EPCAM immunoconjugates and uses thereof

Abstract

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

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 388,486, filed on July 12, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on July 6, 2023, is named CYTX-097-PCT_SL.xml and is 336,140 bytes in size. 3. Background Technology

[0005] The present disclosure generally relates to antibodies and antibody fragments that specifically bind to human EpCAM, EpCAM-activatable antibodies and immunoconjugates thereof, and methods of making and using the antibodies, antibody fragments, activatable antibodies and immunoconjugates for diagnosing and treating diseases such as cancer.

[0006] Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein comprising an extracellular domain, a transmembrane domain, and a single intracellular domain. EpCAM expression in humans is epithelial-specific. Except for squamous epithelium and some specific epithelial cell types (such as epidermal keratinocytes, hepatocytes, gastric parietal cells, and myoepithelial cells), most epithelial cells express EpCAM (Balzar et al., J. Mol. Med. 77: 699-712 (1999); Momburg et al., Cancer Res 47: 2883-2891 (1987)).

[0007] EpCAM is abundantly and uniformly expressed in human cancers of diverse 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 vast majority of epithelial cancers, including, for example, ovarian, colon, gastric, prostate, and lung cancers. In addition, EpCAM has been shown to be expressed on most primary, metastatic, and disseminated NSCLC (non-small cell lung cancer cells) (Passlick, Int. J. Cancer 87: 548-552 (2000)), gastric and gastroesophageal junction adenocarcinomas (Martin, J. Clin. Pathol. 52: 701-704 (1999)), and 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 found that only 4% lacked EpCAM expression. EpCAM is also overexpressed in cancer-initiating cells or cancer cell stem cells isolated from colon, breast, pancreatic, and prostate cancers (O'Brien et al., Nature 445:106-110 (2007); Marhaba et al., Curr. Mol. Med. 8:784-804 (2008)).

[0008] Normal cells express EpCAM on the basolateral side of the epithelial membrane, while cancer cells express EpCAM abundantly on the apical surface. Antibody-based therapeutics have been designed to exploit this feature of EpCAM expression, as normal cell EpCAM is less prominent and less exposed, meaning healthy cells may not be readily bound by therapeutic anti-EpCAM antibodies.

[0009] Camptothecin (CPT) is a pentacyclic alkaloid isolated from the bark and trunk of Camptotheca acuminata (Camptotheca, Happy tree), a tree native to China. Camptothecin inhibits topoisomerase I, which leads to cell death. Due to its cytotoxic mechanism and broad-spectrum antitumor activity, considerable effort has been made to develop clinical analogs of camptothecin. However, poor solubility and inactivity under physiological conditions have limited the clinical development of suitable camptothecin analogs. Camptothecin and most of its derivatives are insoluble in aqueous buffers. In addition, camptothecin is in equilibrium between the active lactone form and the inactive hydrolyzed carboxylate form, which limits its therapeutic efficacy.

[0010] Many antibodies against EpCAM have been used clinically, but have failed for various reasons. The EpCAM antibodies tested have taken a variety of forms, including naked antibodies, immunotoxins, and bi- or tri-specific antibodies (Baeuerle, Br. J. Cancer, 96: 417-423 (2007)). For example, adelimumab (MT201), a naked anti-EpCAM antibody, has been tested in clinical studies for the treatment of colorectal cancer, prostate cancer, and breast cancer. Current safety issues faced by anti-EpCAM antibody-based approaches include systemic tolerance and acute pancreatitis. Therefore, although attempts have been made to develop therapeutic antibodies against EpCAM, there is an urgent need to develop new therapeutic EpCAM immunoconjugates that overcome the shortcomings and limitations of previously developed antibodies or antibody derivatives. 4. Summary of the Invention

[0011] In one aspect, the present disclosure provides an immunoconjugate comprising: (a) an activatable antibody comprising a full-length human IgG1 EpCAM antibody having eight cysteines that form four interchain disulfide bonds when in a native conformation, the full-length human IgG1 EpCAM antibody comprising 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) eight linker-payloads depicted by the following structures:

[0012]

[0013] or a pharmaceutically acceptable salt thereof, wherein each of the eight linker-payloads is individually covalently bound to the activatable antibody via one of the eight cysteines.

[0014] In another aspect, the present disclosure provides an immunoconjugate comprising: (a) an activatable antibody, the activatable antibody comprising: (i) an EpCAM antibody or an EpCAM-binding fragment thereof, comprising: (1) a heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence NYYIH (SEQ ID NO: 13); (2) a heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14); (3) a heavy chain complementary determining region 3 (CDR3) (VH-CDR3) comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15); (4) a light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO: 42); (5) a light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence QTSNLAS (SEQ ID NO: 43); NO:40); (ii) a light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence AQNLELPNT (SEQ ID NO:41); (iii) a cleavable portion coupled to the EpCAM antibody or EpCAM-binding fragment thereof, the cleavable portion comprising the amino acid sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO:168) or the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO:169); and (iv) a masking portion coupled to the EpCAM antibody or EpCAM-binding fragment thereof, the masking portion comprising the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: NO: 155), wherein the masking moiety inhibits binding of the antibody or antibody fragment to EpCAM when the activatable antibody is in an uncleaved state, wherein the activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: (masking moiety)-(cleavable moiety)-(antibody or antibody fragment) or (antibody or antibody fragment)-(cleavable moiety)-(masking moiety); and (b) at least one linker-payload depicted by the following structure:

[0015]

[0016] or a pharmaceutically acceptable salt thereof.

[0017] In some embodiments, the immunoconjugate has a drug to antibody ratio (DAR) of 8.

[0018] In some embodiments, the activatable antibody is a full-length human IgG1 EpCAM antibody having eight cysteines that form four interchain disulfide bonds when in the native conformation, and wherein each linker-payload is individually covalently bound to the activatable antibody via one of the eight cysteines.

[0019] In another aspect, the present disclosure provides an immunoconjugate comprising: (a) an activatable antibody, the activatable antibody comprising: (i) a full-length human IgG1 EpCAM antibody having eight cysteines that form four interchain disulfide bonds when in a native conformation, the full-length human IgG1 EpCAM antibody comprising: (1) a heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence NYYIH (SEQ ID NO: 13); (2) a heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14); (3) a heavy chain complementary determining region 3 (CDR3) (VH-CDR3) comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15); (4) a heavy chain complementary determining region 3 (CDR3) comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO: 16). NO:42); (5) a light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence QTSNLAS (SEQ ID NO:40); (6) a light chain complementary determining region 3 (VL-CDR3) comprising the amino acid sequence AQNLELPNT (SEQ ID NO:41); (ii) a cleavable portion conjugated to the EpCAM antibody or EpCAM-binding fragment thereof, the cleavable portion comprising the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO:169); and (iii) a masking portion conjugated to the EpCAM antibody or EpCAM-binding fragment thereof, the masking portion comprising the amino acid sequence WWPPCQGGAWCEQRI (SEQ ID NO: 155), wherein the masking moiety inhibits binding of the antibody or antibody fragment to EpCAM when the activatable antibody is in an uncleaved state, wherein the activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: (masking moiety)-(cleavable moiety)-(antibody) or (antibody)-(cleavable moiety)-(masking moiety); and (b) eight linkers-payloads depicted by the following structures:

[0020]

[0021] or a pharmaceutically acceptable salt thereof, wherein each of the eight linker-payloads is individually covalently bound to the activatable antibody via one of the eight cysteines.

[0022] 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.

[0023] In another aspect, the disclosure provides an immunoconjugate obtainable by the method of Example 7.4.1 provided herein.

[0024] On the other hand, the present specification provides an immunoconjugate obtainable by the following method, the method comprising: (a) mixing an activatable EpCAM antibody composed of a heterotetramer of two heavy chains and two light chains with a mixture of EPPS buffer and EDTA aqueous solution to form an activatable EpCAM reaction mixture, wherein the two heavy chains are each composed of the amino acid sequence of SEQ ID NO: 103, and the two light chains are each composed of the amino acid sequence of SEQ ID NO: 179; (b) mixing an activatable EpCAM antibody composed of a heterotetramer of two heavy chains and two light chains with a mixture of EPPS buffer and EDTA aqueous solution to form an activatable EpCAM reaction mixture, wherein the two heavy chains are each composed of the amino acid sequence of SEQ ID NO: 103, and the two light chains are each composed of the amino acid sequence of SEQ ID NO: 179; (c) mixing the reduced activatable EpCAM reaction mixture with a camptothecin linker-toxin stock solution comprising a linker-toxin represented by the following structure

[0025]

[0026] The immunoconjugate is thereby formed.

[0027] In another aspect, the present disclosure provides a nucleic acid comprising one or more coding regions of an activatable antibody of an immunoconjugate described herein. A vector comprising such a nucleic acid is also provided. In some embodiments, the vector is a viral vector.

[0028] In another aspect, the present specification provides a method for producing an immunoconjugate, comprising: (a) culturing a host cell comprising a vector of the present disclosure; (b) isolating an activatable antibody from the host cell; and (c) conjugating at least one linker-payload reactant comprising camptothecin or a derivative thereof to the activatable antibody.

[0029] In another aspect, the present disclosure provides a method of producing an immunoconjugate, the method comprising conjugating at least one linker-payload reactant comprising camptothecin or a derivative thereof to an activatable EpCAM antibody described herein.

[0030] In some embodiments, the linker-payload reactant comprises a compound of Formula I:

[0031] EALD (Formula I)

[0032] in:

[0033] EA-Z'-L 1 -D (Formula I),

[0034] in:

[0035] D is represented by the following structural formula:

[0036]

[0037] where R 1 is F and R 2 It is a methyl group;

[0038] -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 of covalent attachment to A;

[0039] Each R 5 are independently H, methyl or benzyl;

[0040] Each R 8 are independently H, methyl or benzyl;

[0041] A is a peptide comprising 2 to 4 amino acids; and

[0042] E is -C(=O)-(C1-C 10 Alkylene)-X 3 , where X 3 yes

[0043]

[0044] In some embodiments, the linker-payload reactant is:

[0045]

[0046] or a pharmaceutically acceptable salt thereof.

[0047] In some embodiments, the linker-payload reactant is:

[0048]

[0049] or a pharmaceutically acceptable salt thereof.

[0050] In certain embodiments, the linker-payload reactant comprises exatecan. In other embodiments, the linker-payload reactant comprises delutecan.

[0051] Also provided are compositions comprising the immunoconjugates of the disclosure and a pharmaceutically acceptable carrier.

[0052] In some aspects, the immunoconjugates or compositions of the present disclosure can be used to treat cancers that express EpCAM. In some embodiments, the cancer that expresses EpCAM is colorectal cancer. 5. Description of the Figures

[0053] Figure 1A Shown are camptothecin derivative linker-payloads used in accordance with certain embodiments of the present disclosure.

[0054] Figure 1B Shown is the conjugation of a camptothecin-derived (CPT66) linker-payload to an activatable EpCAM antibody according to certain embodiments of the present disclosure.

[0055] Figure 2 A sketch depicting an activatable EpCAM antibody immunoconjugate according to certain embodiments of the present disclosure is shown. The depicted embodiment includes an EpCAM antibody, a cleavable substrate linker, and a mask. In the uncleaved (inactive) state, the mask inhibits the binding of the EpCAM antibody to EpCAM. The cleavable substrate linker can be cleaved by a protease. After cleavage, the mask is released and the antibody is free to bind to EpCAM. The depicted embodiment includes 8 conjugated camptothecin-derived (CPT66) linkers-payloads (shown as ovals in the sketch). The conjugation of the linker-payload to the activatable EpCAM antibody is random, wherein conjugation occurs at the interchain cysteines of the antibody.

[0056] Figures 3A-3D OV90 with various EpCAM linker-payloads is shown ( Figure 3A )、SNU182( Figure 3B )、KYSE270( Figure 3C ) and SNU1( Figure 3D ) cancer cell lines. EpCAM-DM21 was conjugated to a DM21 maytansinoid linker payload with a DAR of 4; EpCAM-MMAT was a site-specific conjugation of an auristatin derivative via a valine-citrulline linker with a DAR of 2; EpCAM-mGGFG-DXd was conjugated to a linker-payload for fam-trastuzumab delutocin-nxki with a DAR of 8; EpCAM-mGGFG-CPT66 was a CPT66 payload conjugated to a GGFG linker (SEQ ID NO: 310) from fam-trastuzumab delutocin-nxki with a DAR of 8; and EpCAM-Lala-ala-ala-CPT66 was conjugated to a CPT66 payload using a tripeptide linker (ala-ala-ala) with a DAR of 8. OV90 is an ovarian cancer cell line that expresses approximately 375,149 EpCAM receptors on the cell surface, SNU182 is a liver cancer cell line with 18,898 EpCAM receptors, KYSE270 is an esophageal cancer cell line with 136,409 EpCAM receptors, and SNU1 is a gastric cancer cell line with 425 EpCAM receptors. In cell lines with different EpCAM receptor expression, the potency of EpCAM-CPT was comparable to that of the EpCAM-GGFG-DXd ADC.

[0057] Figures 4A-4E Show use Figures 3A-3D The cytotoxicity of 43 cancer cell lines treated with various EpCAM linker payloads listed in the Table 1 was evaluated. The cytotoxic activity is shown as the area under the curve (AUC), which is the area under the curve. Figures 3A-3D The AUCs of the cytotoxicity curves shown in the table are integrated, and the lower the AUC, the more potent the ADC. The mean AUCs for each cancer indication are plotted, and the number of cell lines tested is indicated in parentheses. Error bars represent standard error measurements. The data demonstrate that breast, CRC, ovarian, and head and neck cancer cell lines are relatively sensitive to the EpCAM-CPT66 ADC, while bladder, uterine, liver, and esophageal cancer cell lines are relatively insensitive to the ADC.

[0058] Figures 5A-5B EpCAM-CPT66 immunoconjugates were shown to be effective in two colorectal cancer cell line-derived xenograft models: HCT116 ( Figure 5A ) and HT29( Figure 5B) in vivo anti-tumor evaluation in a 24-hour RT-PCR assay. The EpCAM-activatable antibody was conjugated to CPT66 with a tripeptide (L-ala-L-ala-L-ala) linker with a DAR of 8 (EpCAM-CPT66) and compared to EpCAM-GGFG-DXd (DAR of 8) conjugated to a linker-payload similar to that of fam-trastuzumab delutec-nxki. All immunoconjugates and isotype control-ADCs were administered as a single dose via intravenous injection on day 0 at the indicated mg / kg (mpk). The anti-tumor activity of EpCAM-CPT66 was comparable to that of EpCAM-GGFG-DXd.

[0059] Figures 6A-6F In vivo anti-tumor evaluation of EpCAM-CPT66 in colorectal patient-derived xenograft (PDX) models is shown. Each model was dosed with 6 mg / kg (mpk) of EpCAM-CPT66 and compared to the same dose of EpCAM-GGFG-DXd. The immunoconjugate was administered once every two weeks for a total of three doses (Q2Wx3). The overall anti-tumor activity of EpCAM-CPT66 was comparable to that of EpCAM-GGFG-DXd.

[0060] Figure 7 Figure 2 shows the evaluation of the anti-tumor activity of EpCAM-CPT66 in various PDX models. The drug dose was 6 mg / kg and administered every 2 weeks for a total of 3 doses (Q2Wx3). The percentage of tumor volume reduction relative to the start of dosing was calculated by tumor volume percentage change = [post-treatment TV - initial TV) / initial TV) x 100. The minimum (highest negative change) percentage was used to compare drug treatment activity. Breast and colorectal PDX tumors were relatively sensitive to EpCAM-CPT66 treatment.

[0061] Figure 8 Figure 2 shows the pharmacokinetic profile of EpCAM-CPT66 from a dose range-finding experiment in cynomolgus monkeys. Cynomolgus monkeys were intravenously administered 10, 30, and 60 mg / kg of EpCAM-CPT66 every two weeks for a total of three doses (Q2W x 3). Total immunoconjugate (activatable antibody + linker-payload) concentrations in monkey plasma were determined by ELISA at different sample collection times. The figure shows that drug exposure was consistent across the three doses, and no significant anti-drug antibodies were observed that affected drug clearance.

[0062] Figure 9 Shown is the in vivo evaluation of the cytotoxicity of EpCAM-CPT66 and EpCAM(-)-CPT in the HCT116 colorectal cancer cell line.

[0063] Figure 10 Depicted is a summary of the cytotoxicity of unmasked EpCAM(-)-CPT66 against 57 cell lines from 11 cancer indications. 6. Specific Implementation Methods

[0064] The present disclosure provides immunoconjugates having antibodies and antigen-binding antibody fragments that specifically bind to human EpCAM, including activatable antibodies (activatable forms of EpCAM antibodies or EpCAM-binding antibody fragments thereof). In some cases, the immunoconjugate comprises a camptothecin derivative linker-payload having the following structure (wherein the following structure represents the linker-payload before conjugation to the antibody, i.e., the linker-payload reactant):

[0065]

[0066] Thus, in some cases, the immunoconjugate comprises a camptothecin derivative linker-payload having the following structure (where ** indicates the position of covalent attachment to the EpCAM antibody or activatable antibody):

[0067]

[0068] EpCAM is known to be associated with cell-cell adhesion in epithelial cells and is involved in cell signaling, differentiation, proliferation and migration. Overexpression of EpCAM has been implicated in the pathogenesis of diseases and conditions such as cancer. For example, EpCAM is highly expressed in a variety of cancer types, such as 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.

[0069] 6.1 Definitions

[0070] To facilitate understanding, several terms and phrases are defined below.

[0071] Unless otherwise indicated, the term "epithelial cell adhesion molecule" or "EpCAM" as used herein refers to any native human EpCAM. The term also encompasses naturally occurring variants of EpCAM, e.g., splice variants, allelic variants, and isoforms. EpCAM polypeptides can be isolated from a variety of sources, such as from human or cynomolgus monkey tissue or other biological samples, or prepared by known recombinant or synthetic methods. EpCAM is also known as CD326, 17-1A antigen, HEA125, MK-1, EGP-2, EGP314, EGP40, GA733-2, KSA, TACSTD1, TROP1, KS1 / 4, M4S1, DIAR5, MIC18, HNPCC8, and ESA. Examples of EpCAM sequences include, but are not limited to, NCBI reference number NP_002345.2 (amino acid residues 24-314 correspond to mature EpCAM, and amino acids 24-265 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 1-36 of SEQ ID NO: 1 (SEQ ID NO: 2)), D2 (amino acids 43-112 of SEQ ID NO: 1 (SEQ ID NO: 3)), and D3 (amino acids 113-243 of SEQ ID NO: 1 (SEQ ID NO: 4)).

[0072] As used herein, the terms "antibody" and "antigen-binding antibody fragment" and the like include any protein- or peptide-containing molecule comprising 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, such as, but not limited to, wherein such an antibody modulates, decreases, increases, antagonizes, agonizes, partially agonizes, partially antagonizes, reduces, alleviates, blocks, inhibits, eliminates, and / or interferes with at least one EpCAM activity or binding in vitro, in situ, in vivo, and / or ex vivo. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof affects at least one EpCAM-mediated activity or function selected from the group consisting of ligand binding, receptor signaling, membrane association, cell migration, cell proliferation, receptor binding activity, RNA, DNA, or protein production and / or synthesis.

