodiments, CM is about 1 x 10 2 ~ about 1 x 10 6 M -1 S -1 (i.e., about 1 x 10 2 ~ about 1 x 10 6 M -1 S -1 ) is specifically cleaved at a rate of.

[0230] For specific cleavage by the enzyme, contact is made between the enzyme and CM. When an EpCAM-activatable antibody (e.g., an EpCAM antibody or an EpCAM-binding antibody fragment thereof) containing Ab, which is coupled to MM and CM, is in the presence of EpCAM and sufficient enzyme activity, CM can be cleaved. Sufficient enzyme activity can refer to the ability of the enzyme to achieve cleavage upon contact with CM. It can be readily envisioned that the enzyme is present in the vicinity of CM but cannot cleave due to protein modification by other cytokines or enzymes.

[0231] For the EpCAM-activatable antibody of the immunoconjugate described herein, a linker suitable for use in the compositions disclosed herein is generally a linker that confers flexibility to the modified Ab (e.g., an EpCAM antibody or an EpCAM-binding antibody fragment thereof) or the EpCAM-activatable antibody and promotes inhibition of the binding of the EpCAM-activatable antibody to human EpCAM. Such linkers are generally referred to as flexible linkers. Suitable linkers can be readily selected and can be any of 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids in length, including suitable different lengths, e.g., 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 in length, 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.

[0232] Exemplary flexible linkers for the activatable antibodies, antibodies, and antibody fragments provided herein include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n). Suitable linkers and linker technologies are known in the art and, in some embodiments of the activatable antibodies provided, can be routinely used to incorporate spacers. See, for example, International Publication No. WO 2016 / 179285, which is incorporated herein by reference in its entirety (e.g., pages 26, 113-116). One of ordinary skill in the art will recognize that the design of an EpCAM-activatable antibody can include a linker that is wholly or partially flexible such that, to provide the desired EpCAM-activatable antibody structure, the linker can include not only flexible linkers but also one or more moieties that impart a less flexible structure.

[0233] In some embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein includes a first linking peptide (LP1) and a second linking peptide (LP2), and the EpCAM-activatable antibody has a structural sequence from the N-terminus to the C-terminus of MM-LP1-CM-LP2-Ab or Ab-LP2-CM-LP1-MM in the non-cleaved state. In some embodiments, the two linking peptides need not be identical to each other.

[0234] In some embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein includes a first linking peptide (LP1) and a second linking peptide (LP2), and the EpCAM-activatable antibody has a structural sequence from the N-terminus to the C-terminus of MM-LP1-CM-LP2-Ab or Ab-LP2-CM-LP1-MM in the non-cleaved state. In some embodiments, the two linking peptides need not be identical to each other.

[0235] In some embodiments, at least one of LP1 or LP2 of the EpCAM-activatable antibody of the immunoconjugate described herein comprises a flexible linker. Suitable linkers and linker technologies are known in the art and, in some embodiments of the activatable antibodies provided, can be routinely used to incorporate a spacer. See, for example, International Publication No. WO 2016 / 179285, which is incorporated herein by reference in its entirety (e.g., pages 26, 113-116).

[0236] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises a light chain having a sequence disclosed in Table 11. In some embodiments, the activatable antibody comprises a light chain having the sequence of SEQ ID NO: 174. In some embodiments, the activatable antibody comprises a light chain having the sequence of SEQ ID NO: 179. [Table 14] TIFF2025524628000038.tif229159TIFF2025524628000039.tif229159TIFF2025524628000040.tif226159

[0237] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate 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-activatable antibody comprises a light chain having a 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-activatable 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-activatable 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.

[0238] In some embodiments, the EpCAM-activatable 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 EpCAM-activatable 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 SEQ ID NO: 103. In some embodiments, the EpCAM-activatable 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 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-activatable 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 SEQ ID NO: 179, 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 percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0239] In some embodiments, the EpCAM-activatable antibody of the 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.

[0240] In certain embodiments, the huEpCAM23 antibody is encoded by a plasmid having ATCC deposit numbers PTA-125343 and PTA-125344 or PTA-125345, which was deposited with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Va. 20110, on October 4, 2018, under the terms of the Budapest Treaty. Examples of EpCAM antibodies, EpCAM-binding antibody fragments thereof, and EpCAM-activatable antibodies, immunoconjugates are provided herein.

[0241] 6.4 Polynucleotides, Vectors, Host Cells, and Recombinant Methods The disclosure further provides polynucleotides comprising nucleotide sequences encoding the EpCAM antibodies, EpCAM-binding antibody fragments thereof, and EpCAM-activatable antibodies disclosed herein.

[0242] Using methods known in the art, polynucleotides can be obtained and the nucleotide sequences of the polynucleotides can be determined. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody can be constructed from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which generally involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of these oligonucleotides, and subsequent amplification of the ligated oligonucleotides by PCR.

[0243] In some embodiments, the polynucleotides of the present disclosure include the sequences set forth in Table 12. [Table 15] TIFF2025524628000042.tif238159TIFF2025524628000043.tif241159TIFF2025524628000044.tif226159

[0244] In some embodiments, the EpBA of the present disclosure includes the heavy-chain nucleic acid sequence of SEQ ID NO: 201 and the light-chain nucleic acid sequence of SEQ ID NO: 202. In some embodiments, the EpBA of the present disclosure includes the heavy-chain nucleic acid sequence of SEQ ID NO: 203 and the light-chain nucleic acid sequence of SEQ ID NO: 204. In some embodiments, the EpBA of the present disclosure includes the heavy-chain nucleic acid sequence of SEQ ID NO: 205 and the light-chain nucleic acid sequence of SEQ ID NO: 204. In some embodiments, the EpBA of the present disclosure includes the heavy-chain nucleic acid sequence of SEQ ID NO: 206 and the light-chain nucleic acid sequence of SEQ ID NO: 204. In some embodiments, the EpBA of the present disclosure includes the heavy-chain nucleic acid sequence of SEQ ID NO: 207 and the light-chain nucleic acid sequence of SEQ ID NO: 204.

[0245] In some embodiments, the EpBA of the present disclosure includes the heavy-chain nucleic acid sequence of SEQ ID NO: 203 and the light-chain nucleic acid sequence of SEQ ID NO: 208. In some embodiments, the EpBA of the present disclosure includes the heavy-chain nucleic acid sequence of SEQ ID NO: 203 and the light-chain nucleic acid sequence of SEQ ID NO: 209.

[0246] In some embodiments, the EpBA of the present disclosure comprises a heavy chain variable region comprising the same amino acid sequence as the amino acid sequence of the heavy chain variable region encoded by the plasmid deposited with the American Type Culture Collection (ATCC®) as PTA - 125343, and a light chain variable region comprising the same amino acid sequence as the amino acid sequence of the light chain variable region encoded by the plasmid deposited with the ATCC® as PTA - 125342. Also included in the present disclosure are methods of making and using an EpCAM antibody comprising the EpBA and its EpCAM - binding antibody fragments.

[0247] In some embodiments, the present disclosure provides an EpCAM antibody comprising a heavy chain comprising the same amino acid sequence as the amino acid sequence of the heavy chain encoded by the plasmid deposited with the ATCC® as PTA - 125343, and a light chain comprising the same amino acid sequence as the amino acid sequence of the light chain variable region encoded by the plasmid deposited with the ATCC® as PTA - 125342. Also included in the present disclosure are methods of making and using the EpCAM antibody.

[0248] In some embodiments, the present disclosure provides an EpCAM - activatable antibody or an EpCAM - binding activatable antibody fragment comprising (i) a heavy chain variable region comprising the same amino acid sequence as the amino acid sequence of the heavy chain variable region encoded by the plasmid deposited with the ATCC® as PTA - 125343, and (ii) a light chain variable region comprising the same amino acid sequence as the amino acid sequence of the light chain variable region encoded by the plasmid deposited with the ATCC® as PTA - 125344. Also included in the present disclosure are methods of making and using the EpCAM - activatable antibody or the EpCAM - binding activatable antibody fragment.

[0249] In other embodiments, the present disclosure provides an EpCAM-activatable antibody or an EpCAM-binding activatable antibody fragment that includes (i) a heavy chain variable region that includes the same amino acid sequence as the amino acid sequence of the heavy chain variable region encoded by the plasmid deposited with the ATCC® as PTA-125343, and (ii) a light chain variable region that includes the same amino acid sequence as the amino acid sequence of the light chain variable region encoded by the plasmid deposited with the ATCC® as PTA-125345. Also included in the present disclosure are methods of making and using the EpCAM-activatable antibody or the EpCAM-binding activatable antibody fragment.

[0250] Methods for constructing recombinant vectors containing antibody coding sequences and appropriate transcriptional and translational control signals are well known in the art. Once the antibody is expressed recombinantly, it can be purified by any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly chromatography by affinity for a specific antigen after protein A, and sizing column chromatography), centrifugation, differences in solubility, or any other standard protein purification technique. In this regard, U.S. Patent No. 7,538,195 is hereby incorporated by reference in its entirety.

[0251] The present disclosure also provides a method of producing the EpCAM antibodies, EpCAM-binding antibody fragments, or EpCAM-activatable antibodies disclosed herein by culturing cells under conditions that result in the expression of the antibody and / or EpCAM-activatable antibody, wherein the cells comprise a nucleic acid molecule encoding the antibody, antibody fragment, or activatable antibody. In some embodiments, the cells are Chinese hamster ovary (CHO) cells.

[0252] The present disclosure also provides a method for producing an EpCAM-activatable antibody that binds to EpCAM in an activated state by: (a) culturing a cell comprising a nucleic acid construct encoding an EpCAM-activatable antibody under conditions that result in the expression of the EpCAM-activatable antibody, wherein the EpCAM-activatable antibody comprises a masking moiety (MM), a cleavable moiety (CM), and an Ab (e.g., and an EpCAM antibody or an EpCAM-binding antibody fragment thereof), (i) the CM is a polypeptide that functions as a substrate for a protease, and (ii) when the EpCAM-activatable antibody is in an uncleaved state, the MM prevents specific binding of the Ab to EpCAM, and when in a cleaved state, the MM does not prevent specific binding of the Ab to EpCAM or the CM is disposed within the EpCAM-activatable antibody such that it does not compete, and (b) recovering the EpCAM-activatable antibody. Suitable Abs, MMs, and / or CMs include any of the Abs, MMs, and / or CMs disclosed herein. In some embodiments, the cell is a Chinese hamster ovary (CHO) cell.

[0253] 6.5 Immunoconjugates The EpCAM immunoconjugates disclosed herein include camptothecin and / or derivatives thereof. Camptothecin and its derivatives are disclosed in U.S. Patent Application Publication No. 2021 / 0077482A1 and U.S. Patent No. 11,229,639B2, the entireties of which are incorporated herein by reference.

[0254] Optionally, the EpCAM immunoconjugate is generated by conjugating at least one linker-payload reactant of a compound of Formula I, or a pharmaceutically acceptable salt thereof, to an EpCAM antibody described in Section 6.2 or an EpCAM-activatable antibody described in Section 6.3: E-A-Z’-L 1 -D (Formula I) Wherein, D is represented by the following structural formula, [Chemical formula] R 1 is F, R 2 is methyl, -L 1 -Z’-* is -(C1-C4 alkylene)-O-CH2-NR 8 -*, -(C1-C4 alkylene)-NR 8 -*, or -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-O-CH2-NR 8 -*, where * is the site covalently bonded to A, each R 5 is independently H, methyl, or benzyl, each R 8 is independently H, methyl, or benzyl, A is a peptide containing 2-4 amino acids, E is C(=O)-(C1-C 10 alkylene)-X 3 where X 3 is as follows. [Chemical formula]

[0255] In some cases, the linker-payload reactant is [Chemical formula] or a pharmaceutically acceptable salt thereof.

[0256] In some cases, the linker-payload reactant is [Chemical formula] or a pharmaceutically acceptable salt thereof.

[0257] When the linker-payload reactant comprises a compound of Formula I, or a pharmaceutically acceptable salt thereof, the maleimide group X in the EpCAM antibody or activatable antibody reacts with a thiol (-SH) group to form a covalent bond in the immunoconjugate as shown below 3 wherein * represents a sulfur-mediated thiol group attachment to the EpCAM antibody or activatable antibody.

Chemical formula

[0258] In some embodiments, the EpCAM antibody or activatable antibody is an IgG1 antibody having eight cysteines that are involved in the formation of four interchain disulfide bonds when in its native higher-order structure (e.g., not reduced). For exemplary identification of such cysteine residues, see, for example, SEQ ID NOs: 103, 140, and 179 in Tables 6, 7, and 11, respectively. In an IgG1 antibody, each light chain is linked to the heavy chain by one covalent disulfide bond, while the two heavy chains are linked together via two disulfide bonds. As provided herein, when these disulfide bonds are reduced, up to eight free cysteines and cysteine thiol groups can be obtained. One linker-payload reactant can react with the thiol group of one cysteine, and up to eight linker-payload reactants can react with each IgG1 EpCAM antibody or activatable antibody. In some embodiments, each EpCAM antibody or activatable antibody reacts with up to eight linker-payload reactants. 3 When the maleimide of group X in the linker-payload reactant reacts with the thiol of the EpCAM antibody or activatable antibody, the maleimide group is reduced to succinimide. Thus, the linker-payload contains succinimide when conjugated to the EpCAM antibody or activatable antibody.

[0259] In some embodiments, the EpCAM antibody or activatable antibody is an IgG1 antibody having eight cysteines that are involved in the formation of four interchain disulfide bonds when in its native higher-order structure (e.g., not reduced). For exemplary identification of such cysteine residues, see, for example, SEQ ID NOs: 103, 140, and 179 in Tables 6, 7, and 11, respectively. In an IgG1 antibody, each light chain is linked to the heavy chain by one covalent disulfide bond, while the two heavy chains are linked together via two disulfide bonds. As provided herein, when these disulfide bonds are reduced, up to eight free cysteines and cysteine thiol groups can be obtained. One linker-payload reactant can react with the thiol group of one cysteine, and up to eight linker-payload reactants can react with each IgG1 EpCAM antibody or activatable antibody. In some embodiments, each EpCAM antibody or activatable antibody reacts with up to eight linker-payload reactants.

[0260] In some cases, the EpCAM immunoconjugate comprises 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 before conjugation to the EpCAM antibody or activatable antibody, E-A-Z’-L 1 -D (Formula I) Wherein, D is represented by the following structural formula,

Chemical formula

Chemical formula

[0261] Thus, in some cases, the EpCAM immunoconjugate comprises at least one linker payload having the structure of formula (Ia) or a pharmaceutically acceptable salt thereof, E-A-Z’-L 1 -D (Formula (Ia)) Wherein, D is represented by the following structural formula,

Chemical formula

Chemical formula

[0262] In some cases, A of formula (I) or A of formula (Ia) is substituted with one or more polyols. In some cases, the polyol is -(C1-C6 alkylene)-X 5 -Y 3 wherein, X 5 is -NR12 C(=O)- or -C(=O)NR 12 - and Y 3 is C6 alkyl substituted with 5 OH groups, and R 12 is -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl.

[0263] In some cases, the polyol of the immunoconjugate comprises the following,

Chemical Structure

[0264] In some cases, the immunoconjugate is the following linker-payload conjugate (i.e., the linker-payload before conjugation to an EpCAM antibody or activatable antibody),

Chemical Structure

[0265] Thus, in some cases, the immunoconjugate is the following linker-payload,

Chemical Structure

[0266] In some cases, the immunoconjugate is the following linker-payload conjugate (i.e., the linker-payload before conjugation to an EpCAM antibody or activatable antibody),

Chemical Structure

[0267] Thus, in some cases, the immunoconjugate is the following linker-payload: [Chemical formula] or a pharmaceutically acceptable salt thereof.

[0268] In this specification, -L 1 -Z'-* in various disclosed embodiments, * is the site covalently bonded to A. In some embodiments, A is a peptide containing 2 to 8 amino acids. In some embodiments, A is a peptide containing 2 to 4 amino acids. In some embodiments, at least one amino acid in the above peptide is an L-amino acid. In some embodiments, each amino acid in the above peptide is an L-amino acid. In some embodiments, at least one amino acid in the above peptide is a D-amino acid. In some embodiments, A is -(AA 1 )-(AA 2 ) a1 -*, where * is the site covalently bonded to E, AA 1 and AA 2 are each independently an amino acid residue, and a1 is an integer from 1 to 9. In some embodiments, -AA 1- (AA 2 ) a1 -* is -Gly-Gly-Gly-*, -Ala-Val-*, -Val-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-N 9 -tosyl-Arg-*, -N U 9 -tosyl-Arg-Phe-*, -Phe-N 9 -nitro-Arg-*, -N 9-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-Val*, -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-Val*, -Val-Ser*, -Ser-Ala*, -Ser-Gly*, -Ala-Ser*, -Gly-Ser*, -Leu-Gln*, -Gln-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, -AA 1 -(AA 2 )a1-* 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-Val*. In some embodiments, -AA 1 -(AA 2)a1-* is -Ala-Ala-*, -Ala-Val-*, -Val-Ala-*, -Gln-Leu-*, -Leu-Gln-*, -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, -AA 1 -(AA 2 )a1-* 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-Leu-L-Gln-*, -L-Leu-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-Gly-*, -Gly-L-Phe-Gly-Gly-*, or Gly-Gly-L-Phe-Gly-*. In some embodiments, -AA 1 -(AA 2 ) a1 -* is -L-Ala-L-Ala-*, -L-Ala-D-Ala-LAla-*, -L-Ala-L-Ala-L-Ala-*, or -L-Ala-L-Ala-L-Ala-L-Ala-*.

[0269] In some embodiments, -AA 1 -(AA 2 ) a1 -* is -L-Ala-L-Ala-L-Ala-*.

[0270] As used herein, -AA 1 -(AA 2 )a1-*, in various embodiments disclosed herein, * is the site covalently attached to E.

[0271] In some embodiments, the EpCAM antibody or activatable antibody is an IgG1 antibody having eight cysteines that are involved in the formation of four disulfide bonds when in its native higher-order structure (e.g., not reduced). For exemplary identification of such cysteine residues, see, for example, SEQ ID NOs: 103, 140, and 179 in Tables 6, 7, and 11, respectively. As provided herein, when these disulfide bonds are reduced, eight free cysteines and cysteine thiol groups can be obtained. One linker-payload reactant can react with the thiol group of one cysteine, and up to eight linker-payload reactants can react with each EpCAM antibody or activatable antibody. In some embodiments, each EpCAM antibody or activatable antibody reacts with up to eight linker-payload reactants to yield a linker-payload to antibody ratio (i.e., drug to antibody ratio (DAR)) in the range of 2-8. In certain embodiments, the DAR represents the average number of cytotoxic linker-payload agents per antibody or activatable 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.

[0272] In some cases, the linker - payload to antibody ratio (i.e., drug - to - antibody ratio (DAR)) ranges from 2 to 12. In some cases, 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 activatable antibody molecule. In some embodiments, the DAR is 8.

[0273] In some embodiments, the present disclosure provides an EpCAM immunoconjugate comprising at least one linker - payload comprising a compound of the following formula, or a pharmaceutically acceptable salt thereof, Z - L 1 - D wherein D is represented by the following structural formula,

Chemical formula

[0274] Additional camptothecin derivatives are disclosed in U.S. Patent Application Publication No. 2021 / 0077482A1 and U.S. Patent No. 11,229,639B2, which are hereby incorporated by reference in their entireties.

[0275] In some embodiments, the linker payload of the immunoconjugate comprises exatecan or a derivative thereof. In some embodiments, the linker payload of the immunoconjugate comprises reduced dextecan, where the maleimide group of dextecan is reduced to succinimide during the conjugation reaction, whereby dextecan is linked to the EpCAM antibody or activatable antibody via a thiol group. Dextecan comprises an exatecan derivative and a maleimide-GGFG linker (SEQ ID NO: 311). Exatecan, its derivatives, and dextecan are described, for example, in International Publication No. WO 2015 / 115091, the entire contents of which are hereby incorporated by reference.

[0276] 6.6 Method for Preparing Immunoconjugate As described in the above embodiments, or in any particular embodiment described herein, an immunoconjugate comprising a cell cytotoxic agent covalently bound via an interchain cysteine residue located on an EpBA (an "EpBA", e.g., an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody) can be prepared according to any method known in the art. See, for example, International Publication No. WO 2020 / 219287, WO 2014 / 197612, WO 2012 / 128868, and WO 2012 / 112687, the entire contents of each of which are incorporated herein by reference.

[0277] A method for making an EpCAM-specific conjugate is disclosed in International Publication No. WO 2020 / 086665, the entire contents of which are incorporated herein by reference.

[0278] In some embodiments, the immunoconjugates of the present disclosure are prepared by a method comprising reacting an EpBA (e.g., an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody disclosed herein) with a cytotoxic agent of the present disclosure having a thiol-reactive group. In certain embodiments, the thiol-reactive group is maleimide. In certain embodiments, the thiol-reactive group of the cytotoxic agent (e.g., a linker payload of the present disclosure) reacts with the thiol group of a cysteine residue of the EpBA.

[0279] In certain embodiments, the thiol-reactive group of the cytotoxic agent (e.g., maleimide) is coupled to the thiol group (e.g., cysteine thiol) of the EpBA via a thiol-Michael addition.

[0280] In some embodiments, conjugation of the linker-payload reactant to the EpCAM-activatable antibody involves partial reduction of the activatable antibody. This typically forms disulfide bridges not only between the heavy chains of the activatable antibody but also between the light and heavy chains, exposing reactive native cysteines that do not interfere with one or more disulfide bonds that occur within the masked portion (MM) of the activatable antibody. Thus, the MM retains its ability to effectively mask the EpCAM antibody or its EpCAM-binding fragment. If the reduction and subsequent conjugation are not properly controlled, the activatable antibody is fully reduced and the masking efficiency of the activatable antibody is impaired.

[0281] In some embodiments, the activatable EpCAM antibody is an IgG1 antibody. In certain embodiments, the activatable EpCAM antibody is a humanized IgG1 antibody. Thus, in certain embodiments, the activatable EpCAM antibody has four disulfide bonds formed by eight cysteine residues. When the activatable antibody is partially reduced, these four disulfide bonds are reduced and the eight cysteines and their corresponding thiol groups become available to react with the thiol-reactive groups (e.g., maleimide) of the linker-payload.

[0282] Examples of reducing agents suitable for use in the partial reduction of the EpCAM-activatable antibody include, but are not limited to, BMS (bis(2-mercaptoethyl)sulfone), cysteamine, cysteine, DMH (dimethyl-bis-mercaptoacetylhydrazine), DTBA (dithiobutylamine), DTT (dithiothreitol), GILT (gamma interferon-inducible lysosomal thiol reductase; for enzymatic reduction), glutathione, β-mercaptoethanol, MEA (2-mercaptoethylamine), pyridine-2-thione, sodium borohydride, sodium thiophosphate, TCEP ((tris(2-carboxyethyl)phosphine)), and thiopropyl agarose. In some embodiments, the reducing agent is DTT, 3-mercaptoethanol, or TCEP.

[0283] In some embodiments, the reducing agent is TCEP. TCEP is often used as a reducing agent for cleaving disulfide bonds within and between proteins. TCEP is highly selective and does not react with other functional groups found within proteins, nor does it react with buried disulfide bonds. Compared to two other common reducing agents, DTT and β-mercaptoethanol, TCEP has the advantages of being a more powerful reducing agent, more hydrophilic, more resistant to oxidation in air, and odorless. Unlike DTT, TCEP is active under both alkaline and acidic conditions. TCEP is particularly useful when reacting cysteine residues with maleimide. TCEP can prevent cysteine from forming disulfide bonds, and unlike DTT and β-mercaptoethanol, TCEP does not readily react with maleimide.