[0073] Antibodies are heterotetrameric glycoproteins consisting of two identical light chains (LC) and two identical heavy chains (HC). Typically, each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has an intrachain disulfide bridge at intervals. Each heavy chain has a variable domain (VH) at one end, followed by multiple 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 is aligned with the first constant domain of the heavy chain, and the light chain variable region is aligned with the variable region of the heavy chain. The light chain of antibodies of any vertebrate species can be designated as one of two distinctly different types (i.e., κ and λ) based on the amino acid sequence of its constant domain. Depending on the heavy chain constant domain amino acid sequence, immunoglobulins can be divided into five major categories, i.e., IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subclassified into isotypes IgAl, IgA2, IgGl, IgG2, IgG3, and IgG4.

[0074] The term "antibody" also includes fragments, designated portions, and variants thereof, including antibody mimics or portions of antibodies comprising the structure and / or function of a simulated 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, either alone or in combination with other antigens. For example, antibody fragments capable of binding to an antigen or portion thereof include, but are not limited to, Fab (e.g., obtained by papain digestion), Fab' (e.g., obtained by pepsin digestion and partial reduction), and F(ab')2 (e.g., obtained by pepsin digestion), facb (e.g., obtained by plasmin digestion), pFc' (e.g., obtained by pepsin or plasmin digestion), Fd (e.g., obtained by pepsin digestion, partial reduction, and reaggregation), Fv, or scFv (e.g., obtained by molecular biology techniques) fragments encompassed by the present disclosure (see, e.g., Colligan, Immunology).

[0075] Such fragments can be produced by enzymatic cleavage, synthesis or recombinant techniques as known in the art and / or as disclosed herein. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combination gene encoding a F(ab')2 heavy chain portion can be designed to include a DNA sequence encoding the CH1 domain and / or hinge region of the heavy chain. The various portions of the antibody can be chemically joined together by conventional techniques, or can be prepared as contiguous proteins using genetic engineering techniques.

[0076] The term "antibody fragment" refers to a portion of an intact antibody, typically the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain (scFv) and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single-Fab arm "one-arm" antibodies, and multispecific antibodies formed from antibody fragments (each having a VH and VL).

[0077] Antibody fragments include any protein or peptide containing molecules that comprise at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one complementarity determining region (CDR) of a heavy or light chain, or a ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof, or at least a portion of an antigen or antigen receptor or binding protein, which can be incorporated into an EpCAM antibody provided herein.

[0078] The term "full-length antibody" refers to an antibody in its substantially intact form, rather than an antibody fragment as defined above. The term particularly refers to antibodies containing heavy chains of a hinge region and an Fc region, including modified Fc regions (e.g., mutated and / or trimmed hinge regions).

[0079] The term "variable" refers to that some parts of the variable region of an antibody in an antibody are very different in sequence and are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed in the variable region of an antibody. In the light chain variable region and the heavy chain variable region, variability is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions. The more highly conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each include four FR regions, mainly using a β-sheet configuration connected by three CDRs, which form a loop connecting the β-sheet structure and form a part of the β-sheet structure in some cases. The CDRs in each chain are kept together closely together by the FR region, and together with the CDRs from another chain, contribute to the formation of the antigen binding site of the antibody. Although the constant domains do not directly participate in the binding of antibodies to antigens, they exhibit various effector functions, such as antibody participation in antibody-dependent cellular toxicity. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., J. Molec. Biol. 273:927-948 (1997)). In addition, a combination of these two methods is sometimes used in the art to determine CDRs.

[0080] The Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest. 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) is generally used when referring to residues in the variable region (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain).

[0081] The amino acid position numbering in Kabat refers to the numbering system of the heavy chain variable region or light chain variable region compiled by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain a shortening of the FR or CDR corresponding to the variable region or several or additional amino acids inserted into the FR or CDR. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (according to Kabat's residue 52a) and an insertion residue after heavy chain FR residue 82 (e.g., according to Kabat's residues 82a, 82b, and 82c, etc.). The Kabat numbering of the residues of a given antibody can be determined by comparing the homology region of the antibody sequence with the "standard" Kabat numbering sequence. Chothia refers to the position of the structural loop (Chothia et al., J. Mol. Biol. 196: 901-917 (1987)). When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if either 35A or 35B is absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0082] The terms "EpCAM antibody," "EpCAM antibody," "antibody that specifically binds to EpCAM," "EpCAM-binding antibody fragments thereof," and "antibody fragment that specifically binds to EpCAM" refer to antibodies that are capable of binding to EpCAM with sufficient affinity to render the antibody useful as a diagnostic and / or therapeutic agent in targeting EpCAM. The extent of binding of the anti-EpCAM antibody to unrelated, non-EpCAM proteins is less than about 10% of the binding of the antibody to EpCAM, as measured, for example, by radioimmunoassay (RIA).

[0083] As in Kabat, amino acid position numbering refers to the numbering system of heavy chain variable domain or light chain variable domain compiled by Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain a shortening of the FR or CDR corresponding to the variable domain or several or other amino acids inserted into the FR or CDR. For example, the heavy chain variable region may include a single amino acid insertion after residue 52 of H2 (according to Kabat's residue 52a) and an insertion residue after heavy chain FR residue 82 (e.g., according to Kabat's residues 82a, 82b, and 82c, etc.). The Kabat numbering of the residues of a given antibody can be determined by comparing the homology region of the antibody sequence with the "standard" Kabat numbering sequence. Chothia refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)), where loops are also identified as light and heavy chain CDRs (e.g., L1 = LC CDR1, etc.). When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if 35A or 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0084] Table 1. Amino acid position numbering system

[0085]

[0086] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. An epitope is typically composed of chemically active surface groups of a molecule (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 by continuous amino acids and non-continuous amino acids juxtaposed by the tertiary folding of the protein. The epitope formed by continuous amino acids is typically retained during protein denaturation, while the epitope formed by the tertiary folding is typically lost during protein denaturation. An epitope typically includes at least 3, and more typically at least 5 or 8-10 amino acids in a unique spatial conformation.

[0087] A "blocking" antibody is an antibody that inhibits or reduces the biological activity of an 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 the activity of the tyrosine kinase associated with EpCAM by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

[0088] An "isolated" antibody is one that is separated and / or recovered from its natural environment. Contaminating components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes. In particular aspects, the antibody will be purified to (1) greater than 95% by weight of the antibody as determined by, for example, the Lowry method, and in some cases greater than 99% by weight of the antibody, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence as determined using a spinning cup sequencer, or (3) to homogeneity as determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) under reducing or non-reducing conditions using Coomassie blue or, in some cases, silver staining. Isolated antibodies include in situ EpCAM antibodies within recombinant cells, as at least one component of the antibody's natural environment will not be present. However, typically, the isolated antibody will be prepared by at least one purification step.

[0089] The term "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 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 monkey EpCAM) binding antibody fragments, and / or antibodies comprising at least one human heavy and / or light chain polypeptide, such as, for example, an antibody comprising a murine light chain and a human heavy chain polypeptide.

[0090] As used herein, the term "chimeric antibody" refers to an antibody in which the sequence of the immunoglobulin molecule is 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 with the desired specificity, affinity, and capacity derived from one mammalian species (e.g., mouse, rat, rabbit, etc.), while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

[0091] As used herein, the term "humanized antibody" refers to a form of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or antigen-binding antibody fragments containing minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins (e.g., Jones et al., Nature 321:522-525 (1986) in which residues from complementary determining regions (CDRs) are replaced with residues from non-human species (e.g., mouse, rat, rabbit, hamster) CDRs with desired specificity, affinity, and ability; 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 human immunoglobulin are replaced with corresponding residues from antibodies from non-human species with desired specificity, affinity, and ability. Humanized antibodies can be further modified to improve and optimize antibody specificity, affinity and / or ability by replacing the extra residues in the Fv framework region and / or the non-human residues of the replacement.In general, antibody will substantially comprise at least one and usually two or three variable regions, and the variable region contains all or substantially all CDR regions corresponding to non-human immunoglobulins, and all or substantially all FR regions are those of human immunoglobulin consensus sequences.Antibody can also comprise at least a portion (Fc) of an immunoglobulin constant region or domain, typically a part for human immunoglobulin.The example of the method for producing humanized antibodies is described in U.S. Patent No. 5,225,539.

[0092] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) of an antibody and vary with the antibody isotype. 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).

[0093] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In certain aspects, the cells express at least FcγRIII and perform ADCC or ADCP effector functions. 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, for example, from blood.

[0094] As used herein, the term "Fc region" includes a polypeptide comprising the constant region of an antibody except the first constant region immunoglobulin domain. Therefore, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. For IgA and IgM, Fc may include a J chain. For IgG, Fc includes immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and a hinge between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as comprising C226 or P230 to its carboxyl-terminal residue, wherein numbering is according to the EU index in Kabat et al., (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). "EU index as set forth in Kabat" refers to the residue numbering of the human IgG1 EU antibody as described in Kabat et al., supra. Fc can refer to this region in isolation or in the context of an antibody, antibody fragment, or Fc fusion protein. An Fc variant protein can be an antibody, an Fc fusion, or any protein or protein domain that comprises an Fc region. Certain proteins comprise variant Fc regions that are non-naturally occurring variants of Fc. Polymorphisms have been observed at many Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, and thus subtle differences may exist between the sequences presented and those in the prior art, and those skilled in the art will understand based on the present teachings.

[0095] As used herein, the terms "conjugate," "immunoconjugate," "ADC," or "AADC" refer to a compound or derivative thereof linked to a cell-binding agent (i.e., an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody) and are defined by the following general formula: CLA, wherein C = compound, L = linker, and A = EpCAM-binding agent (EpBA) (e.g., an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody, as disclosed herein). In some embodiments, the general formula: DLA, wherein D = drug, L = linker, and A = cell-binding agent (e.g., an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody) can also be used in the same manner. In some cases, the immunoconjugates described herein comprise a camptothecin derivative linker-payload.

[0096] A "linker" is a compound (usually a drug such as a maytansinoid, camptothecin derivative, or indolinone benzodiazepine) that can be attached to Compound) is linked to any chemical moiety of a cell binding agent such as an anti-EpCAM antibody or an EpCAM-binding antibody fragment thereof. The linker may be susceptible to or substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups, and esterase-labile groups. Linkers also include peptide linkers and charged linkers and their hydrophilic forms as disclosed herein and known in the art.

[0097] As used herein, unless otherwise indicated, "abnormal cell proliferation" refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition). This includes, for example, the following abnormal growth: (1) tumor cells (tumors) that proliferate by expressing mutant tyrosine kinases or overexpressing receptor tyrosine kinases; (2) benign and malignant cells of other proliferative diseases with aberrant tyrosine kinase activation; (3) any tumor that proliferates due to receptor tyrosine kinases; (4) any tumor that proliferates due to aberrant serine / threonine kinase activation; (5) benign and malignant cells of other proliferative diseases with aberrant serine / threonine kinase activation, and (6) benign and malignant cells of other proliferative diseases.

[0098] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" includes one or more cancerous cells. The terms "cancer" or "cancerous," as defined herein, include "precancerous" conditions that, if left untreated, can progress to cancerous conditions. In some embodiments, the cancer is an epithelial cancer. In some embodiments, the cancer expresses EpCAM. Examples of cancer include breast cancer, lung cancer, non-small cell lung cancer, gastric cancer, colorectal cancer, prostate cancer, bladder cancer, ovarian cancer, colon cancer, rectal cancer, cancers comprising cancer stem cells, uterine cancer, gastric cancer, head and neck cancer, endometrial cancer, and pancreatic cancer.

[0099] The terms "cancer cells," "tumor cells," and grammatical equivalents refer to the total cell population derived from a tumor or precancerous lesion and include both non-tumorigenic cells (which comprise the bulk of the tumor cell population) and tumorigenic stem cells (cancer stem cells).

[0100] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents one or more cellular functions and / or causes cell death.

[0101] As used herein, "treatment" refers to clinical intervention that attempts to alter the natural course of the individual or cell being treated, and can be performed for prevention or during clinical pathology. Desirable therapeutic effects include: preventing the occurrence or recurrence of the disease, alleviating symptoms, eliminating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the methods and compositions provided herein can be used to attempt to delay the development of a disease or condition.

[0102] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both (1) therapeutic measures that cure, alleviate, lessen the symptoms of, and / or arrest the progression of, a diagnosed pathological condition or disorder, and (2) prophylactic or preventative measures that prevent and / or slow the development of a targeted pathological condition or disorder. Thus, subjects in need of treatment include: those already suffering from the disorder; those predisposed to the disorder; and those in whom the disorder is to be prevented. In certain embodiments, a subject's cancer is successfully "treated" according to the methods provided herein if the patient exhibits one or more of the following: a decrease in the number of cancer cells or the complete absence of cancer cells; a decrease in tumor size; inhibition or absence of cancer cell infiltration into surrounding organs, including, for example, spread of cancer into soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; alleviation of one or more symptoms associated with a particular cancer; reduced morbidity and mortality; improved quality of life; a decrease in the tumorigenicity, frequency of tumorigenicity, or tumorigenic capacity of a tumor; a decrease in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non-tumorigenic state; or some combination of these effects.

[0103] An "effective amount" of an antibody as disclosed herein is an amount sufficient to carry out the purpose specifically stated. A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the necessary dosage and for the period of time. A "therapeutically effective amount" of a therapeutic agent (e.g., a conjugate or immunoconjugate) may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.

[0104] "Therapeutic agent" encompasses biological agents such as antibodies, peptides, proteins, enzymes, chemotherapeutic agents, or conjugates or immunoconjugates.

[0105] The terms "subject," "individual," "animal," "patient," and "mammal" refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include, but are not limited to, humans, non-human primates, domestic animals, farm animals, rodents, and the like, who are to be the recipients of a particular treatment.

[0106] The terms "polynucleotide" or "nucleic acid" as used interchangeably herein refer to polymers of nucleotides of any length, and include DNA and RNA. The 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. The polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "capping," substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen), those containing chelators (e.g., metals, radioactive metals, boron, oxidized metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of one or more polynucleotides. In addition, any hydroxyl that is usually present in sugars can be replaced by (for example) phosphonate group, phosphate group, protected by standard protecting groups or activated to provide other linkages with other nucleotides, or can be put together with solid support.5' and 3' terminal OH can be replaced by phosphorylation or by amine or organic end-blocking group part with 1 to 20 carbon atoms.Other hydroxyl groups can also be derivatized into standard protecting groups.Polynucleotides can also contain similar forms of ribose or deoxyribose well known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomer sugars, epimerized sugars (such as arabinose, xylose or lyxose), pyranose, furanose, sedoheptulose, acyclic analogs and no base nucleoside analogs (such as methyl nucleosides).One or more phosphodiester linkages can be replaced by alternative linking groups.These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl group optionally containing an ether (-O-) linkage. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0107] The term "vector" means a construct capable of delivering and optionally expressing one or more target genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (e.g., production cells).

[0108] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0109] As known in the art, the term "identical" or "identity" percentage is a measure of the relationship between two polynucleotides or two polypeptides determined by comparing the sequences of the two polynucleotides or two polypeptides. Sequence identity or similarity is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., identical residues) or similar (i.e., amino acid residues from the same group based on common side chain properties, see below) to the EpCAM antibody residues, after aligning the sequences and introducing spaces (if necessary) to achieve the maximum sequence identity percentage. N-terminal, C-terminal or internal extensions, deletions or insertions into the antibody sequence outside the variable region should not be considered to affect sequence identity or similarity. In general, 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 that give the exact amino acid or nucleotide correspondence between the two sequences is determined, divided by the total length of the alignment and multiplied by 100 to obtain the identity number %. This % identity figure can be determined over the entire length of the sequences to be compared, which is particularly suitable for sequences that are identical or very similar in length and highly homologous, or it can be determined over a shorter, defined length, which is more suitable for sequences that are unequal in length or have a lower level of homology. Similarly, the percentage similarity can be determined in a similar manner based on the presence of both identical and similar residues.

[0110] Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Different algorithms and software that can be used to obtain the comparison of amino acid or nucleotide sequences are known in the art. Such a non-limiting example of a sequence alignment algorithm is described in Karlin et al., Proc.Natl.Acad.Sci.87:2264-2268 (1990), in Karlin et al., Proc.Natl.Acad.Sci.90:5873-5877 (1993) and incorporated into the algorithm of NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1991)). In certain embodiments, gap 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 is another publicly available software program that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software (e.g., using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package that incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either the Blossum 62 matrix or the PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, or 5). Alternatively, in certain embodiments, Myers and Miller (CABIOS 4: 11-17 (1989)) are used to determine the percent identity between nucleotide or amino acid sequences. For example, the ALIGN program (version 2.0) and PAM120 with a residue table, a gap length penalty of 12, and a gap penalty of 4 can be used to determine the percent identity. Appropriate parameters for achieving maximum alignment by specific 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 percent identity "X" of a first amino acid sequence to a second amino acid sequence is calculated as 100 × (Y / Z), where Y is the number of amino acid residues scored as identical matches in the comparison of the first sequence and the second sequence (as compared by visual inspection or a specific 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.

[0111] As non-limiting examples, in certain embodiments, Bestfit program (Wisconsin sequence analysis package, Unix version 8, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711) can be used to determine whether any particular polynucleotide has a certain percentage of sequence identity (for example, 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) with a reference sequence. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the best segment with 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 as provided herein, parameters are set so that on the full length of the reference nucleotide sequence, percent identity is calculated and a homology gap of 5% of the total nucleotides in the reference sequence is allowed.

[0112] " conservative amino acid substitution " is that one of amino acid residues is replaced by another amino acid residue with similar side chain. The family of amino acid residues with similar side chain has been defined in the art, including for example basic side chain (for example, lysine, arginine, histidine), acidic side chain (for example, aspartic acid, glutamic acid), uncharged polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chain (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chain (for example, threonine, valine, isoleucine) and aromatic side chain (for example, tyrosine, phenylalanine, tryptophan, histidine). For example, replacing tyrosine with phenylalanine is conservative substitution. In some embodiments, the conservative substitution in the sequence of polypeptide and antibody provided herein will not eliminate the combination of the polypeptide or antibody containing the amino acid sequence with the antigen to which the polypeptide or antibody is bound. 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)).

[0113] As used herein, "alkyl" refers to a saturated straight or branched monovalent hydrocarbon group of one to twenty carbon atoms. Examples of alkyl groups 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, the alkyl group has one to ten carbon atoms. In some embodiments, an alkyl group has one to four carbon atoms.

[0114] The number of carbon atoms in a group may be designated 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.