[0284] In some embodiments, the ratio of the reducing agent (e.g., TCEP) to the EpCAM-activatable antibody is in the range of about 20:1 to 1:1, about 10:1 to 1:1, about 9:1 to 1:1, about 8:1 to 1:1, about 7:1 to 1:1, about 6:1 to 1:1, about 5:1 to 1:1, about 4:1 to 1:1, about 3:1 to 1:1, about 2:1 to 1:1, about 20:1 to 1:1.5, about 10:1 to 1:1.5, about 9:1 to 1:1.5, about 8:1 to 1:1.5, about 7:1 to 1:1.5, about 6:1 to 1:1.5, about 5:1 to 1:1.5, about 4:1 to 1:1.5, about 3:1 to 1:1.5, about 2:1 to 1:1.5, about 1.5:1 to 1:1.5, or about 1:1 to 1:1.5. In some embodiments, the ratio is in the range of about 5:1 to 1:1. In some embodiments, the ratio is in the range of about 5:1 to 1.5:1. In some embodiments, the ratio is in the range of about 4:1 to 1:1. In some embodiments, the ratio is in the range of about 4:1 to 1.5:1. In some embodiments, the ratio is in the range of about 8:1 to about 1:1. In some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

[0285] The maleimide functional group in the linker payload reactant reacts with the thiol (-SH) group on the activatable antibody to generate a thioester covalent bond. In some embodiments, the native cysteine on the activatable antibody contains a thiol (-SH) group. In some embodiments, the EpCAM activatable antibody is partially reduced with 4.5 molar equivalents of TCEP in solution at room temperature for about 3 hours. The partially reduced activatable antibody is reacted with 10 molar equivalents of the linker payload reactant containing DMAc as a co-solvent of up to about 8.3% (v / v) in an aqueous buffer solution at room temperature for about 1 hour. The reactant is then quenched with 20 molar equivalents of N-acetyl-L-cysteine (NAC) at room temperature for about 1 hour. After quenching, the resulting immunoconjugate can be purified to remove low molecular weight impurities and buffer exchanged into the base formulation buffer by UF / DF. In some embodiments, the base formulation buffer contains one or more pharmaceutically acceptable excipients. The base formulation buffer containing the immunoconjugate can then be sterile filtered into a container.

[0286] Exemplary methods for partially reducing an activatable antibody and conjugating a linker payload thereto are provided herein, for example, from the section "Preparation of huEpCAM23Gv4.2-LalaLalaLala-CPT66" to the section "Preparation of huEpCAM23Gv4.2-GMBS-DM21L" of Example 4.

[0287] In some embodiments, the conjugation methods provided herein using an EpCAM antibody or activatable antibody and a linker payload reactant result in a substantially homogeneous immunoconjugate having 8 linker payloads conjugated to each antibody or activatable antibody. In immunoconjugates prepared using the methods and materials provided herein, the number of linker payloads conjugated to each antibody or activatable antibody is mostly 8. This is in contrast to huEpCAM23Gv4.2-GMBS-DM21L which had a DAR of 3.5 - 5 (see, e.g., the section "Preparation of huEpCAM23Gv4.2-GMBS-DM21L" in Example 4). The consistently high DAR of 8 for the immunoconjugates described herein results in a potent bystander effect.

[0288] An exemplary method for preparing the immunoconjugates of the present disclosure is shown in the section "Preparation of huEpCAM23Gv4.2-LalaLalaLala-CPT66" in Example 4.

[0289] In certain embodiments, the immunoconjugate is prepared by a method comprising reacting an EpBA (e.g., an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM activatable antibody disclosed herein) with a cytotoxic agent having an amine-reactive group.

[0290] In some embodiments, the reaction is carried out in the presence of an imine-reactive reagent such as NaHSO3.

[0291] In certain embodiments, the immunoconjugate is (a) reacting a cytotoxic agent with a linker compound having an amine-reactive group and a thiol-reactive group to form a cytotoxic agent linker compound (i.e., a linker payload reactant) to which the amine-reactive group is attached; and (b) reacting the EpBA with the cytotoxic agent linker compound.

[0292] In one embodiment, the reaction of step (a) is carried out in the presence of an imine-reactive reagent (e.g., NaHSO3). In one embodiment, the cytotoxic agent linker compound is reacted with EpBA without purification. Alternatively, the cytotoxic agent linker compound (i.e., the linker payload reactant) is first purified and then reacted with EpBA.

[0293] In certain embodiments, an immunoconjugate of one embodiment is (a) reacting EpBA with a linker compound having an amine-reactive group and a thiol-reactive group to form a modified EpBA to which the thiol-reactive group is attached; and (b) reacting the modified EpBA with a cytotoxic agent. In one embodiment, the reaction of step (b) is carried out in the presence of an imine-reactive reagent (e.g., NaHSO3).

[0294] In certain embodiments, the immunoconjugate is prepared by a method comprising reacting EpBA, a cytotoxic compound, and a linker compound having an amine-reactive group and a thiol-reactive group. In one embodiment, this reaction is carried out in the presence of an imine-reactive agent (e.g., NaHSO3).

[0295] In a first aspect of the above method, the reaction of step (a) is carried out at a pH of 1.9 to 5.0. More specifically, the pH is 2.5 to 4.9, 1.9 to 4.8, 2.0 to 4.8, 2.5 to 4.5, 2.9 to 4.5, 2.9 to 4.0, 2.9 to 3.7, 3.1 to 3.5, or 3.2 to 3.4. In another particular embodiment, the reaction of step (a) is carried out at a pH of 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0. In yet another particular embodiment, the reaction of step (a) is carried out at a pH of 3.3. As used herein, a particular pH value means the particular value ±0.05.

[0296] In some embodiments, the reaction of step (a) is carried out in the presence of a buffer. Any suitable buffer known in the art can be used in the provided method. Suitable buffers include, for example, citrate buffer, acetate buffer, succinate buffer, phosphate buffer, glycine-containing buffer (e.g., glycine-HCl buffer), phthalate buffer (e.g., buffer containing sodium hydrogen phthalate or potassium hydrogen phthalate), and combinations thereof, but are not limited thereto. In some embodiments, the buffer is a succinate buffer. In some embodiments, the buffer is a phosphate buffer. In some embodiments, the buffer is a citrate-phosphate buffer. In some embodiments, the buffer is a citrate-phosphate buffer containing citrate and Na2HPO4. In other embodiments, the buffer is a citrate-phosphate buffer containing citrate and K2HPO4. In some embodiments, the concentration of the above buffer can range from 10 to 250 mM, 10 to 200 mM, 10 to 150 mM, 10 to 100 mM, 25 to 100 mM, 25 to 75 mM, 10 to 50 mM, or 20 to 50 mM.

[0297] In a second aspect, the reaction step (a) is carried out in the absence of a buffer (e.g., the buffer described in the first aspect). In some embodiments, the method of the present invention comprises (a) reacting the imine moiety in an imine-containing cytotoxic agent having the above thiol-reactive group (i.e., formula (C1a'), (C1a'1), (C1b'), (C1b'1), (C2a"), (C2a"1), (C2b"), or (C2b"1) (wherein the double line between N and C represents a double bond, X is absent, and Y is -H)) with sulfur dioxide, bisulfite, or metabisulfite in an aqueous solution to obtain the following formula:

Chemical formula

[0298] In a third aspect, in the case of the above method or the method of the first or second aspect, in the reaction of step (a), 0.5 to 5.0 equivalents of bisulfite or 0.25 or 2.5 equivalents of metabisulfite are used per equivalent of the imine-containing cytotoxic agent. In some embodiments, 0.5 to 4.5, 0.5 to 4.0, 0.5 to 3.5, 0.5 to 4.0, 0.5 to 3.5, 0.5 to 3.0, 0.5 to 2.5, 0.8 to 2.0, 0.9 to 1.8, 1.0 to 1.7, 1.1 to 1.6, or 1.2 to 1.5 equivalents of bisulfite, or 0.25 to 2.25, 0.25 to 2.0, 0.25 to 1.75, 0.25 to 2.0, 0.25 to 1.75, 0.25 to 1.5, 0.25 to 1.25, 0.4 to 1.0, 0.45 to 0.9, 0.5 to 0.85, 0.55 to 0.8, or 0.6 to 0.75 equivalents of metabisulfite are used per equivalent of the imine-containing cytotoxic agent. In other embodiments, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 4.0, 4.5 or 5.0 equivalents of bisulfite, or 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 2.0, 2.25 or 2.5 equivalents of metabisulfite are used per equivalent of the imine-containing cytotoxic agent. In still other embodiments, 1.4 equivalents of bisulfite or 0.7 equivalents of metabisulfite are used per equivalent of the imine-containing cytotoxic agent. In other embodiments, 1.2 equivalents of bisulfite or 0.6 equivalents of metabisulfite are used per equivalent of the imine-containing cytotoxic agent. As used herein, a particular equivalent means a particular value ± 0.05.

[0299] In the fourth aspect, in the case of the above method, the reaction in step (a) is carried out at a pH of 2.9 to 3.7, and 1.0 to 1.8 equivalents of bisulfite or 0.5 to 0.9 equivalents of metabisulfite are reacted with 1 equivalent of the imine-containing cytotoxic agent. In some embodiments, the reaction in step (a) is carried out at a pH of 3.1 to 3.5, and 1.1 to 1.6 equivalents of bisulfite or 0.55 to 0.8 equivalents of metabisulfite are reacted with 1 equivalent of the imine-containing cytotoxic agent. In other embodiments, the reaction in step (a) is carried out at a pH of 3.2 to 3.4, and 1.3 to 1.5 equivalents of bisulfite or 0.65 to 0.75 equivalents of metabisulfite are reacted with 1 equivalent of the imine-containing cytotoxic agent. In other embodiments, the reaction in step (a) is carried out at a pH of 3.3, and 1.4 equivalents of bisulfite or 0.7 equivalents of metabisulfite are reacted with 1 equivalent of the imine-containing cytotoxic agent. In still other embodiments, the reaction in step (a) is carried out at a pH of 3.3, and 1.4 equivalents of sodium bisulfite are reacted with 1 equivalent of the imine-containing cytotoxic agent.

[0300] In a fifth aspect, in the above method, or in the method of the first, second, third, or fourth aspect, the reaction in step (a) is carried out in a mixture of an organic solvent and water. Any suitable organic solvent can be used. Exemplary organic solvents include, but are not limited to, alcohols (e.g., methanol, ethanol, propanol, etc.), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile, acetone, methylene chloride, etc. In some embodiments, the organic solvent is miscible with water. In other embodiments, the organic solvent is immiscible with water, i.e., the reaction in step (a) is carried out in a two-phase solution. In some embodiments, the organic solvent is dimethylacetamide (DMA). The organic solvent (e.g., DMA) may be present in an amount of 1% to 99% by volume, 1 to 95% by volume, 10 to 80% by volume, 20 to 70% by volume, 30 to 70% by volume, 1 to 60% by volume, 5 to 60% by volume, 10 to 60% by volume, 20 to 60% by volume, 30 to 60% by volume, 40 to 60% by volume, 45 to 55% by volume, 10 to 50% by volume, or 20 to 40% by volume based on the total volume of water and the organic solvent. In some embodiments, the reaction in step (a) is carried out in a mixture of DMA and water, and the volume ratio of DMA to water is 1:1.

[0301] In a sixth aspect, in the above method, or in the method of the first, second, third, fourth, or fifth aspect, the reaction in step (a) can be carried out at any suitable temperature. In some embodiments, the reaction is carried out at a temperature of 0°C to 50°C, 10°C to 50°C, 10°C to 40°C, or 10°C to 30°C. In other embodiments, the reaction is carried out at a temperature of 15°C to 30°C, 20°C to 30°C, 15°C to 25°C, 16°C to 24°C, 17°C to 23°C, 18°C to 22°C, or 19°C to 21°C. In still other embodiments, the reaction can be carried out at a temperature of 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C. In some embodiments, the reaction can be carried out at a temperature of 0°C to 15°C, 0°C to 10°C, 1°C to 10°C, 5°C to 15°C, or 5°C to 10°C.

[0302] In a seventh aspect, in the case of the above method or the method of the first, second, third, fourth, fifth, or sixth aspect, the reaction in step (a) is carried out for 1 minute to 48 hours, 5 minutes to 36 hours, 10 minutes to 24 hours, 30 minutes to 24 hours, 30 minutes to 20 hours, 1 hour to 20 hours, 1 hour to 15 hours, 1 hour to 10 hours, 2 hours to 10 hours, 3 hours to 9 hours, 3 hours to 8 hours, 4 hours to 6 hours, or 1 hour to 4 hours. In some embodiments, the reaction is allowed to proceed for 4 hours to 6 hours. In other embodiments, the reaction is allowed to proceed for 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, etc. In other embodiments, the reaction is allowed to proceed for 4 hours. In still other embodiments, the reaction is allowed to proceed for 2 hours.

[0303] In an eighth aspect, in the case of the method disclosed herein or the method of the first, second, third, fourth, fifth, sixth, or seventh aspect, the reaction in step (b) is carried out at a pH of 4 to 9. In some embodiments, the reaction in step (b) is carried out at a pH of 4.5 to 8.5, 5 to 8.5, 5 to 8, 5 to 7.5, 5 to 7, 5 to 6.5, or 5.5 to 6.5. In other embodiments, the reaction in step (b) is carried out at a pH of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0304] In some embodiments, for the above-described method, or the method of the first, second, third, fourth, fifth, sixth, seventh, or eighth aspect, the reaction of step (b) is carried out in an aqueous solution containing a mixture of water and an organic solvent. Any suitable organic solvent as described above can be used. More specifically, the organic solvent is DMA. In some embodiments, the aqueous solution contains less than 50% by volume, less than 40% by volume, less than 30% by volume, less than 25% by volume, less than 20% by volume, less than 15% by volume, less than 10% by volume, less than 5% by volume, less than 3% by volume, less than 2% by volume, or less than 1% by volume of an organic solvent (e.g., DMA).

[0305] In some embodiments, for the method disclosed herein, or the method of the first, second, third, fourth, fifth, sixth, seventh, or eighth aspect, the bisulfite is sodium bisulfite or potassium bisulfite, and the metabisulfite is sodium metabisulfite or potassium metabisulfite. In certain embodiments, the bisulfite is sodium bisulfite and the metabisulfite is sodium metabisulfite.

[0306] In some embodiments, for the method disclosed herein, or the method of the first, second, third, fourth, fifth, sixth, seventh, or eighth aspect, the modified cytotoxic agent is not purified before reacting with the cell-binding agent in step (b). Alternatively, the modified cytotoxic agent is purified before reacting with the cell-binding agent in step (b). The modified cytotoxic agent can be purified using any suitable method disclosed herein.

[0307] In some embodiments, in the case of the above method, the reaction of step (a) does not substantially result in the sulfonation of the maleimide group. In some embodiments, less than 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the maleimide groups are sulfonated. The percentage of sulfonation of maleimide is equal to the total amount of maleimide-sulfonated cytotoxic agent (cytotoxic agent having sulfonation only on the maleimide) and disulfonated cytotoxic agent (cytotoxic agent having sulfonation on both the maleimide and imine moieties), divided by the starting amount of the imine-containing cytotoxic agent, prior to reacting with bisulfite or metabisulfite.

[0308] In some embodiments, the immunoconjugate prepared by any of the above methods is subjected to a purification step. In this regard, the immunoconjugate can be purified from other components of the mixture using tangential flow filtration (TFF), non-adsorptive chromatography, adsorptive chromatography, adsorptive filtration, selective precipitation, or any other suitable purification method, as well as combinations thereof.

[0309] In some embodiments, the immunoconjugate is purified using a single purification step (e.g., TFF). In some embodiments, the conjugate is purified using a single purification step (e.g., TFF) and exchanged into a suitable formulation. In other embodiments, the immunoconjugate is purified using two consecutive purification steps. For example, the immunoconjugate can first be purified by selective precipitation, adsorptive filtration, absorptive chromatography, or non-absorptive chromatography, followed by purification with TFF. Those skilled in the art will understand that purification of the immunoconjugate enables the isolation of a stable conjugate containing a cell-binding agent chemically coupled to a cytotoxic agent.

[0310] Any suitable TFF system, including the Pellicon type system (Millipore, Billerica, Mass.), the Sartocon Cassette system (Sartorius AG, Edgewood, N.Y.), and the Centrasette type system (Pall Corp., East Hills, N.Y.), can be used for purification.

[0311] Any suitable adsorption chromatography resin can be used for purification. Specific adsorption chromatography resins include hydroxyapatite chromatography, hydrophobic charge induction chromatography (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, mixed mode ion exchange chromatography, immobilized metal affinity chromatography (IMAC), dye ligand chromatography, affinity chromatography, reverse phase chromatography, and combinations thereof. Examples of suitable hydroxyapatite resins include ceramic hydroxyapatite (CHT types I and II, Bio-Rad Laboratories, Hercules, Calif.), HA Ultrogel hydroxyapatite (Pall Corp., East Hills, N.Y.), and ceramic fluoroapatite (CFT types I and II, Bio-Rad Laboratories, Hercules, Calif.). An example of a suitable HCIC resin is the MEP Hypercel resin (Pall Corp., East Hills, N.Y.). Examples of suitable HIC resins include butyl-Sepharose, hexyl-Sepharose, phenyl-Sepharose, and octyl-Sepharose resins (all from GE Healthcare, Piscataway, N.J.), as well as Macro-prep Methyl and Macro-Prep t-Butyl resins (Biorad Laboratories, Hercules, Calif.). Examples of suitable ion exchange resins include SP-Sepharose, CM-Sepharose, and Q-Sepharose resins (all from GE Healthcare, Piscataway, N.J.), as well as Unosphere S resin (Bio-Rad Laboratories, Hercules, Calif.). An example of a suitable mixed mode ion exchanger is the Bakerbond CBAx resin (JT Baker, Phillipsburg N.J.).Examples of suitable IMAC resins include Chelating Sepharose resin (GE Healthcare, Piscataway, N.J.) and Profinity IMAC resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable dye ligand resins include Blue Sepharose resin (GE Healthcare, Piscataway, N.J.) and Affi-gel Blue resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable affinity resins include Protein A Sepharose resin (e.g., MabSelect, GE Healthcare, Piscataway, N.J.) (where the cell binding agent is an antibody), and lectin affinity resins, e.g., Lentil Lectin Sepharose resin (GE Healthcare, Piscataway, N.J.) (where the cell binding agent has a suitable lectin binding site). Alternatively, an antibody specific for the cell binding agent may be used. Such an antibody can be immobilized, for example, on Sepharose 4 Fast Flow resin (GE Healthcare, Piscataway, N.J.). Examples of suitable reverse phase resins include C4, C8, and C18 resins (Grace Vydac, Hesperia, Calif.).

[0312] Any suitable non-adsorbing chromatography resin can be utilized for purification. Examples of suitable non-adsorbing chromatography resins include SEPHADEX™ G-25, G-50, G-100, SEPHACRYL™ resins (e.g., S-200 and S-300), SUPERDEX™ resins (e.g., SUPERDEX™ 75 and SUPERDEX™ 200), BIO-GEL® resins (e.g., P-6, P-10, P-30, P-60, and P-100), and other resins known to those of skill in the art, but are not limited thereto.

[0313] An immunoconjugate comprising EpBA covalently bound to a maytansinoid compound described in the third embodiment of this specification can be prepared according to any suitable method known in the art.

[0314] In some embodiments, the immunoconjugate prepared by any of the methods described above is subjected to a purification process. In this regard, the immunoconjugate can be purified from other components of the mixture using tangential flow filtration (TFF), non-adsorptive chromatography, adsorptive chromatography, adsorption filtration, selective precipitation, or any other suitable purification method, as well as combinations thereof.

[0315] In some embodiments, the immunoconjugate is purified using a single purification step (e.g., TFF). In some embodiments, the conjugate is purified using a single purification step (e.g., TFF) and exchanged into a suitable formulation. In other embodiments of the present invention, the immunoconjugate is purified using two consecutive purification steps. For example, the immunoconjugate can first be purified by selective precipitation, adsorption filtration, absorption chromatography, or non-absorption chromatography, and subsequently purified by TFF. Those skilled in the art will understand that purification of the immunoconjugate enables isolation of a stable conjugate comprising a cell-binding agent chemically coupled to a cytotoxic agent.

[0316] Any suitable TFF system, including the Pellicon type system (Millipore, Billerica, Mass.), the Sartocon Cassette system (Sartorius AG, Edgewood, N.Y.), and the Centrasette type system (Pall Corp., East Hills, N.Y.), can be utilized for purification.

[0317] Any suitable adsorption chromatography resin can be used for purification. Examples of specific adsorption chromatography resins include hydroxyapatite chromatography, hydrophobic charge induction chromatography (HCIC), hydrophobic interaction chromatography (HIC), ion exchange chromatography, mixed-mode ion exchange chromatography, immobilized metal affinity chromatography (IMAC), dye ligand chromatography, affinity chromatography, reverse phase chromatography, and combinations thereof. Examples of suitable hydroxyapatite resins include ceramic hydroxyapatite (CHT types I and II, Bio-Rad Laboratories, Hercules, Calif.), HA Ultrogel hydroxyapatite (Pall Corp., East Hills, N.Y.), and ceramic fluoroapatite (CFT types I and II, Bio-Rad Laboratories, Hercules, Calif.). An example of a suitable HCIC resin is the MEP Hypercel resin (Pall Corp., East Hills, N.Y.). Examples of suitable HIC resins include butyl-Sepharose, hexyl-Sepharose, phenyl-Sepharose, and octyl-Sepharose resins (all from GE Healthcare, Piscataway, N.J.), as well as Macro-prep Methyl and Macro-Prep t-Butyl resins (Biorad Laboratories, Hercules, Calif.). Examples of suitable ion exchange resins include SP-Sepharose, CM-Sepharose, and Q-Sepharose resins (all from GE Healthcare, Piscataway, N.J.), as well as Unosphere S resin (Bio-Rad Laboratories, Hercules, Calif.). An example of a suitable mixed-mode ion exchanger is the Bakerbond ABx resin (JT Baker, Phillipsburg N.J.).Examples of suitable IMAC resins include Chelating Sepharose resin (GE Healthcare, Piscataway, N.J.) and Profinity IMAC resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable dye ligand resins include Blue Sepharose resin (GE Healthcare, Piscataway, N.J.) and Affi-gel Blue resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable affinity resins include Protein A Sepharose resin (e.g., MabSelect, GE Healthcare, Piscataway, N.J.) (where the cell binder is an antibody), and lectin affinity resins, e.g., Lentil Lectin Sepharose resin (GE Healthcare, Piscataway, N.J.) (where the cell binder has a suitable lectin binding site). Alternatively, an antibody specific for the cell binder may be used. Such an antibody can be immobilized, for example, on Sepharose 4 Fast Flow resin (GE Healthcare, Piscataway, N.J.). Examples of suitable reverse phase resins include C4, C8, and C18 resins (Grace Vydac, Hesperia, Calif.).

[0318] Any suitable non-adsorbing chromatography resin can be utilized for purification. Examples of suitable non-adsorbing chromatography resins include SEPHADEX™ G-25, G-50, G-100, SEPHACRYL™ resins (e.g., S-200 and S-300), SUPERDEX™ resins (e.g., SUPERDEX™ 75 and SUPERDEX™ 200), BIO-GEL® resins (e.g., P-6, P-10, P-30, P-60, and P-100), and other resins known to those of skill in the art, but are not limited thereto.

[0319] 6.6.1 Immunoconjugates Produced by the Provided Method This specification also provides immunoconjugates produced by the methods described herein.

[0320] In some embodiments, (a) an activatable EpCAM antibody consisting of a heterotetramer of two heavy chains having the amino acid sequence of SEQ ID NO: 103 and two light chains comprising the amino acid sequence of SEQ ID NO: 179 is mixed with a mixture of an EPPS buffer and an EDTA solution to form an activatable EpCAM reaction mixture; (b) a TCEP solution is added to the activatable EpCAM reaction mixture to form a reduced activatable EpCAM reaction mixture; (c) the reduced activatable EpCAM reaction mixture is mixed with a stock solution of a camptothecin linker toxin comprising a linker toxin having the following structure;

Chemical formula

[0321] In some embodiments, an immunoconjugate obtainable by the method described in the section "Preparation of huEpCAM23Gv4.2-LalaLalaLala-CPT66" of Example 4 is provided herein.

[0322] 6.7 Therapeutic Applications Also included are methods for inhibiting the growth of cells expressing EpCAM (e.g., human EpCAM or cynomolgus monkey EpCAM). As provided herein, the EpCAM immunoconjugates of the disclosure bind to EpCAM present on the surface of cells (e.g., human cells or cynomolgus monkey cells) and have the ability to mediate cell killing. In certain embodiments, the immunoconjugate comprises a cytotoxic payload, e.g., a camptothecin derivative linker payload, is internalized, and mediates cell killing through the activity of the cytotoxic payload. Such cell killing activity can be enhanced by immunoconjugates that induce antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0323] As used herein, the terms "inhibit" and "inhibiting" include any inhibitory effect on cell growth, including cell death. Inhibitory effects include transient effects, persistent effects, and permanent effects.