[0115] The terms "compound" or "cytotoxic compound" or "cytotoxic agent" are used interchangeably. They are intended to include compounds having disclosed herein a structure or formula or any derivative thereof, or compounds having been incorporated by reference into a structure or formula or any derivative thereof. The term also includes stereoisomers, geometric isomers, tautomers, solvates, metabolites and salts (such as pharmaceutically acceptable salts) of compounds of all formulas disclosed herein. The term also includes any of the aforementioned solvates, hydrates and polymorphs. In certain embodiments, the specific description of "stereoisomers," "geometric isomers," "tautomers," "solvates," "metabolites," "salts," "conjugates," "conjugate salts," "solvation," "hydrates" or "polymorphs" provided herein should not be interpreted as an expected omission of these forms in other disclosed embodiments, wherein the term "compound" is used when these other forms are not listed. In some cases, the compound comprises a camptothecin derivative linker-payload.

[0116] The term "imine-reactive reagent" refers to a reagent that is capable of reacting with an imine group. Examples of imine-reactive reagents include, but are not limited to, sulfites (H2SO3, H2SO2, or HSO3 - 、SO3 2- or HSO2 - salts with cations), metabisulfite (H2S2O5, or S2O5 2-salts with cations), monothiophosphates, dithiophosphates, trithiophosphates, and tetrathiophosphates (PO3SH3, PO2S2H3, POS3H3, PS4H3, or PO3S3 - PO2S2 3- POS3 3- or PS4 3- salts formed with cations), phosphorothioates ((RiO)2PS(ORi), RiSH, RiSOH, RiSO2H, RiSO3H), various amines (hydroxylamine (e.g., NH2OH), hydrazine (e.g., NH2NH2), NH2O-Ri, Ri'NH-Ri, NH2-Ri), NH2-CO-NH2, NH2-C(=S)-NH2, thiosulfate (H2S2O3, or S2O3 2- salts with cations), dithionites (H2S2O4, or S2O4 2- with cations), dithiophosphates (P(=S)(ORk)(SH)(OH) or salts thereof with cations), hydroxamic acid (RkC(=O)NHOH or salts thereof with cations), hydrazides (RkCONHNH2), formaldehyde sulfoxylates (HOCH2SO2H, or HOCH2SO2 - Salts formed with cations, such as HOCH2SO2-Na + ), glycosylated nucleotides (such as GDP-mannose), fludarabine, or a mixture thereof, wherein Ri and Ri' are each independently a linear or branched alkyl group having 1 to 10 carbon atoms and are substituted with at least one substituent selected from N(Rj)2, -CO2H, SO3H, and PO3H; Ri and Ri' may be further optionally substituted with a substituent of an alkyl group disclosed herein; Rj is a linear or branched alkyl group having 1 to 6 carbon atoms; and Rk is a linear, branched, or cyclic alkyl group, an alkenyl or alkynyl group, an aryl, a heterocyclic group, or a heteroaryl group having 1 to 10 carbon atoms (in some embodiments, Rk is a linear or branched alkyl group having 1 to 4 carbon atoms; in some embodiments, Rk is a methyl, ethyl, or propyl group). In some embodiments, the cation is a monovalent cation such as Na + or K + In some embodiments, the imine-reactive reagent is selected from the group consisting of sulfite, hydroxylamine, urea, and hydrazine. In some embodiments, the imine-reactive reagent is NaHSO 3 or KHSO 3.

[0117] The term "cation" refers to an ion having a positive charge. A cation can be monovalent (e.g., Na + , K + NH4 + etc.), divalent (e.g., Ca 2+ Mg2+ etc.) or multivalent (e.g., Al 3+ ). In some embodiments, the cation is monovalent.

[0118] 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, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisinates, fumarates, gluconates, glucuronates, sucrose salts, formates, benzoates, glutamates, methanesulfonates (mesylate), ethanesulfonates, benzenesulfonates, p-toluenesulfonates, pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. In addition, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In cases where multiple charged atoms are part of a pharmaceutically acceptable salt, there may be multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0119] If the compound provided herein is a base, the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, for example, by treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, and the like; or an organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidic acid such as glucuronic acid or galacturonic acid, an alpha hydroxy acid such as citric acid or tartaric acid, an amino acid such as aspartic acid or glutamic acid, an aromatic acid such as benzoic acid or cinnamic acid, a sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid, and the like.

[0120] If the compound provided herein is an acid, the desired pharmaceutically acceptable salt 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 oxide or an alkaline earth metal oxide, etc. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids (such as glycine and arginine), ammonia, primary, secondary and tertiary amines, and cyclic amines (such as piperidine, morpholine and piperazine); and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

[0121] As used herein, the term "solvate" refers to a compound that further comprises 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 a compound can be readily prepared by adding at least one molar equivalent of a hydroxylic solvent such as methanol, ethanol, 1-propanol, 2-propanol, or water to the compound to produce solvation or hydration of the imine moiety.

[0122] A "metabolite" or "catabolite" is a product produced by the metabolism or catabolism of a specified compound, derivative thereof, or conjugate thereof, or salt thereof in vivo. Metabolites of a compound, derivative thereof, or conjugate thereof can be identified using conventional techniques known in the art, and their activity can be determined using assays such as those disclosed herein. Such products can be produced, for example, by oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc. of the administered compound. Thus, the present disclosure includes metabolites of a compound, derivative thereof, or conjugate thereof of the disclosed EpCAM compositions disclosed herein, including compounds, derivatives thereof, or conjugates thereof produced by a method comprising contacting a disclosed EpCAM compound, derivative thereof, or conjugate thereof with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0123] The phrase "pharmaceutically acceptable" means that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients comprising the formulation and / or the mammal to be treated therewith.

[0124] The term "protecting group" or "protecting moiety" refers to a substituent that is generally used to block or protect a specific functional group while reacting other functional groups on a compound, its derivative, or its conjugate. For example, an "amino protecting group" is a substituent that blocks or protects the amino functional group in a compound. Such groups are well known in the art (see, for example, P. Wuts and T. Greene, 2007, Protective Groups in Organic Synthesis, Chapter 7, J. Wiley & Sons, NJ), and are exemplified by carbamates such as methyl carbamate and ethyl carbamate, FMOC, substituted ethyl carbamate, carbamates (also referred to as "self-sacrifice") that are cracked by 1,6-β elimination, urea, amides, peptides, alkyl, and aryl derivatives. Suitable amino protecting groups include acetyl, trifluoroacetyl, tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyleneoxycarbonyl (Fmoc). For a general description of protecting groups and their uses, see PGM Wuts and TW Winene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 2007.

[0125] The term "amino acid" refers to naturally occurring amino acids or non-naturally occurring amino acids. In one embodiment, the amino acid is represented by NH2-C(Raa'Raa)-C(=O)OH, where Raa and Raa' are each independently H, an optionally substituted linear, branched, or cyclic alkyl, alkenyl, or alkynyl group having 1 to 10 carbon atoms, an aryl, heteroaryl, or heterocyclic group, or Raa and the N-terminal nitrogen atom may together form a heterocycle (e.g., as in proline). The term "amino acid residue" refers to the corresponding residue when one hydrogen atom is removed from the amine and / or carboxyl terminus of an amino acid, such as -NH-C(Raa'Raa)-C(=O)O-.

[0126] The term "peptide" refers to a short chain of amino acid monomers connected 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 other embodiments, the peptide contains 2 to 5 amino acid residues. As used herein, when a peptide is a part of a cytotoxic agent or a linker disclosed herein represented by a specific sequence of amino acids, the peptide can be connected to the remainder 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.

[0127] The term "reactive ester group" refers to a group of ester groups 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 esters, N-hydroxyphthalimide esters, N-hydroxysulfosuccinimide esters, p-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters, and derivatives thereof, wherein the derivatives promote amide bond formation. In certain embodiments, the reactive ester group is an N-hydroxysuccinimide ester or an N-hydroxysulfo-succinimide ester.

[0128] 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, imidoesters, 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.

[0129] 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.

[0130] As used in this disclosure and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0131] When used in this article, the term "and / or" should be regarded as a specific disclosure of each of two or more specified features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover 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).

[0132] Whenever the language "comprising" is used herein to disclose an embodiment, other similar embodiments described with respect to "consisting of" and / or "consisting essentially of" are also provided.

[0133] 6.2 Immunoconjugates Comprising EpCAM Antibodies and EpCAM-Binding Antibody Fragments Thereof

[0134] Disclosed herein are immunoconjugates comprising EpCAM antibodies and EpCAM-binding antibody fragments thereof. These proteins specifically bind to human EpCAM and are provided herein. In some embodiments, the protein is referred to herein as an "EpCAM binder" or EpBA.

[0135] In other embodiments, the immunoconjugates disclosed herein comprise 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 other embodiments, the EpCAM antibody specifically binds to human EpCAM and cynomolgus monkey EpCAM. In some embodiments, the EpCAM antibody or its EpCAM binding antibody fragment is a mouse, other rodent, chimeric, humanized, or fully human monoclonal antibody.

[0136] In some embodiments, the immunoconjugates disclosed herein comprise an EpCAM antibody that is an EpCAM-binding antibody fragment. In some embodiments, the immunoconjugates disclosed herein comprise a non-human mammalian antibody, a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. In some embodiments, the immunoconjugates disclosed herein comprise a full-length antibody. In some embodiments, the immunoconjugates disclosed herein comprise a human IgG1 antibody.

[0137] In some embodiments, the immunoconjugates disclosed herein comprise Fab, Fab', F(ab')2, scFv, or disulfide-linked Fv (dsFv). In other aspects, the EpCAM-binding antibody fragment is a single-chain Fv (scFv), a disulfide-linked Fv, IgGΔCH2, a minibody, F(ab')3, scAb, a tetrabody, a triabody, a diabody, a DVD-Ig, a Fcab, a mAb 2 , (scFv)2, scFv-Fc or bi-scFv.

[0138] In some cases, the immunoconjugates disclosed herein comprise, optionally with a broad range of affinities (K D ) EpCAM antibodies and EpCAM-binding antibody fragments thereof provided herein that bind to EpCAM (e.g., human EpCAM and / or murine EpCAM). In certain embodiments, the antibodies bind to human EpCAM with high affinity. For example, human or human engineered or humanized or resurfaced mAbs can bind to human antigens with a K D Equal to or less than about 10 -7 M, such as but not limited to 0.1-9.9 (or any range or value therebetween) x10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 or 10 -12 , or any range or value therein, as determined by flow cytometry-based assays, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), or In some embodiments, the EpCAM antibody is administered at about 10 -9 M or lower, more specifically about 10 -9 to 10 - 10 Kd binding of M.

[0139] The affinity or avidity of the antibodies or antibody fragments thereof in the immunoconjugates described herein for EpCAM can be determined experimentally using standard procedures 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). TM Analysis). 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, WE, ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992); and the methods disclosed herein. The measured affinity of a particular antibody-EpCAM interaction may vary if measured under different conditions (e.g., salt concentration, pH, temperature). Therefore, measurements of affinity and other EpCAM binding parameters (e.g., KD or Kd, kon, koff) are performed using standardized solutions of antibody and EpCAM and standardized buffers as known in the art, as well as buffers such as those disclosed herein.

[0140] In one embodiment, the binding assay is performed on cells expressing EpCAM antigen on their surface using flow cytometry. For example, 1 x 10 cells are used per sample in 100 μL FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum). 5 Cells are collected and incubated with different concentrations of EpCAM antibodies. The cells are then pelleted, washed, and incubated with 100 μL of FITC-conjugated goat anti-mouse IgG antibody (such as available from Jackson ImmunoResearch) in FACS buffer for 1 hour. The cells are pelleted again, washed with FACS buffer, and resuspended in 200 μL of PBS containing 1% formaldehyde. For example, a FACSCalibur with an HTS multiwell sampler is used. TM The samples were collected by flow cytometry and used Pro (all from BD Biosciences, San Diego, US) were used for analysis. For each sample, the mean fluorescence intensity (MFI) of FL1 was output and plotted against the antibody concentration in a semi-logarithmic plot to generate a binding curve. Sigmoidal dose-response curves were fitted to the binding curves, and EC values ​​were calculated using a program such as GraphPadPrism v4 (GraphPad software, San Diego, CA) with default parameters. 50 Value. EC 50 The value can be used as the apparent dissociation constant "K" for each antibody. d ” or “K D ” measure.

[0141] 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 properties can be determined by various in vitro assays using methods including, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or kinetics (e.g., BIACORE). TM The determination is made using standard operating procedures known in the art, including ELISA.

[0142] In one embodiment, the EpCAM antibody or EpCAM-binding antibody fragment thereof in any of the immunoconjugates described herein specifically binds to human and / or cynomolgus monkey EpCAM and / or EpCAM antigen fragment thereof with a dissociation constant or KD or Kd (koff / kon) of less than 10 -5 M, or less than 10 -6 M, or less than 10 -7 M, or less than 10 -8 M, or less than 10 -9 M, or less than 10 -10 M, or less than 10 -11 M, or less than 10 -12 M, or less than 10 -13 In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment specifically binds to human and / or cynomolgus monkey EpCAM and / or its EpCAM antigen fragment with a KD of 1.0x10 -9 M or lower, 2.0x10 -9 M or lower, 3.0x10 -9 M or lower, 4.0x10 -9 M or lower, 5.0x10 -9 M or lower, 6.0x10 -9 M or lower, 7.0x10 -9 M or smaller, 8.0x10 -9M or lower, or 9.0x10 -9 In certain embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof is expressed at 3.0 x 10 -9 In some embodiments, the EpCAM antibody or its EpCAM binding antibody fragment binds to human and cynomolgus monkey EpCAM and / or EpCAM antigen fragments with a KD of about 0.4 x 10 -9 In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof binds to human EpCAM with a KD of about 0.8 x 10 -9 In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof binds to human EpCAM with a KD of about 0.8 x 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 x 10 -9 In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment binds to cynomolgus monkey EpCAM with a KD of about 2.8 x 10 -9 The KD of α-glucanase was 0.0447kJ for binding to cynomolgus monkey EpCAM.

[0143] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof in any immunoconjugate 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: D1 (SEQ ID NO: 2), D2 (SEQ ID NO: 3), and D3 (SEQ ID NO: 4). In certain embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof specifically binds to an epitope within the first extracellular domain (D1) of human EpCAM.

[0144] In one embodiment, the EpCAM antibody or EpCAM-binding antibody fragment thereof in any of the immunoconjugates described herein comprises: a VH-CDR1 comprising X1YX3X4H, wherein X1 is selected from N and S, X3 is selected from Y, N, F, S, H, D, L, I and W, and X4 is selected from I and M; a VH-CDR1 comprising WX2X3PGX6VYIQYX 12 X 13 KFX 17 VH-CDR2 of 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 Selected from N and S, X 13 Selected from E and Q, and X 17selected from the group consisting of K and Q (SEQ ID NO: 7); and a VH-CDR3 comprising X1GX3X4FAY, wherein X1 is selected from the group consisting of D and E, X3 is selected from the group consisting of P, A, S, Y, F, G, T and V, and X4 is selected from the group consisting of Y and W (SEQ ID NO: 8).

[0145] In further embodiments, the present disclosure provides an EpCAM antibody or EpCAM-binding antibody fragment thereof in any immunoconjugate described herein, wherein the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises: RSSX4SLLHSX 10 GX 12 TYLX 16 The light chain CDR1 (VL-CDR1) of 10 Selected from N and D, X 12 Selected from F and I, and X 16 selected from the group consisting of Y and S (SEQ ID NO: 10); a light chain VL-CDR2 comprising QTSNLAS (SEQ ID NO: 40); and a VL-CDR3 comprising X1QX3LELPX8T, wherein X1 is selected from the group consisting of A, L, and Q, X3 is selected from the group consisting of S, G, Y, and N, and X8 is selected from the group consisting of N and W (SEQ ID NO: 11). In some embodiments, the present disclosure provides an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising: a heavy chain CDR1 (VH-CDR1) comprising the sequence of SEQ ID NO: 13; a heavy chain CDR2 (VH-CDR2) comprising the sequence of SEQ ID NO: 14; a heavy chain CDR3 (VH-CDR3) comprising the sequence of SEQ ID NO: 15; a light chain CDR1 (VL-CDR1) comprising the sequence of SEQ ID NO: 42; a light chain CDR2 (VL-CDR2) comprising the sequence of SEQ ID NO: 40; and a light chain CDR3 (VL-CDR3) comprising the sequence of SEQ ID NO: 41.

[0146] In some embodiments, the VH-CDR1 comprises the sequence NYX3IH, wherein X3 is selected from the group consisting of Y, N, F, S, H, D, L, I, and W (SEQ ID NO: 6). In some embodiments, the VH-CDR3 comprises the sequence DGPX4FAY, wherein X4 is selected from the group consisting of Y and W (SEQ ID NO: 9). In some embodiments, the VL-CDR3 comprises the sequence AQX3LELPNT, wherein X3 is selected from the group consisting of S, G, Y, and N (SEQ ID NO: 12). In some embodiments, the present disclosure provides an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising: a heavy chain CDR1 (VH-CDR1) comprising the sequence of SEQ ID NO: 13; a heavy chain CDR2 (VH-CDR2) comprising the sequence of SEQ ID NO: 14; a heavy chain CDR3 (VH-CDR3) comprising the sequence of SEQ ID NO: 15; a light chain CDR1 (VL-CDR1) comprising the sequence of SEQ ID NO: 42; a light chain CDR2 (VL-CDR2) comprising the sequence of SEQ ID NO: 40; and a light chain CDR3 (VL-CDR3) comprising the sequence of SEQ ID NO: 41.

[0147] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof in any immunoconjugate 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 heavy chain CDRs are disclosed in Table 2.

[0148] Table 2. Exemplary heavy chain CDR sequences of EpCAM antibodies.

[0149]

[0150]

[0151]

[0152] In some embodiments, the present disclosure provides an EpCAM antibody or EpCAM-binding antibody fragment thereof in any immunoconjugate described herein, wherein the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a single row of heavy chain CDRs in Table 2. In some embodiments, the present disclosure provides an EpCAM antibody or EpCAM-binding antibody fragment thereof, wherein the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises: a VH-CDR1 selected from SEQ ID NOs: 13 and 16-25; a VH-CDR2 selected from SEQ ID NOs: 14 and 26-29; and a VH-CDR3 selected from SEQ ID NOs: 15 and 30-38. In some embodiments, the present disclosure provides an EpCAM antibody or EpCAM-binding antibody fragment thereof, wherein the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH-CDR1 of SEQ ID NO: 13; a VH-CDR2 of SEQ ID NO: 14; and a VH-CDR3 of SEQ ID NO: 15. In some embodiments, the 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: 26; and VH-CDR3 of SEQ ID NO: 15.