[0324] The therapeutic applications provided herein include methods of treating a subject having a disease. The diseases treated by the provided methods are diseases characterized by the expression of EpCAM (e.g., overexpression of EpCAM). Such diseases include, for example, breast cancer, lung cancer, non-small cell lung cancer, stomach cancer, prostate cancer, ovarian cancer, colorectal cancer, colon cancer, esophageal cancer, tracheal cancer, gastric cancer, bladder cancer, uterine cancer, rectal cancer, or cancer of the small intestine, pancreatic cancer, head and neck cancer, endometrial cancer, epithelial cancer, or metastases associated therewith. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is breast cancer. One of ordinary skill in the art will understand that the methods of the disclosure can also be used to treat other diseases not yet described but characterized by the expression (e.g., overexpression) of EpCAM.

[0325] The EpCAM immunoconjugates of the present disclosure are useful for the treatment of cancers expressing EpCAM. In some embodiments, the cancer is an epithelial cancer or a squamous cell carcinoma. In some embodiments, the cancer is breast cancer, lung cancer, non-small cell lung cancer, stomach cancer, prostate cancer, ovarian cancer, colorectal cancer, colon cancer, esophageal cancer, tracheal cancer, gastric cancer, bladder cancer, uterine cancer, rectal cancer or small intestine cancer, pancreatic cancer, head and neck cancer, endometrial cancer, or metastases associated therewith.

[0326] The therapeutic applications provided herein can also be carried out in vitro and ex vivo.

[0327] The present disclosure also provides therapeutic applications of the EpCAM immunoconjugates of the present disclosure, wherein the antibody, antibody fragment, activatable antibody, or conjugate is administered to a subject in a pharmaceutically acceptable dosage form. These can be administered intravenously as a bolus or by continuous infusion over a period of time, or can be administered intramuscularly, subcutaneously, parenterally, intra-articularly, intrasynovially, intrathecally, orally, topically, or by the inhalation route. These can also be administered by the intratumoral, peritumoral, intralesional, or perilesional route in order to exert not only local but also systemic therapeutic effects.

[0328] Also provided is a method for the treatment, prevention, and / or delay or mitigation of the onset or progression of symptoms associated with abnormal expression and / or activity of EpCAM in a subject, using an EpCAM immunoconjugate that binds and neutralizes or otherwise inhibits at least one biological activity of EpCAM and / or EpCAM-mediated signal transduction.

[0329] In some embodiments, the present disclosure provides a method for treating, preventing, and / or delaying the onset or progression, or alleviating symptoms associated with the presence, growth, proliferation, metastasis, and / or activity of cells that express or aberrantly express EpCAM in a subject, using an EpCAM immunoconjugate that binds, targets, neutralizes, kills, or otherwise inhibits at least one biological activity of cells that express EpCAM. The present disclosure also provides a method for treating, preventing, and / or delaying the onset or progression, or alleviating symptoms associated with the presence, growth, proliferation, metastasis, and / or activity of cells that express EpCAM in a subject, using an EpCAM immunoconjugate that binds, targets, neutralizes, kills, or otherwise inhibits at least one biological activity of cells that express EpCAM.

[0330] The present disclosure also provides a method for treating, preventing, and / or delaying the onset or progression, or alleviating symptoms associated with the presence, growth, proliferation, metastasis, and / or activity of cells that aberrantly express EpCAM in a subject, using an EpCAM immunoconjugate that binds, targets, neutralizes, kills, or otherwise inhibits at least one biological activity of cells that aberrantly express EpCAM.

[0331] The present disclosure provides a method for preventing, delaying the progression of, treating, alleviating, or otherwise remitting an EpCAM-mediated disease in a subject by administering a therapeutically effective amount of an EpCAM immunoconjugate disclosed herein to a subject in need thereof.

[0332] The present disclosure also provides a method for preventing, delaying the progression of, treating, alleviating, or otherwise remitting cancer (e.g., epithelial cancer and its metastasis) in a subject by administering a therapeutically effective amount of an EpCAM immunoconjugate disclosed herein to a subject in need thereof. EpCAM is known to be expressed in various cancers, including most cancers of epithelial origin (and metastases).

[0333] In some embodiments, the cancer is an epithelial cancer or a squamous cell cancer.

[0334] In some embodiments, the cancer is a head and neck cancer, breast cancer, lung cancer, non-small cell lung cancer, stomach cancer, prostate cancer, ovarian cancer, colorectal cancer, colon cancer, esophageal cancer, tracheal cancer, gastric cancer, bladder cancer, uterine cancer, endometrial cancer, rectal cancer, pancreatic cancer, or cancer of the small intestine.

[0335] In some embodiments, the cancer is breast cancer, lung cancer, stomach cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the small intestine, ovarian cancer, gastric cancer, or esophageal cancer.

[0336] In some embodiments, the cancer is ovarian cancer, uterine cancer, gastric cancer, pancreatic cancer, or colorectal cancer.

[0337] [[ID=1,7]] In some embodiments, the cancer is ovarian cancer.

[0338] In some embodiments, the cancer is uterine cancer.

[0339] In some embodiments, the cancer is gastric cancer.

[0340] In some embodiments, the cancer is pancreatic cancer.

[0341] In some embodiments, the cancer is colorectal cancer.

[0342] In some embodiments, the cancer is a head and neck cancer.

[0343] In some embodiments, the cancer is breast cancer. In certain embodiments, the cancer is triple-negative breast cancer.

[0344] In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the non-small cell lung cancer is non-squamous non-small cell lung cancer.

[0345] The EpCAM immunoconjugates used in any of these methods and use embodiments can be administered at any stage of the disease. For example, such EpCAM immunoconjugates can be administered to patients suffering from cancer at any stage from early stage to metastasis. The terms subject and patient are used interchangeably herein.

[0346] In some embodiments, the subject is a mammal such as a human, non-human primate, companion animal (e.g., cat, dog, horse), livestock, working animal, or zoo animal. In some embodiments, the subject is a human. In some embodiments, the subject is a companion animal. In some embodiments, the subject is an animal under the care of a veterinarian.

[0347] The EpCAM immunoconjugates and their therapeutic formulations are administered to subjects who have or are susceptible to diseases or disorders associated with abnormal EpCAM expression and / or activity, such as cancer. Subjects who have or are susceptible to diseases or disorders associated with abnormal EpCAM expression and / or activity are identified using any of a variety of methods known in the art. For example, subjects suffering from cancer or other neoplastic conditions are identified using a physical examination to assess their health status, as well as any of a variety of clinical and / or laboratory tests such as blood, urine, and / or stool analysis. For example, subjects suffering from inflammation and / or inflammatory disorders are identified using a physical examination to assess their health status and / or any of a variety of clinical and / or laboratory tests such as body fluid analysis (e.g., analysis of blood, urine, and / or stool).

[0348] The administration of an EpCAM immunoconjugate to a patient suffering from a disease or disorder associated with abnormal EpCAM expression and / or activity (such as cancer, e.g., carcinoma) is considered successful if any of various laboratory or clinical objectives are achieved. For example, the administration of an EpCAM immunoconjugate to a patient suffering from a disease or disorder associated with abnormal EpCAM expression and / or activity is considered successful if one or more of the symptoms associated with the disease or disorder are alleviated, reduced, inhibited, or, further, i.e., do not progress to a worse state. The administration of an EpCAM immunoconjugate to a patient suffering from a disease or disorder associated with abnormal EpCAM expression and / or activity is considered successful if the disease or disorder goes into remission or, further, i.e., does not progress to a worse state.

[0349] In some embodiments, the EpCAM immunoconjugate and its therapeutic formulations are administered to a subject suffering from or susceptible to a disease or disorder, e.g., having cancer or another neoplastic condition, the disease cells of which express EpCAM. In some embodiments, the disease cells are associated with abnormal EpCAM expression and / or activity. In some embodiments, the disease cells are associated with normal EpCAM expression and / or activity. A subject suffering from or susceptible to a disease or disorder in which the disease cells of the subject express EpCAM is identified using any of a variety of methods known in the art. For example, a subject having cancer or another neoplastic condition is identified using either a physical examination to assess health status and / or any of a variety of clinical and / or laboratory tests such as blood, urine, and / or stool analysis. For example, a subject having an inflammation and / or inflammatory disorder is identified using either a physical examination to assess health status and / or a body fluid analysis (e.g., analysis of blood, urine, and / or stool) and / or any of a variety of clinical and / or laboratory tests.

[0350] In some embodiments, the EpCAM immunoconjugate and its therapeutic formulations are administered to a subject suffering from or susceptible to a disease or disorder associated with cells expressing EpCAM, or the presence, growth, proliferation, metastasis, and / or activity of such cells, e.g., a subject suffering from cancer or other neoplastic conditions. In some embodiments, the cells are associated with abnormal EpCAM expression and / or activity. In some embodiments, the cells are associated with normal EpCAM expression and / or activity. Subjects suffering from or susceptible to a disease or disorder associated with cells expressing EpCAM are identified using any of a variety of methods known in the art. For example, a subject suffering from cancer or other neoplastic conditions is identified using any of a physical examination to assess health status, as well as various clinical and / or laboratory tests such as blood, urine, and / or stool analysis. For example, a subject suffering from inflammation and / or an inflammatory disorder is identified using any of a physical examination to assess health status and / or body fluid analysis (e.g., analysis of blood, urine, and / or stool) and other various clinical and / or laboratory tests.

[0351] Administration of an EpCAM immunoconjugate to a patient suffering from a disease or disorder associated with cells expressing EpCAM (such as cancer, e.g., carcinoma) is considered successful if any of a variety of laboratory or clinical objectives are achieved. For example, administration of an EpCAM immunoconjugate to a patient suffering from a disease or disorder associated with cells expressing EpCAM is considered successful if one or more of the symptoms associated with the disease or disorder are alleviated, reduced, inhibited, or, further, i.e., do not progress to a worse condition. Administration of an EpCAM immunoconjugate to a patient suffering from a disease or disorder associated with cells expressing EpCAM is considered successful if the disease or disorder goes into remission or, further, i.e., does not progress to a worse condition.

[0352] The present disclosure provides conjugated EpCAM antibodies useful in methods of treating, preventing, delaying progression, inducing remission, and / or alleviating symptoms of diseases or disorders associated with abnormal EpCAM expression and / or activity. For example, conjugated EpCAM activatable antibodies are used in methods of treating, preventing, delaying progression, inducing remission, and / or alleviating symptoms of cancer or other neoplastic conditions.

[0353] The present disclosure provides conjugated EpCAM antibodies useful in methods of treating, preventing, delaying progression, inducing remission, and / or alleviating symptoms of diseases or disorders associated with cells that express EpCAM. In some embodiments, the cells are associated with abnormal EpCAM expression and / or activity. In some embodiments, the cells are associated with normal EpCAM expression and / or activity. For example, EpCAM activatable antibodies can be used in methods of treating, preventing, delaying progression, inducing remission, and / or alleviating symptoms of cancer or other neoplastic conditions.

[0354] The present disclosure provides EpCAM antibodies and antibody fragments, and / or conjugated EpCAM activatable antibodies useful in methods of treating, preventing, delaying progression, inducing remission, and / or alleviating symptoms of diseases or disorders in which diseased cells express EpCAM. In some embodiments, the diseased cells are associated with abnormal EpCAM expression and / or activity. In some embodiments, the diseased cells are associated with normal EpCAM expression and / or activity. For example, EpCAM conjugated activatable antibodies are used in methods of treating, preventing, delaying progression, inducing remission, and / or alleviating symptoms of cancer or other neoplastic conditions.

[0355] In some embodiments, the conjugated EpCAM antibodies or activatable antibodies provided herein have an excellent therapeutic index. In certain embodiments, the conjugated EpCAM antibodies or activatable antibodies have a therapeutic index of about 3 to about 6. In some embodiments, the conjugated EpCAM antibodies or activatable antibodies have a therapeutic index of about 3. In some embodiments, the conjugated EpCAM antibodies or activatable antibodies have a therapeutic index of about 4. In some embodiments, the conjugated EpCAM antibodies or activatable antibodies have a therapeutic index of about 5. In some embodiments, the conjugated EpCAM antibodies or activatable antibodies have a therapeutic index of about 6. The therapeutic index of the conjugated EpCAM antibodies or activatable antibodies described herein is significantly improved compared to that of other conjugated EpCAM antibodies or activatable antibodies. For example, huEpCAM23Gv4.2-GMBS-DM21L (see, e.g., the section "Preparation of huEpCAM23Gv4.2-GMBS-DM21L" in Example 4) has a therapeutic index of about 1.