[0153] In one embodiment, the present disclosure provides an immunoconjugate having an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody, wherein the EpCAM antibody, the EpCAM-binding antibody fragment thereof, or the EpCAM-activatable antibody comprises: a VH-CDR1 comprising NYYIH (SEQ ID NO: 13), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; a VH-CDR2 comprising WIYPGNVYIQYNEKFKG (SEQ ID NO: 14), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and a heavy chain CDR3 comprising DGPWFAY (SEQ ID NO: 15), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

[0154] In some embodiments, the present disclosure provides an immunoconjugate having an EpCAM antibody or an EpCAM-binding antibody fragment thereof, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising a VH-CDR1 of SEQ ID NO: 22; a VH-CDR2 of SEQ ID NO: 14; and a VH-CDR3 of SEQ ID NO: 15. In some embodiments, the present disclosure provides an EpCAM antibody or an EpCAM-binding antibody fragment thereof, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising a VH-CDR1 of SEQ ID NO: 13; a VH-CDR2 of SEQ ID NO: 14; and a VH-CDR3 of SEQ ID NO: 33. In some embodiments, the present disclosure provides an EpCAM antibody or an EpCAM-binding antibody fragment thereof, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising a VH-CDR1 of SEQ ID NO: 23; a VH-CDR2 of SEQ ID NO: 14; and a VH-CDR3 of SEQ ID NO: 15. In some embodiments, the 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.

[0155] In one embodiment, the present disclosure provides an immunoconjugate having an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody, wherein the EpCAM antibody, the EpCAM-binding antibody fragment thereof, or the EpCAM-activatable antibody comprises: a VH-CDR1 comprising NYHIH (SEQ ID NO: 22), or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; a VH-CDR2 comprising WIYPGNVYIQYNEKFKG (SEQ ID NO: 14), or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; and a heavy chain CDR3 comprising DGPWFAY (SEQ ID NO: 15), or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions. In one embodiment, the present disclosure provides an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody, comprising: a VH-CDR1 comprising NYYIH (SEQ ID NO: 13), or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; a VH-CDR2 comprising WIYPGNVYIQYNEKFKG (SEQ ID NO: 14), or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions; and a heavy chain CDR3 comprising DGYWFAY (SEQ ID NO: 33), or a variant thereof comprising 1, 2, 3 or 4 conservative amino acid substitutions.

[0156] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of any immunoconjugate 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.

[0157] Table 3. Exemplary light chain CDR sequences of EpCAM antibodies.

[0158]

[0159]

[0160] In some embodiments, the present disclosure provides immunoconjugates with EpCAM antibodies or EpCAM-binding antibody fragments thereof, comprising a light chain CDR from a single row in Table 3. In some embodiments, the present disclosure provides immunoconjugates with EpCAM antibodies or EpCAM-binding antibody fragments thereof, comprising a VL-CDR1 selected from the group consisting of SEQ ID NOs: 39 and 42-45; a VL-CDR2 of SEQ ID NO: 40; and a VL-CDR3 selected from the group consisting of SEQ ID NOs: 41 and 46-51. In some embodiments, the present disclosure provides immunoconjugates with EpCAM antibodies or EpCAM-binding antibody fragments thereof, comprising a VL-CDR1 of SEQ ID NO: 42; a VL-CDR2 of SEQ ID NO: 40; and a VL-CDR3 of SEQ ID NO: 41. In some embodiments, the present disclosure provides immunoconjugates having an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising VL-CDR1 of SEQ ID NO:39; VL-CDR2 of SEQ ID NO:40; and VL-CDR3 of SEQ ID NO:41.

[0161] In one embodiment, the present disclosure provides an immunoconjugate having an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody comprising: a VL-CDR1 comprising RSSRSLLHSDGFTYLY (SEQ ID NO: 42), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; a VL-CDR2 comprising QTSNLAS (SEQ ID NO: 40), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and a VL-CDR3 comprising AQNLELPNT (SEQ ID NO: 41), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions. In one embodiment, the present disclosure provides an immunoconjugate having an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody comprising: a VL-CDR1 comprising RSSKSLLHSDGFTYLY (SEQ ID NO: 39), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; a VL-CDR2 comprising QTSNLAS (SEQ ID NO: 40), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions; and a VL-CDR3 comprising AQNLELPNT (SEQ ID NO: 41), or a variant thereof comprising 1, 2, 3, or 4 conservative amino acid substitutions.

[0162] In some embodiments, the present disclosure provides an immunoconjugate having an EpCAM antibody or EpCAM-binding antibody fragment thereof, wherein the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises: 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 immunoconjugates with EpCAM antibodies or EpCAM-binding antibody fragments 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 immunoconjugates with EpCAM antibodies or EpCAM-binding antibody fragments 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 immunoconjugates with EpCAM antibodies or EpCAM-binding antibody fragments 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 immunoconjugates with EpCAM antibodies or EpCAM-binding antibody fragments 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.

[0163] In some embodiments, the present disclosure provides immunoconjugates with EpCAM antibodies or EpCAM-binding antibody fragments 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 immunoconjugates with EpCAM antibodies or EpCAM-binding antibody fragments 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 immunoconjugates with EpCAM antibodies or EpCAM-binding antibody fragments 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 immunoconjugates with EpCAM antibodies or EpCAM-binding antibody fragments 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.

[0164] In some embodiments, the immunoconjugate with the EpCAM antibody or its EpCAM-binding antibody fragment comprises a heavy chain variable region (VH) sequence disclosed in Table 4. In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to the VH sequence disclosed in Table 4. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0165] Table 4. Exemplary heavy chain variable sequences of EpCAM antibodies.

[0166]

[0167]

[0168]

[0169]

[0170] In some embodiments, the immunoconjugates described herein comprise an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH sequence having a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, less than 15, or zero amino acid substitutions, deletions, and / or insertions compared to a reference VH sequence selected from SEQ ID NOs: 53-84. In some embodiments, the insertions, substitutions, deletions, and / or insertions are in one or more framework regions of the reference sequence. In some embodiments, the substitutions are conservative. In other embodiments, the substitutions are non-conservative.

[0171] In some embodiments, the immunoconjugates described herein comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising a VH having a sequence selected from the group consisting of 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 the group consisting of SEQ ID NOs: 53-56. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a VH having a sequence selected from the group consisting of SEQ ID NOs: 53-56. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a VH having a sequence selected from the group consisting of SEQ ID NOs: 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 the group consisting of SEQ ID NOs: 53-56. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to the sequence of SEQ ID NO: 54. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0172] In some embodiments, the immunoconjugates disclosed herein comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising a VH having a sequence selected from the group consisting of SEQ ID NOs: 75-77 and 84. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a VH having a sequence of SEQ ID NO: 75. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a VH having a sequence of SEQ ID NO: 77. 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 the group consisting of SEQ ID NOs: 75-77 and 84. In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to the sequence of SEQ ID NO: 75. In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to the sequence of SEQ ID NO: 77. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0173] In some embodiments, the immunoconjugates disclosed herein comprise an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a light chain variable region (VL) sequence disclosed in Table 5. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a VL sequence disclosed in Table 5. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0174] Table 5. Exemplary light chain variable sequences of EpCAM antibodies.

[0175]

[0176]

[0177]

[0178] In some embodiments, the immunoconjugates disclosed herein comprise an EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VL sequence having a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, less than 15, or zero amino acid substitutions, deletions, and / or insertions compared to a reference VH sequence selected from SEQ ID NOs: 86-99. In some embodiments, the insertions, substitutions, deletions, and / or insertions are in one or more framework regions of the reference sequence. In some embodiments, the substitutions are conservative. In other embodiments, the substitutions are non-conservative.

[0179] In some embodiments, the immunoconjugates disclosed herein comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising a VL having a sequence selected from SEQ ID NOs: 86-99. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a VL having a sequence of SEQ ID NO: 89. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a VL having a 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% 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 a sequence of SEQ ID NO: 89. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to the sequence of SEQ ID NO: 87. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0180] In some embodiments, the immunoconjugates disclosed herein comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising a VH comprising a sequence selected from the group consisting of SEQ ID NOs: 53-84 and a VL comprising a sequence selected from the group consisting of SEQ ID NOs: 86-89. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a VH comprising a sequence of SEQ ID NO: 54 and a VL comprising a sequence of SEQ ID NO: 89. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a VH comprising a sequence of SEQ ID NO: 54 and a VL comprising a sequence of SEQ ID NO: 87. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a VH comprising a sequence of SEQ ID NO: 55 and a VL comprising a sequence of SEQ ID NO: 87. In some embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof comprises a VH comprising a sequence of SEQ ID NO: 56 and a VL comprising a sequence of SEQ ID NO: 88. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH comprising the sequence of SEQ ID NO: 55 and a VL comprising the sequence of SEQ ID NO: 89. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH comprising the sequence of SEQ ID NO: 56 and a VL comprising the sequence of SEQ ID NO: 89.

[0181] In some embodiments, the immunoconjugates disclosed herein comprise an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising 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.

[0182] In some embodiments, the immunoconjugates disclosed herein comprise an EpCAM antibody or EpCAM-binding antibody fragment thereof that competes for binding to human EpCAM with an antibody comprising the VH and VL sequences disclosed in Tables 4 and 5, respectively.

[0183] In one embodiment, the present disclosure provides an immunoconjugate comprising an EpCAM antibody or an EpCAM-binding antibody fragment thereof that competes for binding to human EpCAM with an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL) selected from:

[0184] (a) the heavy chain variable region (VH) of SEQ ID NO: 54 and the light chain variable region (VL) of SEQ ID NO: 89;

[0185] (b) VH of SEQ ID NO: 54 and VL of SEQ ID NO: 87;

[0186] (c) VH of SEQ ID NO: 75 and VL of SEQ ID NO: 89; and

[0187] (d) VH of SEQ ID NO: 77 and VL of SEQ ID NO: 89.

[0188] If an EpCAM antibody or EpCAM-binding antibody fragment thereof (or an immunoconjugate having an EpCAM antibody or EpCAM-binding antibody fragment thereof) binds to human EpCAM to the extent that it blocks, to some extent, the binding of the reference molecule to human EpCAM, then the EpCAM antibody or EpCAM-binding antibody fragment thereof (or an immunoconjugate having an EpCAM antibody or EpCAM-binding antibody fragment thereof) is said to "compete" with the reference molecule for binding to EpCAM. The ability of proteins to compete for binding to EpCAM and thus interfere with, block, or "cross-block" each other's binding to EpCAM can be assayed by any standard competitive binding assay known in the art, including, for example, competition ELISA assays, surface plasmon resonance (SPR); Biosensor, Piscataway, NJ) or according to the method described by Scatchard et al., Ann. NY Acad. Sci. 51: 660-672 (1949). An antibody can be said to competitively inhibit binding of a reference EpCAM antibody to human EpCAM by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0189] In some embodiments, the immunoconjugate with an EpCAM antibody or its EpCAM-binding antibody fragment 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) human IgA constant region or a fragment thereof; (b) human IgD constant region or a fragment thereof; (c) human IgE constant domain or a fragment thereof; (d) human IgG1 constant region or a fragment thereof; (e) human IgG2 constant region or a fragment thereof; (f) human IgG3 constant region or a fragment thereof; (g) human IgG4 constant region or a fragment thereof; and (h) human IgM constant region or a fragment thereof. In certain embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment comprises a heavy chain constant region or a fragment thereof, such as a human IgG constant region or a fragment thereof. In other embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment comprises a heavy chain immunoglobulin constant domain that has or has been mutated to have altered effector function and / or half-life.

[0190] In some embodiments, the immunoconjugate with the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a heavy chain sequence disclosed in Table 6.

[0191] Table 6. Exemplary heavy chain full-length sequences of EpCAM antibodies.

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202] In some embodiments, the immunoconjugate with 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 immunoconjugates described herein comprises a heavy chain (HC) sequence selected from SEQ ID NOs: 102-106. In specific embodiments, the EpCAM antibody or an EpCAM-binding antibody fragment thereof of the immunoconjugates 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 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: 102-106. 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 a 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%.

[0203] In alternative embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises an HC sequence selected from the group consisting of SEQ ID NOs: 125-127 and 134. In specific embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises an HC sequence of SEQ ID NO: 125. In specific embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises an HC sequence of SEQ ID NO: 127. In some embodiments, the EpCAM antibody or 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 the group consisting of SEQ ID NOs: 125-127 and 134. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a HC sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% identical to the sequence of SEQ ID NO: 125. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0204] In a further aspect, the EpCAM antibody or EpCAM binding antibody fragment thereof of the immunoconjugate described herein comprises a light chain immunoglobulin constant region. In another aspect, the antibody comprises a human Igκ constant region or a human Igλ constant region.

[0205] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugate described herein comprises a light chain sequence disclosed in Table 7.

[0206] Table 7. Exemplary full-length light chain sequences of EpCAM antibodies.

[0207]

[0208]

[0209]

[0210] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises a light chain (LC) sequence selected from SEQ ID NOs: 137-150. In a specific embodiment, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises the LC sequence of SEQ ID NO: 140. In another embodiment, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises the LC sequence of SEQ ID NO: 138. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof 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 EpCAM-binding antibody fragment thereof 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 EpCAM-binding antibody fragment thereof comprises an LC sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identical to a sequence of SEQ ID NO: 140. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof 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%.

[0211] In further embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises a HC having a sequence selected from SEQ ID NOs: 102-134 and a LC having a sequence selected from SEQ ID NOs: 137-150. In further embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises a HC having a sequence of SEQ ID NO: 103 and a LC having a sequence of SEQ ID NO: 140. In further embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises a HC having a sequence of SEQ ID NO: 103 and a LC having a sequence of SEQ ID NO: 138. In further embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises a HC having a sequence of SEQ ID NO: 105 and a LC having a sequence of SEQ ID NO: 138. In further embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises a HC having a sequence of SEQ ID NO: 106 and a LC having a sequence of SEQ ID NO: 139. In additional embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 105 and a LC having the sequence of SEQ ID NO: 140. In additional embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 106 and a LC having the sequence of SEQ ID NO: 140. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 125 and a LC having the sequence of SEQ ID NO: 140. In additional embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 127 and a LC having the sequence of SEQ ID NO: 140. In additional embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugates described herein comprises a HC having the sequence of SEQ ID NO: 126 and a LC having the sequence of SEQ ID NO: 140. In further embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugate described herein comprises a HC having the sequence of SEQ ID NO:134 and a LC having the sequence of SEQ ID NO:140.

[0212] In some embodiments, the EpCAM antibody of the immunoconjugate described herein comprises an altered (e.g., mutated or engineered) Fc region. For example, in some aspects, the Fc region has been altered to reduce or enhance the effector function of the antibody, changing the serum half-life or other functional properties of the antibody. In some cases, such as in the case of antibodies whose mechanism of action involves blocking or antagonism but does not kill cells carrying the target antigen, it is desirable to reduce or eliminate the effector function. When directed against undesirable cells, such as tumor cells and foreign cells in which FcγR is expressed at low levels, such as tumor-specific B cells (e.g., non-Hodgkin's lymphoma, CLL, and Burkitt's lymphoma) with low levels of FcγRIIB, it is generally desirable to increase the effector function. The immunoconjugate of the present invention having such an imparted or altered effector function activity can be used to treat and / or prevent diseases, disorders, or infections requiring enhanced effector function activity efficacy. In some aspects, the Fc region is an isotype or other isotype selected from IgM, IgA, IgG, IgE.

[0213] Although the Fc region of an EpCAM antibody or EpCAM-binding antibody fragment thereof may have the ability to bind to one or more Fc receptors (e.g., FcγR), in certain embodiments, the antibody or antibody fragment comprises an antibody or antibody fragment having altered binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a), or FcγRIIIB (CD16b) (relative to the binding exhibited by a wild-type Fc region), for example, having enhanced binding to an activating receptor and / or having significantly reduced or no binding to one or more inhibitory receptors. Thus, the Fc region of an EpCAM antibody or EpCAM-binding antibody fragment thereof may comprise some or all of the CH2 domain and / or some or all of the CH3 domain of an intact Fc region, or may comprise a variant CH2 and / or variant CH3 sequence (which may include, for example, one or more insertions and / or one or more deletions relative to the CH2 or CH3 domain of an intact Fc region). Such an Fc region may comprise a non-Fc polypeptide portion, or may comprise a portion that is not a naturally occurring complete Fc region, or may comprise a non-naturally occurring orientation of the CH2 and / or CH3 domains (such as, for example, two CH2 domains or two CH3 domains, or a CH3 domain connected to a CH2 domain in an N-terminal to C-terminal direction, etc.).

[0214] Fc region modifications identified as altering effector function are known in the art and include modifications that increase binding to activating receptors, such as FcγRIIA (CD16A), and decrease binding to inhibitory receptors, such as 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 of exemplary modifications that increase binding to activating receptors and / or decrease binding to inhibitory receptors (numbering is that of the EU index as in Kabat, and substitutions are relative to the amino acid sequence of SEQ ID NO: 304).

[0215] Table 8. Changes in specific activating Fc regions

[0216]

[0217]

[0218] Exemplary variants of human IgG1 Fc regions that have reduced binding to CD32B and / or increased binding to CD16A contain F243L, R292P, Y300L, V305I, or P396L substitutions, where the numbering is that of the EU index as in Kabat. These amino acid substitutions may be present in any combination in the human IgG1 Fc region. In one embodiment, the variant human IgG1 Fc region contains F243L, R292P, and Y300L substitutions. In another embodiment, the variant human IgG1 Fc region contains F243L, R292P, Y300L, V305I, and P396L substitutions.

[0219] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugate described herein comprises an immunoglobulin heavy chain constant region containing modifications that reduce 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 54:17-18 (2001); 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; U.S. Appl. Pub. 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), the contents of each of which are incorporated herein by reference in their entirety).

[0220] In some embodiments, the Fc region of the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugate described herein exhibits reduced binding (or substantially no binding) to an effector receptor 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); relative to the binding exhibited by a wild-type IgG Fc region (SEQ ID NO: 304). In some embodiments, the effector receptor binding affinity of the EpCAM antibody or EpCAM-binding antibody fragment Fc region variant is reduced by a factor of 10 or less, 50 or less, or 100 or less compared to the binding affinity of the corresponding antibody or antibody binding fragment comprising a wild-type Fc region of the corresponding immunoglobulin.

[0221] In a specific embodiment, the EpCAM antibody or EpCAM-binding antibody fragment of the immunoconjugate described herein comprises an IgG Fc region that exhibits reduced effector function (e.g., reduced ADCC) and comprises a modification at one or more 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 amino acid position numbering is according to the EU index as set forth in Kabat. In one embodiment, the CH2-CH3 domain of the EpCAM antibody comprises any 1, 2, 3 or 4 of the following substitutions: L234A, L235A, D265A, N297Q, N297A and N297G, wherein the numbering is that of the EU index as in Kabat. In another embodiment, the CH2-CH3 domain contains an N297Q substitution, an N297A substitution, or both an L234A and L235A substitution, as these mutations abolish FcR binding. Alternatively, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugate described herein comprises a naturally occurring CH2-CH3 domain of an Fc region that inherently exhibits reduced (or substantially no) binding to FcγRIIIA (CD16a) and / or reduced effector function relative to the binding and effector function exhibited by a wild-type IgG1 Fc region (SEQ ID NO: 304). In a specific embodiment, the Fc constant region of the EpCAM antibody comprises an IgG2 Fc region (SEQ ID NO: 305) or an IgG4 Fc region (SEQ ID NO: 306). Because the N297A, N297G, N297Q, L234A, L235A, and D265A substitutions abrogate effector function, these substitutions will not be employed in some cases where effector function is desired.