[0356] The EpCAM antibodies or activatable antibodies provided herein have relatively low systemic toxicity. In cynomolgus monkeys, for example, EpCAM-CPT66 was tolerated up to 60 mg / kg (see Example 7.7). This is an immunoconjugate that contains the same EpCAM-activatable antibody as EpCAM-CPT66 but contains the auristatin-based DM21 linker payload, and was significantly contrasted with EpCAM-DM21, which was only tolerated up to 6 mg / kg. The tolerance of EpCAM-CPT66 is not inferior even when compared to fam-trastuzumab deruxtecan-nxki, in which HNSTD in cynomolgus monkeys was measured to be 30 mg / kg.

[0357] 6.7.1 Pharmaceutical Composition The provided composition includes a pharmaceutical composition (e.g., an impure or non-sterile composition), and a drug substance composition useful for the manufacture of a pharmaceutical composition that can be used to prepare a unit dosage form (i.e., a composition suitable for administration to a subject or patient). Such a composition includes a prophylactically or therapeutically effective amount of the provided immunoconjugate and a pharmaceutically acceptable carrier.

[0358] In some embodiments, the provided composition includes a prophylactically or therapeutically effective amount of the immunoconjugate of the present disclosure and a pharmaceutically acceptable carrier.

[0359] In certain embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and, more specifically, for use in humans. The term "carrier" means a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which a therapeutic agent is administered. Generally, the components of the compositions provided herein are provided in unit dosage form, separately or mixed together, as a lyophilized powder or anhydrous concentrate in a sealed container, such as an ampoule or sachet, indicating the amount of the active agent. When the composition is administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0360] The present disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with the immunoconjugates provided herein, alone or together with such pharmaceutically acceptable carriers. The present disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the pharmaceutical compositions provided herein. Optionally, such containers may be accompanied by a notice in a form prescribed by a governmental agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products, which notice indicates that the agency has approved the manufacture, use, or sale for human administration.

[0361] The present disclosure provides a kit that can be used in the methods described above. The kit can include any of the immunoconjugates disclosed herein.

[0362] 6.7.2 Administration Method The compositions of the present disclosure can be provided to treat, prevent, and alleviate one or more symptoms associated with a disease or disorder by administering a therapeutically effective amount of the immunoconjugates provided herein to a subject. In certain embodiments, such compositions are substantially purified (i.e., substantially free of substances that limit their effect or cause undesirable side effects). In certain embodiments, the subject is a mammal such as an animal, optionally a non - primate (e.g., cow, horse, cat, dog, rodent, etc.) or a primate (e.g., monkey such as cynomolgus monkey, human, etc.). In certain embodiments, the subject is a human.

[0363] Examples of the administration methods of the immunoconjugates provided herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., nasal and oral routes). In certain embodiments, the immunoconjugates provided herein are administered intramuscularly, intravenously, or subcutaneously. The composition may be administered by any convenient route, such as by infusion or bolus injection, and may be administered together with other biologically active agents. The administration may be systemic or local.

[0364] The present disclosure also provides that the preparations of the immunoconjugates of the present disclosure are packaged in a sealed container such as an ampoule or sachet displaying the amount of the molecule. In one embodiment, such molecules are provided as a dry-sterilized lyophilized powder or anhydrous concentrate in a sealed container and can be reconstituted, for example, with water or saline to a concentration appropriate for administration to a subject. In some embodiments, the immunoconjugate is provided as a dry-sterilized lyophilized powder in a sealed container.

[0365] The lyophilized preparations of the immunoconjugates provided herein should be stored at 2°C to 8°C in their original containers, and the molecules should be administered within within 12 hours, optionally within 6 hours, within 5 hours, within 3 hours, or within 1 hour after reconstitution. In alternative embodiments, such molecules are provided in liquid form in a sealed container displaying the amount and concentration of the molecule, fusion protein, or conjugate molecule. In some embodiments, when provided in liquid form, such immunoconjugates are provided in a sealed container.

[0366] As used herein, a "therapeutically effective amount" of a pharmaceutical composition refers to an amount sufficient to achieve beneficial or desirable results, including, but not limited to, clinical outcomes such as reduction of cancer symptoms (e.g., cancer cell proliferation, presence of a tumor, tumor metastasis, etc.), thereby improving the quality of life of a person suffering from the disease, reducing the dosage of other drugs required for treatment of the disease, enhancing the effect of another drug such as by targeting and / or internalization, delaying the progression of the disease, and / or extending the survival period of an individual.

[0367] A therapeutically effective amount can be administered in one or more administrations. For the purposes of the present disclosure, a therapeutically effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly reduce the growth (or its effects) of the presence of a virus and reduce and / or delay the onset of a viral disease.

[0368] 6.8 Diagnostic and research uses In addition to the therapeutic uses of the immunoconjugates discussed herein, the immunoconjugates provided herein can be used in many known diagnostic and research applications. The provided EpCAM immunoconjugates can be used, for example, for the purification, detection, and targeting of EpCAM involved in both in vitro and in vivo diagnostic methods. For example, the antibody and / or fragment can be used in immunoassays to qualitatively and quantitatively measure the level of EpCAM (e.g., human EpCAM or cynomolgus monkey EpCAM) expressed by cells in a biological sample. See, e.g., Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), the entire contents of which are incorporated herein by reference.

[0369] The provided EpCAM immunoconjugates can be used, for example, in competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)).

[0370] Detectably labeling an EpCAM immunoconjugate can be achieved by conjugation to an enzyme for use in an enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA). The conjugated enzyme reacts with an exposed substrate to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric, or visual means. Examples of enzymes that can be used to detectably label an EpCAM immunoconjugate of the present disclosure include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0371] Detecting EpCAM using radioimmunoassay (RIA) is possible by radiolabeling the EpCAM immunoconjugate (see, for example, Work, et al., Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, N.Y. (1978)). The radioisotope can be detected by means such as the use of a gamma counter or scintillation counter, or by autoradiography. Isotopes particularly useful for the purposes of the present disclosure are 3 H, 125 I, 131 I, 35 S, 14 C and, in some cases 125I is.

[0372] It is also possible to label the EpCAM immunoconjugate with a fluorescent compound. When a fluorescently labeled antibody, antibody fragment, or activatable antibody is exposed to light of an appropriate wavelength, its presence can be detected by fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

[0373] The EpCAM immunoconjugate can also be detectably labeled using a fluorescent-emitting metal such as 125Eu or other lanthanides. These metals can be bound to the EpCAM antibody, its EpCAM-binding antibody fragment, and the EpCAM activatable antibody using a metal chelate group such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0374] In additional embodiments, the EpCAM immunoconjugate is detectably labeled by coupling it to a chemiluminescent compound. Next, the presence of the chemiluminescently labeled antibody, or antibody fragment, is determined by detecting the presence of luminescence that occurs during a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, isoluminol, acridinium ester, imidazole, acridinium salts, and oxalate esters. Similarly, bioluminescent compounds can be used to label the EpCAM antibody, its EpCAM-binding antibody fragment, the EpCAM activatable antibody, or derivatives thereof. Bioluminescence is a type of chemiluminescence found in biological systems where a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of the bioluminescent protein is determined by detecting the presence of luminescence. Bioluminescent compounds important for labeling purposes are luciferin, luciferase, and aequorin.

[0375] EpCAM immunoconjugates are useful for in vivo imaging diagnosis, where an EpCAM immunoconjugate labeled with a detectable moiety such as a radiopaque agent or a radioisotope is administered to a subject, optionally in the bloodstream, and the presence and location of the labeled antibody or antibody fragment in the host are assayed. This imaging diagnostic technique is useful for staging and treating malignant tumors. The EpCAM immunoconjugate can be labeled with any moiety detectable in the host by any of nuclear magnetic resonance, radiology, or other detection means known in the art.

[0376] The label used in accordance with the methods of the present disclosure can be any detectable moiety capable of generating a detectable signal either directly or indirectly. For example, the label can be a biotin label, an enzyme label (e.g., luciferase, alkaline phosphatase, beta-galactosidase, and horseradish peroxidase), a radiolabel (e.g., 3 H, 14 C, 32 P, 35 S, and 125 I), a fluorophore such as a fluorescent or chemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine), a contrast agent (e.g., Tc-m 99 and indium ( 111 In), and metal ions (e.g., gallium and europium).

[0377] Any method known in the art for conjugating an EpCAM immunoconjugate to a label can be used, examples of which are described by Hunter et al., Nature 144:945 (1962); David et al., Biochemistry 13:1014 (1974); Pain et al., J. Immunol. Meth. 40:219 (1981); Nygren, Histochem. and Cytochem. 30:407 (1982).

[0378] 7. Examples 7.1 Example 1. EPCAM Antibodies and Fragments Thereof The generation and selection of monoclonal antibodies against human and cynomolgus (cyno) EpCAM are described in detail in International Publication No. WO 2020 / 086665, the entire disclosure of which is incorporated herein by reference for all purposes. The generation, selection, and humanization of the EpCAM antibodies and fragments thereof of the present disclosure are summarized below.

[0379] To generate monoclonal antibodies, three different immunization protocols were used. In the first immunization protocol, wild-type BALB / c female mice were subcutaneously injected three times with the cynomolgus EpCAM-expressing 300-19 cell line, a pre-B cell derived from BALB / c, followed by two injections with the human EpCAM-expressing 300-19 cell line. In the second immunization protocol, wild-type BALB / c female mice were subcutaneously injected four times with the NSCLC cell line H1568, followed by four injections with cynomolgus primary kidney epithelial cells. In the third immunization protocol, FcgammaR2b ko / ko BALB / c female mice (Model #579, Taconic) were subcutaneously injected three times with human EpCAM-expressing 300-19 cells, followed by two injections with cynomolgus-EpCAM-expressing 300-19 cells. In all three protocols, the cells were prepared in PBS and injected into the mice at a dose of 5×10 6 cells / mouse / injection, with a two-week interval between injections. To enhance the immune response, anti-GITR Ab (clone DTA-1) was injected one week after the first immunization. Three days before sacrifice for hybridoma generation, the immunized mice were intraperitoneally injected with another dose of human EpCAM-expressing 300-19 cells. Hybridoma clones were prepared for antibody screening.

[0380] Other immunization and hybridoma production techniques may also be used, including those described in J. Langone and H. Vunakis (Eds., Methods in Enzymology, Vol. 121, Immunochemical Techniques, Part I, Academic Press, Florida); and E. Harlow and D. Lane (Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory Press, New York, NY).

[0381] Hybridoma screening was performed using a flow cytometry binding assay with human EpCAM-expressing 300-19 cells and wild-type 300-19 cells. Hybridomas that were positive for binding to human and cynomolgus monkey EpCAM antigens but negative for wild-type 300-19 cells were further subcloned by limiting dilution. One subclone was selected from each hybridoma that showed specific binding to human and cynomolgus monkey EpCAM antigens for subsequent analysis.

[0382] During this investigation, a total of 20 fusions were performed. Sixty-three hybridomas specific for human and cynomolgus monkey EpCAM antigens were generated and 29 hybridomas were subcloned. Stable subclones were cultured and the isotype of the monoclonal antibody was identified using commercially available mouse IgG isotype identification reagents.

[0383] Mouse antibodies were purified and the binding affinity was assayed by a flow cytometry binding assay using the purified antibody mEpCAM23, performed with HSC2 cells. The apparent K d of the mEpCAM23 antibody ranged from 3.8×10 -10 to 7.9×10 -10 . The sequence of mEpCAM23 was identified and the mEpCAM23 clone was selected for chimerization, humanization, and further evaluation.

[0384] The VL and VH chains of the EpCAM hybridoma were cloned, sequenced, and subsequently, the variable region amino acid sequences of the mouse EpCAM antibody were codon-optimized, synthesized, and cloned in-frame with the human IgG1 constant region by GenScript (New Jersey) to construct a chimeric version of the EpCAM antibody.