[0222] A specific IgG1 sequence of the CH2 and CH3 domains of the Fc-region-containing EpCAM antibody or EpCAM-binding antibody fragment thereof with reduced or abolished effector function of the immunoconjugates described herein comprises the substitutions L234A / L235A (underlined) (SEQ ID NO: 307):

[0223]

[0224] A specific IgG1 sequence of the CH2 and CH3 domains of the Fc-region-containing EpCAM antibody or EpCAM-binding antibody fragment thereof with reduced or abolished effector function of the immunoconjugates described herein comprises the substitution N297A (underlined) (SEQ ID NO: 308):

[0225]

[0226] A specific IgG1 sequence of the CH2 and CH3 domains of the Fc-region-containing EpCAM antibody or EpCAM-binding antibody fragment thereof with reduced or abolished effector function of the immunoconjugates described herein comprises the substitution N297Q (underlined) (SEQ ID NO: 309):

[0227]

[0228] In some embodiments, the EpCAM antibodies or EpCAM-binding antibody fragments thereof of the immunoconjugates described herein comprise 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, P331 S; IgG1-L234F, L235E, P331S; IgG1-H268Q, A330S, P331S; IgG1-G236R, L328R; IgG1-L235G, G236R, IgG1-N 297A; 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; wherein position numbering is according to the EU index as in Kabat.

[0229] In some embodiments, the EpCAM antibody or its EpCAM-binding antibody fragment of the immunoconjugate described herein comprises a heavy chain immunoglobulin constant domain with reduced CDC activity. In specific aspects, the EpCAM antibody or its EpCAM-binding antibody fragment of the immunoconjugate described herein comprises an IgG1 heavy chain constant region containing a mutation that reduces CDC activity (see, e.g., WO 1997 / 11971 and WO 2007 / 106585; U.S. 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 spp. 11:509-517 (2009); Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and Sazinsky et al., PNAS USA 105:20167-20172 (2008); the contents of each of which are herein incorporated by reference in their entirety). Examples of heavy chain constant domain sequence modifications that reduce CDC include one or more modifications corresponding to: 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 IgG2 - H268Q, V309L, A330S, A331S according to EU index.

[0230] In some embodiments, the EpCAM antibodies or EpCAM-binding antibody fragments thereof provided by the immunoconjugates described herein comprise a heavy chain immunoglobulin constant domain containing one or more amino acid modifications (e.g., substitutions) that extend half-life. Many mutations capable of increasing the half-life of molecules containing an Fc region are known in the art and are encompassed as components of the EpCAM antibodies and EpCAM-binding antibody fragments thereof provided herein. See, for example, U.S. Patent Nos. 6,277,375; 7,083,784; 7,217,797 and 8,088,376; U.S. Publication Nos. 2002 / 0147311; and 2007 / 0148164; and PCT Publication Nos. WO 1998 / 23289; WO 2009 / 058492; and WO 2010 / 033279, the contents of each of which are incorporated herein by reference in their entirety.

[0231] The serum half-life of proteins comprising an Fc region can be extended by increasing the binding affinity of the Fc region to FcRn. As used herein, the term "half-life" refers to the pharmacokinetic properties of a molecule, which is a measure of the average survival time of a molecule after its administration. Half-life can be expressed as the time required to eliminate fifty percent (50%) of a known amount of a molecule from the body of a subject (e.g., a human patient or other mammal) or a specific compartment thereof (e.g., as measured in serum (i.e., circulating half-life) or in other tissues). In general, an increase in half-life results in an increase in the mean residence time (MRT) of the administered molecule in the circulation.

[0232] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugate 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 amino acid position numbering is according to the EU index. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof contains one or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, wherein amino acid position numbering is according to the EU index. In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises one or more of the following: substitution of the amino acid at Kabat position 252 with Tyr, Phe, Trp, or Thr; substitution of the amino acid at Kabat position 254 with Thr; substitution of the amino acid at Kabat position 256 with Ser, Arg, Gln, Glu, Asp, or Thr; substitution of the amino acid at Kabat position 257 with Leu; substitution of the amino acid at Kabat position 309 with Pro; substitution of the amino acid at Kabat position 311 with Ser; substitution of the amino acid at Kabat position 428 with Thr, Leu, Phe, or Ser; substitution of the amino acid at Kabat position 433 with Arg, Ser, Iso, Pro, or Gln; or substitution of the amino acid at Kabat position 434 with Trp, Met, Ser, His, Phe, or Tyr. More specifically, the EpCAM antibody or EpCAM-binding antibody fragment domain thereof may contain amino acid substitutions relative to the wild-type human IgG constant domain, including substitution of the amino acid at Kabat position 252 with Tyr, substitution of the amino acid at Kabat position 254 with Thr, and substitution of the amino acid at Kabat position 256 with Glu.

[0233] In some embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugate described herein comprises at least one substitution selected from the group consisting of T250Q, M252Y, S254T, T256E, K288D, T307Q, V308P, A378V, M428L, N434A, N434S, N434H, N434Y, H435K and Y436I, wherein the numbering is that of the EU index as in Kabat. In other embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises substitutions selected from the group consisting of: (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.

[0234] In certain embodiments, the EpCAM antibody or EpCAM-binding antibody fragment thereof of the immunoconjugate described herein contains a variant IgG Fc region comprising any one, two, or three of the following substitutions: M252Y, S254T, and T256E. The present disclosure further provides EpCAM antibodies or EpCAM-binding antibody fragments thereof having a variant Fc region, wherein the EpCAM antibody or EpCAM-binding antibody fragment comprises: (a) one or more mutations that alter effector function and / or FcγR; and (b) one or more mutations that extend serum half-life.

[0235] 6.3 Immunoconjugates Containing EpCAM-Activatable Antibodies

[0236] In other 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 its EpCAM binding antibody fragment that specifically binds to EpCAM (e.g., human EpCAM) coupled to a masking portion (MM) such that the 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, which is, for example, a protease that is active in diseased tissue and / or a protease that is co-localized with EpCAM at the treatment site of the subject. In some embodiments, the immunoconjugate comprising an EpCAM activatable antibody comprises the EpCAM antibody described in section 6.2 coupled to the MM via a protease substrate.

[0237] EpCAM-activatable antibodies are, in some cases, stable in circulation, activated at the intended site of therapy and / or diagnosis, but unstable in normal tissue, such as healthy tissue or other tissue not targeted for therapy and / or diagnosis, and upon activation exhibit at least comparable binding to EpCAM as the corresponding unmodified antibody. Also provided are immunoconjugates comprising the EpCAM-activatable antibodies, nucleic acids or nucleic acid groups encoding the EpCAM-activatable antibodies, and vectors and host cells comprising the nucleic acids. Also provided are pharmaceutical compositions comprising the activatable antibodies, immunoconjugates, nucleic acids, vectors, and host cells.

[0238] In some embodiments, the immunoconjugate with the EpCAM-activatable antibody or antibody fragment further comprises:

[0239] (a) a cleavable moiety coupled to the antibody or antibody fragment, wherein the cleavable moiety is a polypeptide that serves as a substrate for a protease; and

[0240] (b) a masking moiety coupled to the antibody or antibody fragment, wherein the masking moiety inhibits binding of the antibody or antibody fragment to EpCAM when the activatable antibody is in an uncleaved state,

[0241] The activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: (masking moiety)-(cleavable moiety)-(antibody or antibody fragment) or (antibody or antibody fragment)-(cleavable moiety)-(masking moiety).

[0242] In some cases, an immunoconjugate disclosed herein comprises an EpCAM-activatable antibody comprising:

[0243] (a) an EpCAM antibody or an EpCAM-binding antibody fragment thereof, comprising a VH-CDR1, a VH-CDR2, a VH-CDR3, a VL-CDR1, a VL-CDR2 and a VL-CDR3 having a sequence selected from a member of the group consisting of:

[0244] (i) SEQ ID NOs: 13-15, 42, 40, and 41, respectively;

[0245] (ii) SEQ ID NOs: 13-15 and 39-41, respectively;

[0246] (iii) SEQ ID NOs: 13, 26, 15, and 39-41, respectively; and

[0247] (iv) SEQ ID NOs: 13, 24, 15, 42, 40, and 41, respectively;

[0248] (b) a masking moiety coupled to the EpCAM antibody or EpCAM-binding antibody fragment thereof, wherein the masking moiety inhibits binding of the antibody or antibody fragment to EpCAM when the activatable antibody is in an uncleaved state; and

[0249] (c) a cleavable moiety coupled to the EpCAM antibody or EpCAM-binding antibody fragment thereof, wherein the cleavable moiety is a polypeptide that serves as a substrate for a protease;

[0250] The activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: (masking moiety)-(cleavable moiety)-(antibody or antibody fragment) or (antibody or antibody fragment)-(cleavable moiety)-(masking moiety).

[0251] The EpCAM-activatable antibody of the immunoconjugate described herein in an activated state binds to human EpCAM and includes (i) an EpCAM antibody or EpCAM-binding antibody fragment thereof (Ab) of the immunoconjugate described herein that specifically binds to human EpCAM (as disclosed herein, for example, in Section 6.2); (ii) a masking portion (MM) that inhibits binding of the EpCAM-activatable antibody to EpCAM when the EpCAM-activatable antibody is in an uncleaved state; and (c) a cleavable portion (CM) coupled to the EpCAM antibody or EpCAM-binding antibody fragment thereof, wherein the CM is a polypeptide that serves as a substrate for a protease. In some embodiments, the EpCAM-activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-CM-Ab or Ab-CM-MM. In some embodiments, the EpCAM-activatable antibody comprises a connecting peptide between the MM and the CM. In some embodiments, the EpCAM-activatable antibody comprises a connecting peptide between the CM and the Ab.

[0252] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein in the uncleaved state specifically binds to mammalian EpCAM with a dissociation constant of less than or equal to 1 nM, less than or equal to 5 nM, less than or equal to 10 nM, less than or equal to 15 nM, less than or equal to 20 nM, less than or equal to 25 nM, less than or equal to 50 nM, less than or equal to 100 nM, less than or equal to 150 nM, less than or equal to 250 nM, less than or equal to 500 nM, less than or equal to 750 nM, less than or equal to 1000 nM, and 122. / or less than or equal to 2000 nM.

[0253] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein in the uncleaved state specifically binds to mammalian EpCAM (e.g., human EpCAM or cynomolgus monkey EpCAM) with a dissociation constant of greater than or equal to 1 nM, greater than or equal to 5 nM, greater than or equal to 10 nM, greater than or equal to 15 nM, greater than or equal to 20 nM, greater than or equal to 25 nM, greater than or equal to 50 nM, greater than or equal to 100 nM, greater than or equal to 150 nM, greater than or equal to 250 nM, greater than or equal to 500 nM, greater than or equal to 750 nM, greater than or equal to 1000 nM, and 122. / or greater than or equal to 2000 nM.

[0254] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein in an uncleaved state is expressed in an amount 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 to 15nM, 5nM to 10nM, 10nM to 2000nM, 10nM to 1000nM, 10nM to 750nM, 10nM to 500nM, 10nM to 250nM, 10nM to 150nM, 10nM to 100nM, 10nM to 50nM, 10nM to 25nM, 10nM to 15nM M, 15nM to 2000nM, 15nM to 1000nM, 15nM to 750nM, 15nM to 500nM, 15nM to 250nM, 15nM to 150nM, 15nM to 100nM, 15nM to 50nM, 15nM to 25nM, 25nM to 2000nM, 25nM to 1000nM , 25nM to 750nM, 25nM to 500nM, 25nM to 250nM, 25nM to 150nM, 25nM to 100nM, 25nM to 50nM, 50nM to 2000nM, 50nM to 1000nM, 50nM to 750nM, 50nM to 500nM, 50nM to 250nM, 50nM to 150nM, 50nM to 100nM, 100nM to 2000nM, 100nM to 1000nM, 100nM to 750nM, 100nM to 500nM, 100nM to 250nM, 100nM to 150nM, 150nM to 2000nM, 150nM to 1000nM, 15 The invention further comprises a dissociation constant that specifically binds to mammalian EpCAM (e.g., EpCAM 500, EpCAM 700, EpCAM 800, EpCAM 900, EpCAM 1000, EpCAM 1000, EpCAM 2000, EpCAM 2000, EpCAM 3000, EpCAM 3000, EpCAM 4000, EpCAM 5000, EpCAM 6000, EpCAM 7000, EpCAM 8000, EpCAM 2000, EpCAM 3000, EpCAM 4000, EpCAM 5000, EpCAM 6000, EpCAM 2000, EpCAM 3000, EpCAM 5000, EpCAM 2000, EpCAM 3000, EpCAM 4000, EpCAM 5000, EpCAM 6000, EpCAM 2000, EpCAM 3000, EpCAM 5000, EpCAM 2000, EpCAM 3000, EpCAM 5000, EpCAM 2000, EpCAM 3000, EpCAM 4000, EpCAM 5000, EpCAM 5000, EpCAM 6000, EpCAM 2000, EpCAM 3000, EpCAM 5000, EpCAM 2000, EpCAM 3000, EpCAM 5000, EpCAM 5000human EpCAM or cynomolgus monkey EpCAM).

[0255] 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 less than or equal to 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, or 10 nM. In some embodiments, the EpCAM-activatable antibody in the activated state specifically binds to mammalian EpCAM with a dissociation constant of greater than or equal to 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 5 nM, or 10 nM.

[0256] In some embodiments, the EpCAM-activating antibody in the activated state is expressed 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 The invention specifically binds to mammalian EpCAM (e.g., human EpCAM or cynomolgus monkey EpCAM) with a dissociation constant ranging from 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.

[0257] 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 human EpCAM and cynomolgus monkey EpCAM with a dissociation constant of less than 1 nM.

[0258] In some embodiments, the serum half-life of the EpCAM-activatable antibody of the immunoconjugate described herein is longer than the serum half-life of the corresponding antibody; for example, the pK of the EpCAM-activatable antibody is longer than the pK of the corresponding antibody. In some embodiments, the serum half-life of the EpCAM-activatable antibody is similar to the serum half-life of the corresponding antibody.

[0259] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising a VH-CDR1, a VH-CDR2, and a VH-CDR3 having the sequences listed in a row of Table 2. In some embodiments, the EpCAM-activatable antibody comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3 having the sequences listed in a row of Table 3.

[0260] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof, wherein the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises 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.

[0261] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof, wherein the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 having a sequence selected from the group consisting of: (i) SEQ ID NOs: 13-15, 42, 40 and 41, respectively; (ii) SEQ ID NOs: 13-15 and 39-41, respectively; (iii) SEQ ID NOs: 13, 26, 15 and 39-41, respectively; and (iv) SEQ ID NOs: 13, 26, 15, 42, 40 and 41, respectively. In some embodiments, the EpCAM-activatable antibody comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof, wherein the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises 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.

[0262] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof, wherein the EpCAM antibody or EpCAM-binding antibody fragment thereof comprises a VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 having a sequence selected from the group consisting of: (i) SEQ ID NO: 22, 14, 15, 42, 40 and 41, respectively; (ii) SEQ ID NO: 13, 14, 33, 42, 40 and 41, respectively; (iii) SEQ ID NO: 23, 14, 15, 42, 40 and 41, respectively; and (iv) SEQ ID NO: 25, 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: 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.

[0263] In some embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein comprises a VH disclosed in Table 4. In some embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein comprises a VL disclosed in Table 5. In some embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein comprises a VH having the sequence of SEQ ID NO: 54 and a VL having the sequence of SEQ ID NO: 89. In some embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof of the immunoconjugates described herein, the EpCAM antibody or EpCAM-binding antibody fragment thereof comprising a VH having the sequence of SEQ ID NO: 75 and a VL having the sequence of SEQ ID NO: 89. In some embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein comprises an EpCAM antibody or an EpCAM-binding antibody fragment thereof comprising a VH having the sequence of SEQ ID NO: 77 and a VL having the sequence of SEQ ID NO: 89.

[0264] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises a VH sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from SEQ ID NOs: 54, 75, and 77. In some embodiments, the EpCAM-activatable antibody comprises a VL sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence comprising SEQ ID NO: 89. In some embodiments, the percent identity in each instance is 95%. In some embodiments, the percent identity in each instance is 98%.

[0265] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises a HC disclosed in Table 6. In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises a LC disclosed in Table 7. In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises a HC having a sequence of SEQ ID NO: 103 and a LC having a sequence of SEQ ID NO: 140. In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises a HC having a sequence of SEQ ID NO: 125 and a LC having a sequence of SEQ ID NO: 140. In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises a HC having a sequence of SEQ ID NO: 127 and a light chain having a sequence of SEQ ID NO: 140.

[0266] In some embodiments, the EpCAM-activatable antibody of the immunoconjugates described herein comprises 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 antibody of the immunoconjugates described herein comprises 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).

[0267] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises: a VH-CDR1 comprising X1YX3X4H, wherein X1 is selected from N and S, X3 is selected from Y, N, F, S, H, D, L, I, and W, and X4 is selected from I and M; a VH-CDR1 comprising WX2X3PGX6VYIQYX 12 X 13 KFX 17 VH-CDR2 of 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 Selected from N and S, X 13 Selected from E and Q, and X 17 selected from K and Q (SEQ ID NO: 7); and a VH-CDR3 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 (SEQ ID NO: 8). In some embodiments, the EpCAM activatable antibody comprises: comprising RSSX4SLLHSX 10 GX 12TYLX 16 VL-CDR1, wherein X4 is selected from R and K, X 10 Selected from N and D, X 12 Selected from F and I, and X 16 selected from the group consisting of Y and S (SEQ ID NO: 10); a light chain VL-CDR2 comprising QTSNLAS (SEQ ID NO: 40); and a VL-CDR3 comprising X1QX3LELPX8T, wherein X1 is selected from the group consisting of A, L, and Q, X3 is selected from the group consisting of S, G, Y, and N, and X8 is selected from the group consisting of N and W (SEQ ID NO: 11). 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.

[0268] In some embodiments, the VH-CDR1 of the EpCAM-activatable antibody of the immunoconjugate described herein comprises the sequence NYX3IH, wherein X3 is selected from the group consisting of 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, wherein X4 is selected from the group consisting of Y and W (SEQ ID NO: 9). In some embodiments, the VL-CDR3 of the EpCAM-activatable antibody comprises the sequence AQX3LELPNT, wherein X3 is selected from the group consisting of 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.

[0269] Suitable components of the disclosed EpCAM-activatable antibodies of the immunoconjugates described herein also include EpCAM antibodies or EpCAM-binding antibody fragments thereof that cross-compete for binding to human EpCAM and / or cynomolgus monkey EpCAM with an EpCAM antibody comprising a VH having the sequence of SEQ ID NO: 54 and a VL having the sequence of SEQ ID NO: 89. Additionally, suitable EpCAM-activatable antibodies cross-compete for binding to human EpCAM and / or cynomolgus monkey EpCAM with an EpCAM antibody comprising a VH having the sequence of SEQ ID NO: 75 and a VL having the sequence of SEQ ID NO: 89. Additionally, suitable EpCAM-activatable antibodies cross-compete for binding to human EpCAM and / or cynomolgus monkey EpCAM with an EpCAM antibody comprising a VH having the sequence of SEQ ID NO: 77 and a VL having the sequence of SEQ ID NO: 89.