[0385] Humanization of the antibody was performed essentially as described in Jones et al., Nature 321:604-608 (1986), Verhoeyen et al., Science 239:1534-1536 (1988), U.S. Patent No. 5,225,539, and No. 5,585,089, using the complementarity-determining region (CDR) grafting method. CDR grafting generally consists of replacing the Fv framework region (FR) of the mouse antibody with the human antibody Fv framework region while preserving the mouse CDR residues essential for the specific antigen-binding properties of the parental antibody. Exemplary CDRs of the mouse EpCAM-23 antibody according to the Kabat definition are shown in Table 13 below.

Table 16

[0386] The initial CDR grafts of muEpCAM-23 were generated by transplanting Kabat positions 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L2) of VL, and Kabat positions 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) of VH into the corresponding human germline IGKV2D-29*01 and IGHV1-3*01 frameworks. The initial CDR grafted version contained 12 framework residue substitutions in V L and 27 framework residue changes in V H In addition, variants containing one or more revertant mutations of the Buriani zone residues were generated and subsequently evaluated for EpCAM binding.

[0387] Some humanized versions of EpCAM23 retained binding to human HSC2 cells. However, only versions Gv2.2 (VL Gv2 and VH Gv2) and Gv4.2 (VL Gv4 and VH Gv2) also retained binding to cynomolgus primary kidney epithelial cells. Affinity variants of the humanized antibody Gv4.2 were generated. Mutagenesis of either CDR residues or VH / VL framework residues exposed to the solvent was used to generate variants that rarely occur (<5% frequency) in the mouse / human natural repertoire.

[0388] An initial set of humanized anti-EpCAM23 Gv4.2 affinity variants was tested by enzyme-linked immunosorbent assay (ELISA) using huEpCAM or cynoEpCAM protein tagged with mFc. Two affinity variants, huEpCAM23 Gv4.2-1361-H and huEpCAM23 Gv4.2-1565-Y, were selected for further evaluation. Specifically, these variants were selected due to their somewhat lower binding affinity (i.e., approximately 3-fold lower Kd compared to the parental antibody on HSC2 cells) and their different mutation sites on HSC2 cells.

[0389] 7.2 Example 2. Discovery of the Mask The identification and characterization of a masking moiety (MM or "mask") for use with the activatable anti-EpCAM antibodies and fragments thereof ("activatable antibodies") of the present disclosure are described in detail in International Publication No. WO 2020 / 086665, which is hereby incorporated by reference in its entirety and is described below.

[0390] Using a method similar to that described in PCT International Publication No. WO 2010 / 081173, published July 15, 2010, the entire disclosure of which is hereby incorporated by reference herein, a humanized anti-human EpCAM monoclonal antibody (EpCAM23Gv4.2) that cross-reacts with human and cynomolgus EpCAM was randomly X 15A peptide library (where X is any amino acid) was used for screening. The screening consisted of 1 round of MACS selection and 3 rounds of FACS selection. The first MACS selection was performed using protein-A Dynabeads (Invitrogen) with anti-EpCAM antibody. For MACS, the anti-EpCAM antibody was conjugated with DyLight-488 (ThermoFisher) to confirm EpCAM binding activity, and anti-EpCAM-488 was used as a fluorescent probe in all FACS rounds. Individual peptide clones were identified by sequence analysis from each FACS round.

[0391] Two heavy chain variants of a humanized anti-human EpCAM monoclonal antibody (EpCAM23(1361-H) and EpCAM23(1565-Y)) were generated using a method similar to that described in PCT International Publication No. WO 2010 / 081173 published on July 15, 2010, and a random X 15 A peptide library (where X is any amino acid) was used for screening. Mask EP101 - EP104 were identified using the EpCAM23(1565-Y) heavy chain variant. Mask EP105 - EP110 were identified using the EpCAM23(1361-H) heavy chain variant. Mutations of lysine residues in the Ep107 masking portion were also generated.

[0392] The sequences of the selected anti-EpCAM masking portions are listed in Table 14.

Table 17

[0393] Using these masking peptides, anti-EpCAM activatable antibodies of the present disclosure were generated. The amino acid sequences of specific ones of these anti-EpCAM activatable antibodies are shown in Table 15 below. In some embodiments, these anti-EpCAM activatable antibodies of the present disclosure include ISSGLLSGRSDNH (SEQ ID NO: 312), AVGLLAPPGGLSGRSDNH (SEQ ID NO: 313), ISSGLLSGRSDIH (SEQ ID NO: 314), ISSGLLSGRSDQH (SEQ ID NO: 315), ISSGLLSGRSDNP (SEQ ID NO: 316), ISSGLLSGRSANP (SEQ ID NO: 317), ISSGLLSGRSANI (SEQ ID NO: 318), ISSGLLSGRSDNI (SEQ ID NO: 169), AVGLLAPPGGLSGRSDIH (SEQ ID NO: 319), AVGLLAPPGGLSGRSDQH (SEQ ID NO: 320), AVGLLAPPGGLSGRSDNP (SEQ ID NO: 321), AVGLLAPPGGLSGRSANP (SEQ ID NO: 322), AVGLLAPPGGLSGRSANI (SEQ ID NO: 323), or AVGLLAPPGGLSGRSDNI (SEQ ID NO: 168). In some embodiments, a specific anti-EpCAM activatable antibody of the present disclosure includes a non-cleavable portion "NSUB" that is not expected to be cleaved.

[0394] The specific light chain sequences of the activatable antibodies of the present disclosure shown below include an N-terminal spacer sequence of QGQSGQG (SEQ ID NO: 324), but those skilled in the art will understand that the activatable anti-EpCAM antibodies of the present disclosure can include any suitable spacer sequence, such as a spacer sequence selected from the group consisting of QGQSGQ (SEQ ID NO: 325), QGQSG (SEQ ID NO: 326), QGQS (SEQ ID NO: 327), QGQ, QG, GQSGQG (SEQ ID NO: 328), QSGQG (SEQ ID NO: 329), SGQG (SEQ ID NO: 330), GQG, G, or Q. In some embodiments, the light chain of the activatable anti-EpCAM antibody of the present disclosure cannot have a spacer sequence attached to the N-terminus.

Table 18

[0395] Exemplary anti-EpCAM activatable antibodies having the following heavy and light chains are shown in Table 16 below. [Table 19] TIFF2025524628000070.tif239159TIFF2025524628000071.tif33159

[0396] The binding of the anti-human EpCAM antibodies of the present disclosure was demonstrated using a solid-phase binding assay. Briefly, recombinant human EpCAM-mFc protein (Immunogen) was coated on an ELISA plate (50 μL of 1 μg / mL), followed by incubation with serially diluted anti-EpCAM antibodies (starting from 62.5 nM) or activatable anti-EpCAM antibodies (starting from 1 μM), where the activatable antibodies were assayed in their non-cleaved form. The amount of bound antibody was detected using anti-human IgG (anti-Fab) conjugated to horseradish peroxidase (Sigma) with Ultra TMB-ELISA reagent (Thermo Fisher Scientific), and the OD was measured at 450 nM. The K D of each antibody and activatable antibody was measured, and the ELISA masking efficiency (ME) of each activatable antibody relative to the unmasked antibody was calculated. Exemplary results are shown in Tables 17 and 18. Collectively, these data demonstrate that the anti-human EpCAM activatable antibodies of the present disclosure exhibit a shifted binding affinity to recombinant EpCAM protein compared to the parental anti-EpCAM antibodies of the present disclosure. [Table 20] TIFF2025524628000073.tif28159 [Table 21]

[0397] As shown in Tables 17 and 18, these exemplary results demonstrated that the anti-human EpCAM activatable antibodies exhibit a range of masking efficiencies compared to unmasked anti-EpCAM antibodies.

[0398] 7.3 Example 3. Epitope Mapping The human CD326 antigen, EpCAM (epithelial cell adhesion molecule), consists of 314 amino acids and includes a 265-amino acid extracellular domain, a 23-amino acid transmembrane domain, and a 26-amino acid cytoplasmic tail. The extracellular domain can be further divided into three domains (D1, D2, and D3). The extracellular domain contains two cysteine-rich epidermal growth factor-like (EGF-like) repeats, which include a first domain encompassing the region from glutamine at position 24 to cysteine at position 59 of the mature protein (i.e., before signal peptide cleavage), and a second domain encompassing the region from cysteine at position 66 to cysteine at position 135. Next, in series with the first two domains, there is also a third domain (D3) that does not contain cysteine and includes amino acid residues 136-243. [Table 22]

[0399] The epitope for the humanized EpCAM antibody Gv4.2 was mapped by engineering chimeric proteins using combinations of the extracellular domains of human and mouse EpCAM. Since human and mouse EpCAM share more than 82% amino acid sequence identity and 85% similarity, the topology of the chimeric proteins should remain unchanged.

[0400] The extracellular region of human EpCAM (residues 1 - 265) was codon-optimized, synthesized, and cloned in-frame into a vector (pGSmuFc2ANL) containing the mouse IgG2a Fc region using the HindIII and BamHI restriction enzyme recognition sites at Genscript. Similarly, other expression vectors containing various chimeric variants of the human / mouse EpCAM extracellular domain were synthesized by substituting the corresponding residues corresponding to human EpCAM domain D1 (24 - 59), domain D2 (66 - 135), domain D3 (136 - 265), or combinations thereof with the corresponding mouse residues. These expression constructs were transiently produced in suspension-adapted HEK-293T cells using PEI as a transfection reagent in shake flasks. The transfection was incubated for 1 week, harvested, and the filtered supernatant was purified using a combination of protein A and CHT chromatography.

[0401] The humanized EpCAM antibody huEpCAM23Gv4.2 was tested in an enzyme-linked immunosorbent assay (ELISA) format for binding to the above EpCAM protein. Briefly, each mFc-tagged EpCAM protein was purified using a combination of protein A and CHT chromatography. Each mFc-tagged EpCAM protein was diluted to 0.5 μg / mL in 50 mM sodium bicarbonate buffer pH 9.6, and 100 μL was added to each well. After incubation at 4°C for 16 hours, the plates were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST), and subsequently blocked with 200 μL of blocking buffer (TBS containing 1% BSA). Next, 100 μL of the primary antibody huEpCAM23Gv4.2, serially diluted in blocking buffer, was added to the ELISA wells in duplicate and incubated at room temperature for 1 hour. The plates were then washed 3 times with TBST, after which 100 μL of anti-human IgG (H+L)-HRP was added to each well. The plates were incubated again at room temperature for 1 hour and subsequently washed 3 times with TBST. Finally, 100 μL of TMB one component HRP microwell substrate was added to each well and incubated for 5 minutes. The reaction was stopped by adding 100 μL of stop solution, and the absorbance was read at 450 nm using a multi-well plate reader. OD450 was plotted against antibody concentration on a semi-logarithmic plot. A dose-response curve was generated by non-linear regression, and the EC 50 values of each curve were calculated using GraphPad Prism v6.

[0402] The binding of the huEpCAM2...

Claims

1. (a) An activatable antibody comprising a full-length human IgG1 EpCAM antibody having eight cysteines that form four interchain disulfide bonds in its natural higher-order structure, wherein the full-length human IgG1 EpCAM antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 103 and a light chain containing the amino acid sequence of SEQ ID NO: 179, (b) Structure: 【Chemistry 1】 An immunoconjugate comprising eight linker payloads or pharmaceutically acceptable salts thereof, wherein each of the eight linker payloads is individually covalently bound to the activatable antibody via one of the eight cysteines.

2. (a) An activatable antibody, (i) An EpCAM antibody or an EpCAM-binding fragment thereof (1) Heavy chain complementarity determination region 1 (VH-CDR1) containing the amino acid sequence NYYIH (SEQ ID NO: 13), (2) Heavy chain complementarity determination region 2 (VH-CDR2) containing the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14), (3) Heavy chain complementarity determination region 3 (CDR3) (VH-CDR3) containing the amino acid sequence DGPWFAY (SEQ ID NO: 15), (4) Light chain complementarity determination region 1 (VL-CDR1) containing the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO: 42), (5) Light chain complementarity determination region 2 (VL-CDR2) containing the amino acid sequence QTSNLAS (SEQ ID NO: 40), (6) The EpCAM antibody or its EpCAM-binding fragment, comprising: a light chain complementarity determining region 3 (VL-CDR3) containing the amino acid sequence AQNLELPNT (SEQ ID NO: 41); (ii) A cleavable portion coupled to the EpCAM antibody or its EpCAM-binding fragment, comprising the amino acid sequence AVGLLAAPPGGLSGRSDNI (SEQ ID NO: 168) or the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169), (iii) A masking portion coupled to the EpCAM antibody or its EpCAM binding fragment, comprising the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein when the activatable antibody is in an uncleaved state, the masking portion inhibits the binding of the antibody or antibody fragment to EpCAM, The activatable antibody, when in an uncleaved state, has a structural sequence from the N-terminus to the C-terminus: (masking portion) - (cleavable portion) - (antibody or antibody fragment), or (antibody or antibody fragment) - (cleavable portion) - (masking portion), and (b) Structure: 【Chemistry 2】 An immunoconjugate comprising at least one linker payload or a pharmaceutically acceptable salt thereof, as shown by [the relevant source].