[0270] The EpCAM activatable antibodies of the immunoconjugates described herein provided herein comprise a masking moiety (MM). In some embodiments, the masking moiety (or "mask") is an amino acid sequence that is coupled or otherwise attached to the EpCAM antibody and is located 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 WO2009 / 025846, the contents of which are incorporated herein by reference in their entirety.

[0271] In some embodiments, the dissociation constant of the MM pair of the activatable antibody to bind to the Ab is greater than the dissociation constant of the Ab to EpCAM. In some embodiments, the dissociation constant of the MM pair to bind to the Ab is no greater than the dissociation constant of the Ab to EpCAM.

[0272] In some embodiments, the dissociation constant of the MM for binding to the Ab is less than the dissociation constant of the Ab for binding to EpCAM.

[0273] In some embodiments, the dissociation constant (K) of the MM for the Ab is d) is at most 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 1-5, 5-10, 10-100, 10-1,000, 10-10,000, 10-1,000,000, 10-10,000,000, , 100-1,000, 100-10,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,000, 1,000-10,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 100,000-1,000,000 or 100,000-10,000,000 times larger.

[0274] In some embodiments, when the EpCAM-activatable antibody of the immunoconjugate described herein is in a cleaved state, the MM does not interfere with or compete with the Ab for binding to EpCAM. In some embodiments, the MM is a polypeptide of about 2 to 40 amino acids in length. In some embodiments, the MM is a polypeptide of up to about 40 amino acids in length.

[0275] In some embodiments, the MM polypeptide sequence is different from the sequence of EpCAM. In some embodiments, the MM polypeptide sequence is at most 50% identical to any natural binding partner of Ab. In some embodiments, the MM polypeptide sequence is different from the sequence of EpCAM and is at most 40%, 30%, 25%, 20%, 15%, or 10% identical to any natural binding partner of Ab.

[0276] In some embodiments, conjugation of the MM to the Ab reduces the ability of the Ab to bind EpCAM such that the dissociation constant (IQ) of the Ab for EpCAM when conjugated to the MM is at least 2, 5, 10, 20, 40, 100, 1,000, 10,000 times greater than the Kd of the Ab for EpCAM when not conjugated to the MM.

[0277] In some embodiments, in the presence of EpCAM, the MM reduces the ability of the Ab to bind to EpCAM by at least 90% when the CM is not cleaved, compared to when the CM is cleaved, when assayed in vitro using a target displacement assay, e.g., such as the assay described in WO 2010 / 081173 (the contents of which are hereby incorporated by reference in their entirety).

[0278] 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 Ab not modified with MM or the specific binding of the parent Ab to human EpCAM.

[0279] The Kd of the Ab modified with MM 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 times greater, or 5-10, 10-100, 10-1,000, 10-10,000, 10-1,000,000, 10-100,000, 10-1,000,000, 10-100,000, 10-1000,000, 10-1000,000, 10-1000,000, 10-1000,000, 10-1000,000, 10-1000,000, 10-1000,000, 10-1000,000, 10-1000,000, 10-1000,000, 10-1000,000, 10-1000,000, 10-1000 times larger than 10,000,000, 100-1,000, 100-10,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,000, 1,000-10,000, 1,000-10,000,000, 1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 100,000-1,000,000 or 100,000-10,000,000 times larger. In contrast, the binding affinity of the Ab modified with MM to 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 times, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000, or less than the binding affinity of the Ab not modified with MM or the parental Ab to human EpCAM. ,000, 10-10,000,000, 100-1,000, 100-10,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 1000-10,000,000, 10,000-10,000,000, 10,000-1,000,000, 100,000-10,000,000 or 100,000-10,000,000 times.

[0280] In some embodiments, the dissociation constant (Kd) of the MM for the Ab is approximately equal to the Kd of the Ab for human EpCAM. In some embodiments, the dissociation constant (Kd) of the MM for the Ab is no greater than the dissociation constant of the Ab for human EpCAM. In some embodiments, the dissociation constant (Kd) of the MM for the Ab is less than the dissociation constant of the Ab for human EpCAM. In some embodiments, the dissociation constant (Kd) of the MM for the Ab is greater than the dissociation constant of the Ab for human EpCAM.

[0281] In some embodiments, the Kd of the MM for binding to the Ab does not exceed the Kd for binding of the Ab to human EpCAM.

[0282] In some embodiments, the Kd of the MM pair binding to the Ab is no less than the Kd of the binding of the Ab to human EpCAM. In some embodiments, the Kd of the MM pair binding to the Ab is approximately equal to the Kd of the binding of the Ab to human EpCAM. In some embodiments, the Kd of the MM pair binding to the Ab is less than the Kd of the binding of the Ab to human EpCAM. In some embodiments, the Kd of the MM pair binding to the Ab is greater than the Kd of the binding of the Ab to human EpCAM. In some embodiments, the Kd of the MM pair binding to the Ab is at most 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times greater than the Kd of the binding of the Ab to human EpCAM. In some embodiments, the Kd of the MM for binding to the Ab is between 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1000, or 100-1,000 times greater than the Kd of the Ab for binding to human EpCAM.

[0283] In some embodiments, the affinity of the MM pair binding to the Ab is less than the affinity of the Ab binding to human EpCAM. In some embodiments, the affinity of the MM pair binding to the Ab is no greater than the affinity of the Ab binding to human EpCAM. In some embodiments, the affinity of the MM pair binding to the Ab is approximately equal to the affinity of the Ab binding to human EpCAM. In some embodiments, the affinity of the MM pair binding to the Ab is no less than the affinity of the Ab binding to human EpCAM. In some embodiments, the affinity of the MM pair binding to the Ab is greater than the affinity of the Ab binding to human EpCAM.

[0284] In some embodiments, the affinity of the MM for binding to the Ab is 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or 1,000 times lower than the affinity of the Ab for binding to human EpCAM. In some embodiments, the affinity of the MM for binding to the Ab is between 1-5, 2-5, 2-10, 5-10, 5-20, 5-50, 5-100, 10-100, 10-1,000, 20-100, 20-1000, or 100-1,000 times lower than the affinity of the Ab for binding to human EpCAM. In some embodiments, the affinity of the MM for binding to the Ab is 2 to 20 times lower than the affinity of the Ab for binding to human EpCAM. In some embodiments, the MM not covalently linked to the Ab and at equimolar concentrations with the EpCAM-activatable antibody does not inhibit the binding of the Ab to human EpCAM.

[0285] 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 Ab not modified with MM or the specific binding of the parent Ab to human EpCAM. When modified with MM, the ability of the Ab to bind to human EpCAM may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and even 100% compared to the binding of the Ab not modified with MM to human EpCAM or the binding of the parental Ab to human EpCAM when measured in vivo or in an 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.

[0286] MM inhibits the binding of Ab to human EpCAM. MM binds to the antigen binding domain of Ab and inhibits the binding of said Ab to human EpCAM. MM can sterically inhibit the binding of Ab to human EpCAM. MM can allosterically inhibit the binding of Ab to its target. In these embodiments, when the Ab is modified with MM or coupled to MM and in the presence of the target, the Ab does not bind or substantially does not bind to human EpCAM, or there is at most 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50% binding persists 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.

[0287] When an Ab is conjugated to or modified by an MM, the MM "masks" or reduces or otherwise inhibits the specific binding of the Ab to human EpCAM. When an Ab is conjugated to or modified by an MM, such conjugation or modification may cause structural changes that reduce or inhibit the ability of the Ab to specifically bind to its target.

[0288] Abs 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):

[0289] (MM)-(Ab)

[0290] (Ab)-(MM)

[0291] (MM)-L-(Ab)

[0292] (Ab)-L-(MM)

[0293] wherein MM is a masking moiety, 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 provide flexibility.

[0294] In certain embodiments, the MM is not a natural binding partner of the Ab. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of the Ab. In some embodiments, the MM is at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of the Ab. In some embodiments, the MM is at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of the Ab. In some embodiments, the MM is at most 25% identical to any natural binding partner of the Ab. In some embodiments, the MM is at most 50% identical to any natural binding partner of the Ab. In some embodiments, the MM is at most 20% identical to any natural binding partner of the Ab. In some embodiments, the MM is at most 10% identical to any natural binding partner of the Ab.

[0295] In some embodiments, the MM comprises a sequence disclosed in Table 9. In some embodiments, the MM comprises a sequence selected from SEQ ID NOs: 151-157. In some embodiments, the MM comprises a sequence selected from SEQ ID NOs: 158-161. In some embodiments, the MM comprises a sequence selected from SEQ ID NOs: 162-167. In some embodiments, the MM comprises a sequence selected from SEQ ID NOs: 155.

[0296] Table 9. Exemplary masking moiety sequences.

[0297]

[0298]

[0299] The EpCAM activatable antibodies of the immunoconjugates described herein provided herein comprise a cleavable portion. In some embodiments, the cleavable portion (or "substrate") comprises an amino acid sequence that is a substrate for a protease (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 WO 2014 / 026136, the contents of each of which are incorporated herein by reference in their entirety (see also, Boulware et al., Biotechnol Bioeng. 106(3):339-346(2010)).

[0300] In some embodiments, the EpCAM activatable antibody of the immunoconjugate described herein comprises an Ab modified by a MM and further comprises one or more cleavable moieties (CM). Such EpCAM activatable antibodies exhibit activatable / convertible binding to human EpCAM. EpCAM activatable antibodies generally comprise an antibody or antigen-binding antibody fragment (Ab) modified or coupled to a masking moiety (MM) and a modifiable or cleavable moiety (CM). In some embodiments, the CM contains an amino acid sequence that serves as a substrate for at least one protease. In some embodiments, the MM and the cleavable moiety are 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).

[0301] The elements of the EpCAM-activatable antibody of the immunoconjugate described herein are arranged such that the MM and CM are positioned so that the EpCAM-activatable antibody binds to human EpCAM in a cleaved (or relatively active) state and in the presence of human EpCAM, but when the EpCAM-activatable antibody is in an uncleaved (or relatively inactive) state in the presence of human EpCAM, the specific binding of the EpCAM-activatable antibody to human EpCAM is reduced or inhibited. Because the MM inhibits or masks the ability of the EpCAM-activatable antibody to specifically bind to human EpCAM, the specific binding of the EpCAM-activatable antibody to human EpCAM can be reduced.

[0302] The Kd of the EpCAM-activatable antibody of the immunoconjugate described herein modified with MM and CM 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 times greater, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000 times greater than the Kd of the EpCAM-activatable antibody or parent Ab not modified with MM and CM for human EpCAM. , 10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 10,000-10,000,000, 10,000-1,000,000, 10,000-10,000,000 or 100,000-10,000,000 times larger. In contrast, the binding affinity of the Ab modified with MM and CM to 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, 10-100,000, 10-100,000, 10-100,000, 10-100,000, 10-100,000, 10-100,000, 10-100,000, 10-100,000, 10-100,000, 10-100,000, 10-100,000, 10-100 ,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 10,000-10,000,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times.

[0303] When 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 the EpCAM-activatable antibody or the parent Ab not modified with MM and CM to human EpCAM. When modified with MM and CM, the ability of the EpCAM-activatable antibody to bind to human EpCAM may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and even 100% compared to the binding of the parent Ab to human EpCAM or the binding of the EpCAM-activatable antibody not modified with MM and CM to human EpCAM when measured in vivo or in an 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.

[0304] As used herein, the term "cleavage state" refers to the condition of the EpCAM-activatable antibody of the immunoconjugate described herein after modification of the CM by at least one protease. As used herein, the term uncleaved or intact state refers to the condition of the EpCAM-activatable antibody in the absence of cleavage of the CM by a protease. As described above, the term "activatable antibody" or "activatable antibody" is used herein to refer to the EpCAM-activatable antibody in its uncleaved (native or intact) state and its cleaved state. It is obvious to those of ordinary skill that in some embodiments, the cleaved EpCAM-activatable antibody may lack the MM due to cleavage of the CM by a protease, thereby resulting in the release of at least the MM (e.g., wherein the MM is not attached to the EpCAM-activatable antibody by a covalent bond (e.g., a disulfide bond between cysteine ​​residues).

[0305] Activatable or convertible means that when the EpCAM activatable antibody is in an inhibited, masked, intact or uncleaved state (i.e., a first conformation), the EpCAM activatable antibody of the immunoconjugate described herein exhibits a first level of binding to the target, and when in an uninhibited, unmasked and / or cleaved state (i.e., a second conformation), exhibits a second level of binding to human EpCAM, wherein the second target binding level is greater than the first binding level. Generally speaking, in the presence of a cleaving agent (i.e., a protease) capable of cleaving the CM, human EpCAM has greater access to the Ab of the EpCAM activatable antibody than in the absence of such a cleaving agent. Thus, when the EpCAM activatable antibody is in the uncleaved state, the Ab is inhibited from binding to human EpCAM and can be masked from binding to human EpCAM (i.e., the first conformation is such that the Ab cannot bind to human EpCAM), and in the cleaved state, the Ab is not inhibited or masked from target binding.

[0306] The CM and Ab of the EpCAM activatable antibodies of the immunoconjugates described herein are selected so that the Ab represents the binding portion for a given target and the CM represents the substrate for the protease. In some embodiments, the protease is co-localized with human EpCAM at the treatment site or diagnostic site of the subject. As used herein, co-localization refers to being in the same site or relatively close proximity. In some embodiments, the protease cleaves the CM, thereby producing an activated antibody that binds to the target located near the cleavage site. The EpCAM activatable antibodies disclosed herein have special uses, where, for example, a protease (i.e., a protease) capable of cleaving a site in the CM is present at relatively higher levels in target-containing tissues at the treatment site or diagnostic site than in tissues at non-treatment sites (e.g., in healthy tissue). In some embodiments, the CM of the present disclosure is also cleaved by one or more other proteases. In some embodiments, it is precisely one or more other proteases that are co-localized with human EpCAM and are responsible for the cleavage of the CM in vivo.

[0307] In some embodiments, the EpCAM-activatable antibodies of the immunoconjugates described herein provide reduced toxicity and / or adverse side effects that may otherwise result from binding of the EpCAM-activatable antibodies at non-therapeutic sites if the EpCAM-activatable antibodies are not masked or otherwise inhibited from binding to human EpCAM.

[0308] Generally, the EpCAM-activatable antibody of the immunoconjugate described herein can be designed by selecting the target Ab and constructing the remaining portion of the EpCAM-activatable antibody so that when conformationally constrained, the MM can mask the EpCAM-activatable antibody or reduce the binding of the EpCAM-activatable antibody to human EpCAM. Structural design criteria can be considered to provide this functional characteristic.

[0309] EpCAM activatable antibodies of the immunoconjugates described herein that exhibit a switchable phenotype of the desired dynamic range of target binding in an inhibited conformation versus an uninhibited conformation are provided. Dynamic range generally refers to the ratio of (a) the maximum detection level of a parameter under a first set of conditions to (b) the minimum detection value of the parameter under a second set of conditions. For example, in the case of EpCAM activatable antibodies, dynamic range refers to the ratio of (a) the maximum detection level of binding of the target protein 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 detection level of binding of the target protein to the EpCAM activatable antibody in the absence of the protease. The dynamic range of the EpCAM activatable antibody can be calculated as the ratio of the dissociation constant of the EpCAM activatable antibody cleavage agent (e.g., enzyme) treatment to the dissociation constant of the EpCAM activatable antibody cleavage agent treatment. The greater the dynamic range of the EpCAM activatable antibody, the better the switchable phenotype of the EpCAM activatable antibody.

[0310] EpCAM-activatable antibodies of the immunoconjugates described herein having relatively high dynamic range values ​​(e.g., greater than 1) exhibit a more desirable switching phenotype, such that target protein cleavage by the EpCAM-activatable antibody occurs to a greater extent (e.g., predominantly) in the presence of a cleavage agent (e.g., an enzyme) capable of cleaving the CM of the EpCAM-activatable antibody than in the absence of the cleavage agent.

[0311] The EpCAM-activatable antibodies of the immunoconjugates described herein can be provided in a variety of structural configurations. Exemplary formulas of EpCAM-activatable antibodies are provided below. It is specifically contemplated that within an EpCAM-activatable antibody, the N-terminus to C-terminus order of the Ab, MM, and CM can be reversed. It is also specifically contemplated that the CM and MM can overlap in amino acid sequence, such that the CM is contained within the MM.

[0312] For example, the EpCAM-activatable antibody of the immunoconjugate described herein can be represented by the following formula (in order from the amino (N) terminal region to the carboxyl (C) terminal region):

[0313] (MM)-(CM)-(Ab)

[0314] (Ab)-(CM)-(MM)

[0315] wherein MM is a masking moiety, CM is a cleavable moiety, and Ab is an EpCAM antibody or an EpCAM-binding antibody fragment thereof. It should be noted that although MM and CM are indicated as distinct components in the above formula, in all exemplary embodiments disclosed herein (including formulas), it is contemplated that the amino acid sequences of MM and CM may overlap, for example, such that CM is fully or partially contained within MM. Furthermore, the above formula provides for additional amino acid sequences that may be located at the N-terminus or C-terminus of the EpCAM-activatable antibody element.

[0316] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises a CM that is cleavable by a protease. In some embodiments, the protease that cleaves the CM is active in diseased tissue, e.g., upregulated or otherwise unregulated, and when the EpCAM-activatable antibody is exposed to the protease, the protease cleaves the CM in the EpCAM-activatable antibody.

[0317] In some embodiments, the protease is co-localized with EpCAM in the tissue, and when the EpCAM-activatable antibody is exposed to the protease, the protease cleaves the CM in the EpCAM-activatable antibody.

[0318] In some embodiments, the CM is positioned in the EpCAM-activatable antibody of the immunoconjugate described herein such that when the EpCAM-activatable antibody is in the uncleaved state, binding of the EpCAM-activatable antibody to EpCAM is reduced to occur with a dissociation constant that is at least 2, 5, 10, 20, 40, 50, 100, or 200 greater than the dissociation constant for binding of the unmodified Ab to EpCAM, whereas in the cleaved state (i.e., when the EpCAM-activatable antibody is in the cleaved state), the Ab binds to EpCAM.

[0319] In some embodiments, the CM is a polypeptide of up to 15 amino acids in length.

[0320] In some embodiments, the CM is a polypeptide comprising a first cleavable portion (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and a second cleavable portion (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 of up to 15 amino acids in length.

[0321] In some embodiments, CM is a substrate for at least one protease that is believed to be upregulated in cancer or otherwise unregulated. In some embodiments, CM is a substrate for at least one protease that is believed to be upregulated in inflammation. In some embodiments, CM is a substrate for at least one protease that is believed to be upregulated in autoimmunity or otherwise unregulated.

[0322] In some embodiments, the CM is a substrate for at least one protease selected from the group consisting of matrix metalloproteinases (MMPs), thrombin, neutrophil elastase, cysteine ​​proteases, legumin, and serine proteases such as MT-SP1 and urokinase (uPA). Without being bound by theory, it is believed that these proteases are upregulated or otherwise unregulated in at least one of cancer, inflammation, and / or autoimmunity.

[0323] Exemplary substrates include, but are not limited to, substrates that can be cleaved by one or more of the following enzymes or proteases: ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM10, ADAM 12, ADAM 15, ADAM 17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5); aspartic proteases (e.g., BACE, renin); aspartic cathepsins (e.g., cathepsin D and cathepsin E); caspases (e.g., caspase 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., Cruzipain, legumin, and otubain-2); KLKs (e.g., KLK4-8, KLK10, KLK11, KLK 13 and KLK14); Metalloproteinases (e.g., Meprin, Neprilysin, PSMA, and BMP1); MMPs (e.g., MMP1-3, MMP 7-17, MMP 19, MMP 20, MMP 23, MMP 24, MMP 26, and MMP 27); 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; guanidinobenzoatase; HtrA1; lactoferrin; marapsin; NS3 / 4A; PACE4; plasmin; PSA, tPA; thrombin; tryptase; uPA; type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, hepsin, matriptase-2, MT-SP1 / matriptase, and TMPRSS2-4).

[0324] In some embodiments, the CM is selected for use with a specific protease, such as a protease known to co-localize with the target of the EpCAM-activatable antibody.

[0325] In some embodiments, the CM is a substrate for at least one MMP. Examples of MMPs include MMPs 1-3, MMPs 7-17, MMP19, MMP20, MMP23, MMP24, MMP26, and MMP27. In some embodiments, the CM is a substrate for a protease selected from the group consisting of MMP 9, MMP 14, MMP1, MMP3, MMP13, MMP 17, MMP11, and MMP19. In some embodiments, the CM is a substrate for MMP9. In some embodiments, the CM is a substrate for MMP14.

[0326] Suitable CMs that can be routinely incorporated into the provided activatable antibodies are known in the art. See, for example, WO2016 / 179285 (e.g., at pages 40-47), the contents of which are incorporated herein by reference in their entirety.

[0327] In some embodiments, CM is a substrate for neutrophil elastase. In some embodiments, CM is a substrate for serine proteases. In some embodiments, CM is a substrate for legumin. In some embodiments, CM is a substrate for matrix proteases. In some embodiments, CM is a substrate for cysteine ​​proteases. In some embodiments, CM is a substrate for cysteine ​​proteases such as cathepsins. In other embodiments, CM is a substrate for uPA.

[0328] In certain embodiments, the CM is a substrate for uPA. In some embodiments, the CM comprises a sequence disclosed in Table 10.

[0329] Table 10. Exemplary CM sequences.

[0330] <![CDATA[ Substrate name ]]> <![CDATA[ sequence ]]> 3014 AVGLLAPPGGLSGRSDNI (SEQ ID NO: 168) 2014 ISSGLLSGRSDNI(SEQ ID NO:169)

[0331] 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).

[0332] In some embodiments, the CM is a substrate for at least two proteases. In some embodiments, each protease is selected from the group consisting of: ADAMS / ADAMTS (e.g., ADAM8, ADAM9, ADAM 10, ADAM 12, ADAM 15, ADAM 17 / TACE, ADAMDEC1, ADAMTS1, ADAMTS4, ADAMTS5); aspartic proteases (e.g., BACE, renin); aspartic cathepsins (e.g., cathepsin D and cathepsin E); caspases (e.g., caspase 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., Cruzipain, legumin, and otubain-2); KLKs (e.g., KLK4-8, KLK10, KLK11, KLK 13 and KLK14); Metalloproteinases (e.g., Meprin, Neprilysin, PSMA and BMP1); MMPs (e.g., MMP1-3, MMP 7-17, MMP 19, MMP 20, MMP 23, MMP 24, MMP 26 and MMP 27); 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; Guanidinobenzoate esterase; HtrA1; Lactoferrin; Marapsin; NS3 / 4A; PACE4; Plasmin; PSA, tPA; Thrombin; Tryptase; uPA; Type II transmembrane serine proteases (TTSPs) (e.g., DESC1, DPP-4, FAP, serine transmembrane elastase); In some embodiments, the CM is a substrate for at least two proteases, one of which is selected from the group consisting of MMPs, thrombin, neutrophil elastase, cysteine ​​proteases, uPA, leguminase, and matriptase, and the other is selected from the group consisting of those listed above. In some embodiments, the CM is a substrate for at least two proteases selected from the group consisting of MMPs, thrombin, neutrophil elastase, cysteine ​​proteases, uPA, leguminase, and matriptase.

[0333] 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 polypeptides no longer than 15 amino acids. In some embodiments, the first and second CMs in the uncleaved EpCAM-activatable antibody have the following structural arrangement from N-terminus to C-terminus: MM-CM1-CM2-Ab or Ab-CM2-CM1-MM. In some embodiments, at least one of the first and second CMs is a polypeptide that serves as a substrate for a protease selected from the group consisting of MMPs, thrombin, neutrophil elastase, cysteine ​​proteases, uPA, myristases, and matriptase. In some embodiments, the first CM is cleaved by a first cleavage agent selected from the group consisting of MMPs, thrombin, neutrophil elastase, cysteine ​​proteases, uPA, myristases, and matriptase in the target tissue, and the second CM is cleaved by a second cleavage agent in the target tissue. In some embodiments, the second protease is selected from the list set forth in the previous paragraph. In some embodiments, the first and second cleaving agents are the same protease selected from a MMP, thrombin, neutrophil elastase, cysteine ​​proteases, uPA, leguminase, and malate, and the first CM and the second CM are different substrates for the enzymes. In some embodiments, the first and second cleaving agents are the same protease selected from the list in the previous paragraph. In some embodiments, the first and second cleaving agents are different proteases. In some embodiments, the first and second cleaving agents are co-localized in the target tissue. In some embodiments, the first and second cleaving agents are cleaved by at least one cleaving agent in the target tissue.

[0334] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein further 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 a signal peptide. In some embodiments, the spacer is directly linked to the MM of the EpCAM-activatable antibody. In some embodiments, the spacer is directly linked to the MM of the EpCAM-activatable antibody in a structural arrangement from N-terminus to C-terminus of Spacer-MM-CM-Ab.

[0335] Suitable spacers and spacer technology are known in the art and can be routinely used to incorporate spacers in some embodiments of the provided activatable antibodies. See, for example, WO 2016 / 179285 (e.g., at pages 52-53), the contents of which are incorporated herein by reference in their entirety.

[0336] In many embodiments, it may be desirable to insert one or more linkers (e.g., flexible linkers) into the EpCAM-activatable antibody construct to provide flexibility at the MM-CM junction, the CM-Ab junction, 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 required flexibility. Thus, the switchable phenotype of such EpCAM-activatable antibody constructs may benefit from the introduction of one or more amino acids to provide a flexible linker. Furthermore, as described below, when the EpCAM-activatable antibody is provided as a conformationally constrained construct, a flexible linker may be operably inserted to promote the formation and maintenance of a ring structure in the uncleaved EpCAM-activatable antibody.

[0337] For example, in certain embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises one of the following formulae (wherein the following formulae represent an amino acid sequence in the N-terminal to C-terminal direction or the C-terminal to N-terminal direction):

[0338] (MM)-L1-(CM)-(Ab)

[0339] (MM)-(CM)-L2-(Ab)

[0340] (MM)-L1-(CM)-L2-(Ab)

[0341] Wherein MM, CM and Ab are as defined above; wherein LI and L2 are each independently and optionally present or absent, the same or different flexible linkers comprising at least one flexible amino acid (e.g., Gly). In addition, the above formula provides additional amino acid sequences that can be located at the N-terminus or C-terminus of the EpCAM activatable antibody element. Examples include, but are not limited to, targeting moieties (e.g., ligands for receptors present on cells in the target tissue) and serum half-life extending moieties (e.g., polypeptides that bind serum proteins, such as immunoglobulins (e.g., IgG) or serum albumins (e.g., human serum albumin (HSA))).

[0342] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein is exposed to and cleaved by a protease such that, in the activated or cleaved state, the activated antibody comprises a light chain sequence that comprises at least a portion of the LP2 and / or CM sequence after the protease has cleaved the CM.

[0343] CM is treated with at least one protease at a concentration of about 0.001-1500 x 10 4 M -1 S -1or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500 x 10 4 M -1 S -1 In some embodiments, the CM is specifically cleaved at a rate of about 100,000 M -1 S -1 In some embodiments, the CM is specifically cleaved at a rate of about 1x10 2 to about 1x 10 6 M -1 S -1 (i.e., approximately 1x10 2 to about 1x l0 6 M -1 S -1 ) is specifically cleaved at a rate of .

[0344] For specific enzymatic cleavage, contact is established between the enzyme and the CM. When an EpCAM-activatable antibody comprising an Ab (e.g., an EpCAM antibody or EpCAM-binding antibody fragment thereof) coupled to both the CM and the CM is present in the presence of EpCAM and sufficient enzymatic activity, the CM can be cleaved. Sufficient enzymatic activity refers to the ability of the enzyme to contact the CM and achieve cleavage. It is readily foreseeable that the enzyme may be in the vicinity of the CM but unable to cleave it due to protein modification by other cytokines or the enzyme.

[0345] The EpCAM activatable antibodies suitable for use in the immunoconjugates described herein, the linkers in the compositions disclosed herein are generally those that provide flexibility to the modified Ab (e.g., EpCAM antibodies or EpCAM binding antibody fragments thereof) or EpCAM activatable antibodies to help inhibit the binding of EpCAM activatable antibodies to human EpCAM. Such linkers are generally referred to as flexible linkers. Suitable linkers can be easily selected and can have any suitable different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and the length 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.

[0346] Examples of 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 technology are known in the art and can be routinely used to incorporate spacers in some embodiments of the activatable antibodies provided. See, for example, WO2016 / 179285 (e.g., at pages 26, 113-116), the contents of which are incorporated herein by reference in their entirety. One of ordinary skill will recognize that the design of the EpCAM activatable antibody may include a linker that is fully or partially flexible, such that the linker may include a flexible linker as well as one or more portions that impart a less flexible structure to provide the desired EpCAM activatable antibody structure.

[0347] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the EpCAM-activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-Ab or Ab-LP2-CM-LP1-MM. In some embodiments, the two connecting peptides need not be identical to each other.

[0348] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises a first connecting peptide (LP1) and a second connecting peptide (LP2), and the EpCAM-activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: MM-LP1-CM-LP2-Ab or Ab-LP2-CM-LP1-MM. In some embodiments, the two connecting peptides need not be identical to each other.

[0349] In some embodiments, at least one of LP1 or LP2 of the EpCAM activatable antibody of the immunoconjugates described herein comprises a flexible linker. Suitable linkers and linker technology are known in the art and can be routinely used to incorporate a spacer in some embodiments of the provided activatable antibodies. See, for example, WO 2016 / 179285 (e.g., at pages 26, 113-116), the contents of which are incorporated herein by reference in their entirety.

[0350] 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 a sequence of SEQ ID NO: 174. In some embodiments, the activatable antibody comprises a light chain having a sequence of SEQ ID NO: 179.

[0351] Table 11. Exemplary light chain activatable antibody sequences.

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises a light chain having a sequence selected from the group consisting of SEQ ID NOs: 170-180 and a heavy chain having a sequence selected from the group consisting of SEQ ID NOs: 103, 125, and 127. In some embodiments, the EpCAM-activatable antibody comprises a light chain having a sequence selected from the group consisting of SEQ ID NOs: 170-180 and a heavy chain having a sequence of SEQ ID NO: 103. In some embodiments, the EpCAM-activatable antibody comprises a light chain having a sequence selected from the group consisting of SEQ ID NOs: 174 and a heavy chain having a sequence of SEQ ID NO: 103. In some embodiments, the EpCAM-activatable antibody comprises a light chain having a sequence of SEQ ID NO: 179 and a heavy chain having a sequence of SEQ ID NO: 103.

[0359] 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%.

[0360] In some embodiments, the EpCAM-activatable antibody of the immunoconjugate described herein comprises a light chain having a sequence selected from SEQ ID NOs: 181-188 and a heavy chain having a 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 a sequence of SEQ ID NO: 125.

[0361] In certain embodiments, the huEpCAM23 antibody is encoded by a plasmid deposited with the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, Va 20110 under the Budapest Treaty on October 4, 2018, and having ATCC deposit numbers PTA-125343, PTA-125344, or PTA-125345. Examples of EpCAM antibodies, EpCAM-binding antibody fragments thereof, and EpCAM-activatable antibodies and immunoconjugates are provided herein.

[0362] 6.4 Polynucleotides, Vectors, Host Cells, and Recombinant Methods

[0363] The present disclosure further provides polynucleotides comprising nucleotide sequences encoding the EpCAM antibodies, EpCAM-binding antibody fragments thereof, and EpCAM-activatable antibodies disclosed herein.

[0364] Polynucleotides can be obtained using methods known in the art, and the nucleotide sequence of the polynucleotides can be determined. For example, if the nucleotide sequence of an antibody is known, the polynucleotides encoding the antibody can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)). In short, this involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and connecting those oligonucleotides, and then amplifying the connected oligonucleotides by PCR.

[0365] In some embodiments, the polynucleotides of the present disclosure comprise the sequences listed in Table 12.

[0366] Table 12. Exemplary nucleic acid sequences.

[0367]

[0368]

[0369]

[0370]

[0371] In one embodiment, the EpBA of the present disclosure comprises a heavy chain nucleic acid sequence of SEQ ID NO: 201 and a light chain nucleic acid sequence of SEQ ID NO: 202. In one embodiment, the EpBA of the present disclosure comprises a heavy chain nucleic acid sequence of SEQ ID NO: 203 and a light chain nucleic acid sequence of SEQ ID NO: 204. In one embodiment, the EpBA of the present disclosure comprises a heavy chain nucleic acid sequence of SEQ ID NO: 205 and a light chain nucleic acid sequence of SEQ ID NO: 204. In one embodiment, the EpBA of the present disclosure comprises a heavy chain nucleic acid sequence of SEQ ID NO: 206 and a light chain nucleic acid sequence of SEQ ID NO: 204. In one embodiment, the EpBA of the present disclosure comprises a heavy chain nucleic acid sequence of SEQ ID NO: 207 and a light chain nucleic acid sequence of SEQ ID NO: 204.

[0372] In one embodiment, the EpBA of the present disclosure comprises a heavy chain nucleic acid sequence of SEQ ID NO: 203 and a light chain nucleic acid sequence of SEQ ID NO: 208. In one embodiment, the EpBA of the present disclosure comprises a heavy chain nucleic acid sequence of SEQ ID NO: 203 and a light chain nucleic acid sequence of SEQ ID NO: 209.

[0373] In some embodiments, the EpBAs of the present disclosure comprise (i) a heavy chain variable region comprising a heavy chain variable region comprising a heavy chain variable region comprising a heavy chain variable region deposited with the American Type Culture Collection as PTA-125343. and (ii) a light chain variable region comprising an amino acid sequence identical to the amino acid sequence of the heavy chain variable region encoded by the plasmid deposited at The present disclosure also encompasses methods for preparing and using EpCAM antibodies and EpCAM-binding antibody fragments thereof comprising the EpBA.

[0374] In some embodiments, the present disclosure provides an EpCAM antibody comprising (i) a heavy chain comprising the same heavy chain as that deposited at and (ii) a light chain comprising an amino acid sequence identical to the amino acid sequence of the heavy chain encoded by the plasmid deposited at The present disclosure also encompasses methods of making and using EpCAM antibodies.

[0375] In some embodiments, the present disclosure provides EpCAM-activatable antibodies or EpCAM-binding activatable antibody fragments comprising (i) a heavy chain variable region comprising the same heavy chain variable region as that deposited at and (ii) a light chain variable region comprising an amino acid sequence identical to the amino acid sequence of the heavy chain variable region encoded by the plasmid deposited at The present disclosure also encompasses methods of making and using EpCAM-activatable antibodies or EpCAM-binding, activatable antibody fragments.

[0376] In other embodiments, the present disclosure provides EpCAM-activatable antibodies or EpCAM-binding activatable antibody fragments comprising (i) a heavy chain variable region comprising a heavy chain variable region deposited at and (ii) a light chain variable region comprising an amino acid sequence identical to the amino acid sequence of the heavy chain variable region encoded by the plasmid deposited at The present disclosure also encompasses methods of making and using EpCAM-activatable antibodies or EpCAM-binding, activatable antibody fragments.

[0377] The method for building the recombinant vector containing antibody coding sequence and suitable transcription and translation control signal is well known in the art.Once recombinantly expressed antibody, the antibody can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (for example, ion exchange chromatography, affinity chromatography, particularly by affinity for specific antigens after protein A, and size fractionation column chromatography), centrifugal, differential solubility or by any other standard technique for purifying protein.In this respect, reference has been made to U.S. Patent number 7,538,195 in the present disclosure, and the content of the patent is hereby incorporated by reference in its entirety.

[0378] The present disclosure also provides methods for producing the EpCAM antibodies, EpCAM-binding antibody fragments thereof, or EpCAM-activatable antibodies disclosed herein by culturing cells under conditions that result in expression of the antibodies and / or EpCAM-activatable antibodies, wherein the cells comprise a nucleic acid molecule encoding the antibodies, antibody fragments, or activatable antibodies. In some embodiments, the cells are Chinese Hamster Ovary (CHO) cells.

[0379] The present disclosure also provides a method for producing an EpCAM-activatable antibody that binds to EpCAM in an activated state by: (a) culturing cells comprising a nucleic acid construct encoding the EpCAM-activatable antibody under conditions that produce 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., an EpCAM antibody or an EpCAM-binding antibody fragment thereof), (i) wherein the CM is a polypeptide that serves as a substrate for a protease; and (ii) wherein the CM is located in the EpCAM-activatable antibody such that when the EpCAM-activatable antibody is in an uncleaved state, the MM interferes with the specific binding of the Ab to EpCAM, and when in a cleaved state, the MM does not interfere with or compete with the specific binding of the Ab to EpCAM; and (b) recovering the EpCAM-activatable antibody. Suitable Abs, MMs, and / or CMs include any Abs, MMs, and / or CMs disclosed herein. In some embodiments, the cells are Chinese hamster ovary (CHO) cells.

[0380] 6.5 Immunoconjugates

[0381] EpCAM immunoconjugates as disclosed herein comprise camptothecin and / or its derivatives. Camptothecin and its derivatives have been disclosed in U.S. Application Publication No. 2021 / 0077482A1 and U.S. Patent No. 11,229,639B2, each of which is incorporated herein by reference in its entirety.