3. The immunoconjugate according to claim 2, wherein the cleavable portion includes the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169).

4. The immunoconjugate according to claim 2, wherein the cleavable portion includes the amino acid sequence AVGLAPPGGLSGRSDNI (SEQ ID NO: 168).

5. The immunoconjugate according to any one of claims 2 to 4, wherein the immunoconjugate has a drug-to-antibody ratio (DAR) of 2 to 8.

6. The immunoconjugate according to claim 5, wherein the immunoconjugate has at least 5 DARs.

7. The immunoconjugate according to claim 5, wherein the immunoconjugate has at least 6 DARs.

8. The immunoconjugate according to claim 5, wherein the immunoconjugate has 8 DARs.

9. The immunoconjugate according to claim 8, wherein the activatable antibody is a full-length human IgG1 EpCAM antibody having eight cysteines that form four interchain disulfide bonds when in its natural higher-order structure, and each linker payload is individually covalently bound to the activatable antibody via one of the eight cysteines.

10. (a) an activatable antibody, (i) A full-length human IgG1 EpCAM antibody having eight cysteine ​​molecules that form four interchain disulfide bonds in its natural higher-order structure, (1) Heavy chain complementarity determination region 1 (VH-CDR1) containing the amino acid sequence NYYIH (SEQ ID NO: 13), (2) Heavy chain complementarity determination region 2 (VH-CDR2) containing the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14), (3) Heavy chain complementarity determination region 3 (CDR3) (VH-CDR3) containing the amino acid sequence DGPWFAY (SEQ ID NO: 15), (4) Light chain complementarity determination region 1 (VL-CDR1) containing the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO: 42), (5) Light chain complementarity determination region 2 (VL-CDR2) containing the amino acid sequence QTSNLAS (SEQ ID NO: 40), (6) The full-length human IgG1 EpCAM antibody comprising a light chain complementarity determining region 3 (VL-CDR3) containing the amino acid sequence AQNLELPNT (SEQ ID NO: 41), (ii) A cleavable portion coupled to the EpCAM antibody or its EpCAM-binding fragment, comprising the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169), (iii) A masking portion coupled to the EpCAM antibody or its EpCAM binding fragment, comprising the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein when the activatable antibody is in an uncleaved state, the masking portion inhibits the binding of the antibody or antibody fragment to EpCAM, The activatable antibody, when in an uncleaved state, has a structural sequence from the N-terminus to the C-terminus: (masking portion) - (cleavable portion) - (antibody) or (antibody) - (cleavable portion) - (masking portion), and (b) Each has its own structure: 【Transformation 3】 An immunoconjugate comprising eight linker payloads or pharmaceutically acceptable salts thereof, wherein each of the eight linker payloads is individually covalently bound to the activatable antibody via one of the eight cysteines.

11. The immunoconjugate according to claim 10, wherein the activatable antibody comprises a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 54 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO:

89.

12. (a) an activatable antibody, (i) A full-length human IgG1 EpCAM antibody having eight cysteines that form four interchain disulfide bonds in its natural higher-order structure, wherein the full-length human IgG1 EpCAM antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 103 and a light chain containing the amino acid sequence of SEQ ID NO: 140, (ii) A cleavable portion coupled to the EpCAM antibody or its EpCAM-binding fragment, comprising the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169), (iii) A masking portion coupled to the EpCAM antibody or its EpCAM binding fragment, comprising the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein when the activatable antibody is in an uncleaved state, the masking portion inhibits the binding of the antibody or antibody fragment to EpCAM, The activatable antibody, when in an uncleaved state, has a structural sequence from the N-terminus to the C-terminus: (masking portion) - (cleavable portion) - (antibody) or (antibody) - (cleavable portion) - (masking portion), and (b) Each has its own structure: 【Chemistry 4】 Eight linker payloads or pharmaceutically acceptable salts thereof, wherein each of the eight linker payloads is individually covalently bound to the activatable antibody via one of the eight cysteines, An immunoconjugate that includes this.

13. (a) an activatable antibody, (i) A full-length human IgG1 EpCAM antibody having cysteine ​​that forms an interchain disulfide bond when in its natural higher-order structure, wherein the full-length human IgG1 EpCAM antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 103 and a light chain containing the amino acid sequence of SEQ ID NO: 140, (ii) A cleavable portion coupled to the EpCAM antibody or its EpCAM-binding fragment, comprising the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169), (iii) A masking portion coupled to the EpCAM antibody or its EpCAM binding fragment, comprising the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein when the activatable antibody is in an uncleaved state, the masking portion inhibits the binding of the antibody or antibody fragment to EpCAM, The activatable antibody, when in an uncleaved state, has a structural sequence from the N-terminus to the C-terminus: (masking portion) - (cleavable portion) - (antibody) or (antibody) - (cleavable portion) - (masking portion), and (b) Structure: 【Transformation 5】 A linker payload or a pharmaceutically acceptable salt thereof, wherein each linker payload is individually covalently bound to the activatable antibody via a cysteine ​​amino acid residue, An immunoconjugate that includes this.

14. (a) an activatable antibody, (i) A full-length human IgG1 EpCAM antibody having cysteine ​​that forms an interchain disulfide bond when in its natural higher-order structure, wherein the full-length human IgG1 EpCAM antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 103 and a light chain containing the amino acid sequence of SEQ ID NO: 140, (ii) A cleavable portion coupled to the EpCAM antibody or its EpCAM-binding fragment, comprising the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169), (iii) A masking portion coupled to the EpCAM antibody or its EpCAM binding fragment, comprising the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein when the activatable antibody is in an uncleaved state, the masking portion inhibits the binding of the antibody or antibody fragment to EpCAM, The activatable antibody, when in an uncleaved state, has a structural sequence from the N-terminus to the C-terminus: (masking portion) - (cleavable portion) - (antibody) or (antibody) - (cleavable portion) - (masking portion), and (b) Each has its own structure: 【Transformation 6】 A linker payload or a pharmaceutically acceptable salt thereof, wherein the linker payload is individually covalently bound to the activatable antibody via cysteine ​​amino acid residues, An immunoconjugate comprising, wherein the immunoconjugate has 2 to 8 DARs.

15. The immunoconjugate according to claim 14, wherein the immunoconjugate has at least 5 DARs.

16. The immunoconjugate according to claim 14, wherein the immunoconjugate has at least 6 DARs.

17. The immunoconjugate according to claim 14, wherein the immunoconjugate has 8 DARs.

18. An immunoconjugate obtainable by the method described in the section "7,4,1 Preparation of HUEPCAM23GV4.2-LALALALALALA-CPT66" of Example 4.

19. Immunoconjugates that can be obtained by methods including the following: (a) To form an activatable EpCAM reaction reaction by mixing an activatable EpCAM antibody, which is a heterotetramer consisting of two heavy chains each having the amino acid sequence of SEQ ID NO: 103 and two light chains each having the amino acid sequence of SEQ ID NO: 179, with a mixture of EPPS buffer and EDTA aqueous solution, (b) Adding a TCEP solution to the activatable EpCAM reaction mixture to form a reduced activatable EpCAM reaction reaction, (c) The reduced Activatable EpCAM reaction mixture is mixed with a camptothecin linker toxin stock solution containing a linker toxin represented by the following structure, 【Transformation 7】 An immunoconjugate that can be obtained by a method including forming an immunoconjugate thereafter.

20. A nucleic acid comprising one or more coding regions of an immunoconjugate-activatable antibody according to any one of claims 1, 2, and 10 to 14.

21. A vector comprising the nucleic acid described in claim 20.

22. The vector according to claim 21, which is a viral vector.

23. (a) Culturing host cells containing the vector described in claim 22, (b) Isolating the activatable antibody from the host cell, (c) A method for producing an immunoconjugate, comprising conjugating at least one linker payload reaction product containing camptothecin or a derivative thereof to the activatable antibody.

24. The linker payload reactant comprises the compound of formula I, E-A-LD (Formula I) During the ceremony, E-A-Z'-L １ -D (Formula I) During the ceremony, D is represented by the following structural formula: 【Transformation 8】 In the formula, R １ F is R ２ It is methyl, -L １ -Z'-* is -(C １ ~C ４ alkylene)-O-CH ２ -NR ８ -*, -(C １ ~C ４ alkylene)-NR ８ -*, or -(C １ ~C ５ alkylene)-NR ５ C(=O)-(C １ ~C ５ alkylene)-O-CH ２ -NR ８ -*, where * is the site covalently bonded to A, Each R ５ These are independently H, methyl, or benzyl. Each R ８ These are independently H, methyl, or benzyl. A is a peptide containing 2 to 4 amino acids. E is -C (=O) - (C １ ~C １０ Alkylene)-X ３ And in the formula, X ３ teeth, 【Chemistry 9】 The method according to claim 23.

25. The linker payload reactant is 【Chemistry 10】 The method according to claim 23, or a pharmaceutically acceptable salt thereof.

26. The linker payload reactant is 【Chemistry 11】 The method according to claim 23, or a pharmaceutically acceptable salt thereof.

27. The method according to claim 23, wherein the linker payload reactant comprises exatecan.

28. The method according to claim 23, wherein the linker payload reactant comprises deruxtecan.

29. A method for producing an immunoconjugate, comprising conjugating at least one linker payload reaction product containing camptothecin or a derivative thereof to an activatable antibody as described in claim 8.

30. The linker payload reactant comprises the compound of formula I, E-A-Z'-L １ -D (Formula I) During the ceremony, D is represented by the following structural formula: 【Chemistry 12】 In the formula, R １ F is R ２ It is methyl, -L １ -Z'-* is -(C １ ~C ４ Alkylene)-O-CH ２ -NR ８ -*, -(C １ ~C ４ Alkylene)-NR ８ -*, or -(C １ ~C ５ Alkylene)-NR ５ C (= O) - (C １ ~C ５ Alkylene)-O-CH ２ -NR ８ -*, where * is the site covalently bonded to A, Each R ５ These are independently H, methyl, or benzyl. Each R ８ These are independently H, methyl, or benzyl. A is a peptide containing 2 to 4 amino acids. E is -C (=O) - (C １ ~C １０ Alkylene)-X ３ And in the formula, X ３ teeth, 【Chemistry 13】 The method according to claim 29.

31. The linker payload reactant is 【Chemistry 14】 The method according to claim 29, wherein the salt is pharmaceutically acceptable or a pharmaceutically acceptable salt thereof.

32. The linker payload reactant is 【Chemistry 15】 The method according to claim 29, wherein the salt is pharmaceutically acceptable or a pharmaceutically acceptable salt thereof.

33. The method according to claim 29, wherein the linker payload reactant comprises exatecan.

34. The method according to claim 29, wherein the linker payload reactant comprises deruxtecan.

35. A method for producing an immunoconjugate, comprising conjugating a full-length human IgG1 EpCAM antibody with a linker toxin represented by the following structure: 【Chemistry 16】 A method wherein the full-length human IgG1 EpCAM antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 103 and a light chain containing the amino acid sequence of SEQ ID NO: 179, and the immunoconjugate has a drug-to-antibody ratio (DAR) of 2 to 8.

36. The method according to claim 35, wherein the immunoconjugate has at least 5 DARs.

37. The method according to claim 35, wherein the immunoconjugate has at least 6 DARs.

38. The method according to claim 35, wherein the immunoconjugate has 8 DARs.

39. (a) The immunoconjugate described in claim 8, (b) A composition comprising a pharmaceutically acceptable carrier.

40. The immunoconjugate according to claim 8 for use in the treatment of cancer expressing EpCAM.

41. The composition according to claim 39 for use in the treatment of cancer expressing EpCAM.

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