[0382] In some cases, the EpCAM immunoconjugate is produced by conjugating at least one linker-payload reactant comprising a compound of Formula I or a pharmaceutically acceptable salt thereof to an EpCAM antibody as described in Section 6.2 or an EpCAM-activatable antibody as described in Section 6.3:

[0383] EA-Z'-L 1 -D (Formula I)

[0384] in:

[0385] D is represented by the following structural formula:

[0386]

[0387] R 1 It is F;

[0388] R 2 It is a methyl group;

[0389] -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 of covalent attachment to A;

[0390] Each R 5 are independently H, methyl or benzyl;

[0391] Each R 8 are independently H, methyl or benzyl;

[0392] A is a peptide comprising 2 to 4 amino acids; and

[0393] E is C(=O)-(C1-C 10 Alkylene)-X 3 , where X 3 yes

[0394]

[0395] In some cases, the linker-payload reaction comprises:

[0396]

[0397] or a pharmaceutically acceptable salt thereof.

[0398] In some cases, the linker-payload reaction comprises:

[0399]

[0400] or a pharmaceutically acceptable salt thereof.

[0401] When the linker-payload reactant comprises a compound of Formula I or a pharmaceutically acceptable salt thereof, the maleimide group X 3 Reacts with the thiol (-SH) group of the EpCAM antibody or activatable antibody to form a covalent bond in the immunoconjugate as follows:

[0402]

[0403] wherein ** indicates linkage to the EpCAM antibody or activatable antibody via the sulfur of the thiol group.

[0404] In some embodiments, the thiol (-SH) group of the EpCAM antibody or activatable antibody is a cysteine ​​thiol group. One skilled in the art will recognize that once the group X in Formula I of the above linker-payload reactant is 3The maleimide of the linker-payload reacts with the thiol of the EpCAM antibody or the activatable antibody, and the maleimide group is reduced to a succinimide. Thus, the linker-payload comprises a succinimide when conjugated to the EpCAM antibody or the activatable antibody.

[0405] In some embodiments, the EpCAM antibody or activatable antibody is an IgG1 antibody having eight cysteines that participate in the formation of four interchain disulfide bonds when in a native conformation (e.g., unreduced). 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 a heavy chain via one covalent disulfide bond, while the two heavy chains are linked together via two disulfide bonds. As provided herein, these disulfide bonds can be reduced, thereby generating up to eight free cysteines and cysteine ​​thiol groups. One linker-payload reactant can react with the thiol group of one cysteine, and up to eight linkers-payload reactants react with each IgG1 EpCAM antibody or activatable antibody. In some embodiments, each EpCAM antibody or activatable antibody reacts with up to eight linkers-payload reactants.

[0406] In some cases, the EpCAM immunoconjugate comprises at least one linker-payload comprising a compound of Formula I or a pharmaceutically acceptable salt thereof (wherein the compound of Formula I or a pharmaceutically acceptable salt thereof represents the linker-payload reactant, i.e., the linker-payload prior to conjugation to the EpCAM antibody or activatable antibody):

[0407] EA-Z'-L 1 -D (Formula I)

[0408] in:

[0409] D is represented by the following structural formula:

[0410]

[0411] R 1 It is F;

[0412] R 2 It is a methyl group;

[0413] -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 of covalent attachment to A;

[0414] Each R 5 are independently H, methyl or benzyl;

[0415] Each R 8 are independently H, methyl or benzyl;

[0416] A is a peptide comprising 2 to 4 amino acids; and

[0417] E is C(=O)-(C1-C 10 Alkylene)-X 3 , where X 3 yes

[0418]

[0419] Thus, in some cases, the EpCAM immunoconjugate comprises at least one linker-payload comprising a structure of Formula (Ia) or a pharmaceutically acceptable salt thereof:

[0420] EA-Z'-L 1 -D (Formula (Ia))

[0421] in:

[0422] D is represented by the following structural formula:

[0423]

[0424] R 1 It is F;

[0425] R 2 It is a methyl group;

[0426] -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 of covalent attachment to A;

[0427] Each R 5 are independently H, methyl or benzyl;

[0428] Each R 8 are independently H, methyl or benzyl;

[0429] A is a peptide comprising 2 to 4 amino acids; and

[0430] E is C(=O)-(C1-C 10 Alkylene)-X 3 , where X 3 yes

[0431]

[0432] where ** is the site for covalent attachment to the EpCAM antibody or activatable antibody.

[0433] In some cases, A in Formula (I) or A in Formula (Ia) is substituted with one or more polyols. In some cases, the polyol is -(C1-C6 alkylene)-X 5 -Y 3 , where X 5 Yes-NR 12 C(=O)- or -C(=O)NR 12 -;Y 3 is a C6 alkyl group substituted with 5 OH groups; and R 12 It is -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl.

[0434] In some cases, the polyol of the immunoconjugate comprises

[0435]

[0436] wherein R12 is H or methyl.

[0437] In some cases, the immunoconjugate comprises the following linker-payload conjugate (i.e., linker-payload prior to conjugation to the EpCAM antibody or activatable antibody):

[0438]

[0439] or a pharmaceutically acceptable salt thereof.

[0440] Thus, in some cases, the immunoconjugate comprises the following linker-payload:

[0441]

[0442] or a pharmaceutically acceptable salt thereof.

[0443] In some cases, the immunoconjugate comprises the following linker-payload conjugate (i.e., linker-payload prior to conjugation to the EpCAM antibody or activatable antibody):

[0444]

[0445] or a pharmaceutically acceptable salt thereof.

[0446] Thus, in some cases, the immunoconjugate comprises the following linker-payload:

[0447]

[0448] or a pharmaceutically acceptable salt thereof.

[0449] In this article, the 1 In various embodiments of -Z'-*, * is a site of covalent attachment to A. In some embodiments, A is a peptide comprising 2 to 8 amino acids. In some embodiments, A is a peptide comprising 2 to 4 amino acids. In some embodiments, at least one amino acid in the peptide is an L amino acid. In some embodiments, each amino acid in the peptide is an L amino acid. In some embodiments, at least one amino acid in the peptide is a D amino acid. In some embodiments, A is -(AA 1 )-(AA 2 ) a1 -*, where * is the site of covalent attachment 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 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-A la-*, -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 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-Ar g-D-Arg-*, -L-Ala-Ala-*, -L-Ala-D-Ala-*, -Ala-D-Ala-*, -Val-D-Cit-*, -L-Al a-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-*.

[0450] In some embodiments, -AA 1 -(AA 2 )a1 -*Yes-L-Ala-L-Ala-L-Ala-*.

[0451] Disclosed herein -AA 1 -(AA 2 )a1-*, * is the site of covalent attachment to E.

[0452] In some embodiments, the EpCAM antibody or activatable antibody is an IgG1 antibody with eight cysteines that participate in the formation of four disulfide bonds when in a native conformation (e.g., unreduced). 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, these disulfide bonds can be reduced to produce eight free cysteines and cysteine ​​thiol groups. One linker-payload reactant can react with the thiol group of one cysteine, and up to eight linkers-payload reactants react with each EpCAM antibody or activatable antibody. In some embodiments, each EpCAM antibody or activatable antibody reacts with up to eight linkers-payload reactants, thereby generating a linker-payload to antibody ratio (i.e., drug to antibody ratio (DAR)) in the range of 2 to 8. In certain embodiments, DAR represents the average number of cytotoxic linkers-payload agents per antibody or activatable antibody molecule. In some embodiments, DAR is 2. In some embodiments, 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.

[0453] In some cases, the linker-payload to antibody ratio (i.e., drug to antibody ratio (DAR)) is in the range of 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.

[0454] In some embodiments, the present disclosure provides EpCAM immunoconjugates comprising at least one linker-payload comprising a compound of the formula:

[0455] ZL 1 -D

[0456] Wherein D is represented by the following structural formula:

[0457]

[0458] R 1 is -F, -CH3 or -CF3;

[0459] R 2 Yes -H, -F, -OR 3 ,—SR 3 、—S(O)R 4 ,—S(O)2R 4 , C1-C6 alkyl or C1-C6 fluoroalkyl; or R 1 and R 2 Together with the carbon atoms to which they are attached, they form a methylenedioxy or difluoromethylenedioxy ring;

[0460] R 3 is H or C1-C6 alkyl;

[0461] R 4 is a C1-C6 alkyl group;

[0462] L 1 does not exist, is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 —(C1-C6 alkylene)-, —X 1′ —(C1-C6 alkylene)-* or —(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z;

[0463] X 1 It’s —O—, —S—, —S(O)—, —S(O)2—, —C(═O)—, —NR 5 —、—NR 5 C(═O)—or—C(═O)NR 5 —;

[0464] X 1′ is —O—, —S—, —S(O)—, or —S(O)2—;

[0465] L 2 is phenylene;

[0466] Each R 5 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0467] Z is -H or -X 2 ;

[0468] X 2 Yes—OR6 ,—SR 6 、—S(O)R 6 ,—S(O)2R 6 ,—SSR 6 or—N(R 6 )2;

[0469] Each R 6 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0470] L 1 and L 2 Each independently optionally substituted by 1-4 substituents selected from the group consisting of halogen, -CN, -SR 7 、—N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 heterocycloalkyl, aryl, or heteroaryl; and

[0471] Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0472] The condition is that if R 1 If it is F, then L 1 is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 —(C1-C6 alkylene)-, —X 1′ —(C1-C6 alkylene)-* or —(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z; and Z is -X 2 ;

[0473] The condition is that if R 1 is F and R 2 is —OMe, then it cannot be NH2; and

[0474] The condition is that if R 1 is F and R 2 If it is -Me, then -L1-Z cannot be -CH2OH.

[0475] Additional camptothecin derivatives are disclosed in U.S. Application Publication No. 2021 / 0077482A1 and U.S. Patent No. 11,229,639B2, each of which is herein incorporated by reference in its entirety.

[0476] 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 delutecan, wherein the maleimide group of delutecan is reduced to succinimide during the conjugation reaction, thereby linking delutecan to the EpCAM antibody or activatable antibody via the thiol group. Delutecan comprises an exatecan derivative and a maleimide-GGFG linker (SEQ ID NO: 311). Exatecan, its derivatives, and delutecan are described, for example, in WO2015 / 115091, the contents of which are incorporated herein by reference in their entirety.

[0477] 6.6 Methods for Preparing Immunoconjugates

[0478] The immunoconjugates comprising an EpCAM-binding agent ("EpBA," e.g., an EpCAM antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody) covalently linked to a cytotoxic agent via an interchain cysteine ​​residue located on the EpBA as described in the above embodiments or any specific embodiments described therein can be prepared according to any method known in the art, see, e.g., 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 in their entirety.

[0479] Methods for preparing EpCAM-specific conjugates are disclosed in WO 2020 / 086665, the entire contents of each of which are incorporated herein by reference in their entirety.

[0480] In some embodiments, the immunoconjugates of the present disclosure are prepared by a method comprising the steps of 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 specific embodiments, the thiol-reactive group of the cytotoxic agent (e.g., a linker-payload disclosed herein) reacts with a thiol group of a cysteine ​​residue of the EpBA.

[0481] In certain embodiments, a thiol-reactive group (eg, maleimide) of a cytotoxic agent is coupled to a thiol group (eg, cysteine ​​thiol) of EpBA via a thiol-Michael addition.

[0482] In some embodiments, the conjugation of the linker-payload reactant to the EpCAM activatable antibody includes partial reduction of the activatable antibody. This exposes reactive native cysteines that typically form disulfide bridges between the heavy chains and between the light and heavy chains of the activatable antibody, but does not interfere with one or more disulfide bonds present within the masking portion (MM) of the activatable antibody. Thus, the MM retains the ability to effectively mask the EpCAM antibody or its EpCAM binding fragment. When reduction and subsequent conjugation are not properly controlled, the activatable antibody is completely reduced and the masking efficiency of the activatable antibody is / will be compromised.

[0483] 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 respective thiol groups can react with the thiol-reactive group (e.g., maleimide) of the linker-payload.

[0484] Examples of reducing agents suitable for partial reduction of EpCAM-activatable antibodies include, but are not limited to, BMS (bis(2-mercaptoethyl)sulfone), cysteamine, cysteine, DMH (dimethyl-bis-mercaptoacetic hydrazide), DTBA (dithiobutylamine), DTT (dithiothreitol), GILT (gamma interferon-inducible lysosomal thiol reductase; for enzyme 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.

[0485] In some embodiments, the reducing agent is TCEP. TCEP is often used as a reducing agent to cleave disulfide bonds within and between proteins. TCEP has strong selectivity and does not react with other functional groups present in proteins, nor with embedded disulfides. Compared to two other common reducing agents, DTT and β-mercaptoethanol, TCEP has the advantages of being a stronger reducing agent, being more hydrophilic, more resistant to oxidation in air, and being odorless. Unlike DTT, TCEP is active under both alkaline and acidic conditions. TCEP is particularly useful when reacting cysteine ​​residues with maleimide. TCEP prevents cysteine ​​from forming disulfide bonds, and unlike DTT and β-mercaptoethanol, TCEP does not readily react with maleimide.

[0486] In some embodiments, the ratio of reducing agent (e.g., TCEP) to EpCAM-activatable antibody will be 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 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 1:1. 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 4:1 to 1:1. 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 8:1 to about 1:1. In some embodiments, the ratio is in the range of about 2.5:1 to 1:1.

[0487] The maleimide functional group in the linker-payload reactant reacts with a thiol (-SH) group on the activatable antibody to form a covalent thioester linkage. In some embodiments, the native cysteine ​​on the activatable antibody comprises a thiol (-SH) group. In some embodiments, the EpCAM activatable antibody is partially reduced with 4.5 molar equivalents of TCEP solution at room temperature for about 3 hours. The partially reduced activatable antibody is reacted with 10 molar equivalents of the linker-payload reactant in an aqueous buffer solution with up to about 8.3% (v / v) DMAc as a cosolvent at room temperature for about 1 hour. The reaction 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 small molecule impurities and the buffer exchanged into a base formulation buffer by UF / DF. In some embodiments, the base formulation buffer comprises one or more pharmaceutically acceptable excipients. The base formulation buffer containing the immunoconjugate can then be sterile filtered into a container.

[0488] Exemplary methods for partially reducing activatable antibodies and conjugating linker-payloads thereto are provided herein, e.g., in Examples 7.4.1-7.4.5.

[0489] In some embodiments, the conjugation methods provided herein using EpCAM antibodies or activatable antibodies and linker-payload reactants produce generally homogeneous immunoconjugates having 8 linker-payloads conjugated to each antibody or activatable antibody. Few immunoconjugates prepared using the methods and materials provided herein have a linker-payload number other than 8 that is conjugated to each antibody or activatable antibody. This is in contrast to huEpCAM23Gv4.2-GMBS-DM21L (see, e.g., Example 7.4.5), which has a DAR between 3.5 and 5. The consistently higher DAR of 8 for the immunoconjugates described herein provides a strong bystander effect.

[0490] Representative methods for preparing the immunoconjugates of the disclosure are provided in Example 7.4.1.

[0491] In certain embodiments, the immunoconjugate is prepared by a method comprising reacting an EpBA (eg, an EpCAM antibody, EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody disclosed herein) with a cytotoxic agent having an amine-reactive group.

[0492] In some embodiments, the reaction is carried out in the presence of an imine-reactive reagent such as NaHSO 3 .

[0493] In certain embodiments, the immunoconjugate is prepared by a method comprising the steps of:

[0494] (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 having the amine-reactive group attached thereto (i.e., a linker-payload reactant); and

[0495] (b) reacting the EpBA with the cytotoxic agent-linker compound.

[0496] In one embodiment, the reaction in step (a) is carried out in the presence of an imine-reactive reagent (e.g., NaHSO 3 ). In one embodiment, the cytotoxic agent-linker compound is reacted with EpBA without purification. Alternatively, the cytotoxic agent-linker compound (i.e., linker-payload reactant) is first purified prior to reaction with EpBA.

[0497] In certain embodiments, the immunoconjugate of the embodiments is prepared by a method comprising the steps of:

[0498] (a) reacting EpBA with a linker compound having an amine-reactive group and a thiol-reactive group to form a modified EpBA having a thiol-reactive group bound thereto; and

[0499] (b) reacting the modified EpBA with a cytotoxic agent.

[0500] In one embodiment, the reaction in step (b) is carried out in the presence of an imine-reactive reagent (eg, NaHSO 3 ).

[0501] In certain embodiments, the immunoconjugate is prepared by a method comprising the following steps: reacting EpBA, a cytotoxic compound, and a linker compound having an amine reactive group and a thiol reactive group. In one embodiment, the reaction is carried out in the presence of an imine reactive agent (e.g., NaHSO 3 ).

[0502] In the first aspect, for 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 specific 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 another specific embodiment, the reaction of step (a) is carried out at pH 3.3. As used herein, a specific pH value refers to the specific value ± 0.05.

[0503] In some embodiments, the reaction of step (a) is carried out in the presence of a buffer solution. Any suitable buffer solution known in the art can be used in the provided method. Suitable buffer solutions include, for example, but not limited to, citrate buffer, acetate buffer, succinate buffer, phosphate buffer, glycine-containing buffer (for example, glycine-HCl buffer), phthalate buffer (for example, a buffer solution comprising sodium hydrogen phthalate or potassium hydrogen phthalate) and combinations thereof. In some embodiments, the buffer solution is a succinate buffer. In some embodiments, the buffer solution is a phosphate buffer. In some embodiments, the buffer is a citrate-phosphate buffer. In some embodiments, the buffer is a citrate-phosphate buffer comprising citric acid and Na2HPO4 citrate-phosphate buffer. In other embodiments, the buffer is a citrate-phosphate buffer comprising citric acid and K2HPO4. In some embodiments, the concentration of the buffer solution may be in the range of 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.

[0504] In the second aspect, the reaction step (a) is carried out in the absence of a buffer solution (e.g., the buffer described in the first aspect). In some embodiments, the method of the present invention comprises the following steps: (a) reacting an imine-containing cytotoxic agent having a thiol-reactive group as described above (i.e., an imine moiety in 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 aqueous solution to form a modified cytotoxic agent comprising a modified imine moiety represented by the following formula:

[0505]

[0506] or a pharmaceutically acceptable salt thereof, wherein the aqueous solution does not contain a buffer; and (b) reacting the modified cytotoxic agent with an EpCAM-binding agent disclosed herein (e.g., an EpCAM-binding antibody, an EpCAM-binding antibody fragment thereof, or an EpCAM-activatable antibody disclosed herein) to form an immunoconjugate. In some embodiments, the reaction of step (a) is carried out in a mixture of an organic solvent and water. More specifically, the reaction of step (a) is carried out in a mixture of dimethylacetamide (DMA) and water. In some embodiments, the mixture of DMA and water contains less than 60% DMA by volume. Even more specifically, the volume ratio of DMA to water is 1:1.

[0507] In the third aspect, for the method described above or in aspect 1 or aspect 2, 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 for every 1 equivalent of the imine-containing cytotoxic agent. In some embodiments, for every 1 equivalent of imine-containing cytotoxic agent, 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. In other embodiments, for every 1 equivalent of the imine-containing cytotoxic agent, 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, 0.75, 0.85, 0.90, 100, 150, 150, 160, 170, 180, 190, 210, 211, 220, 221 In some embodiments, the present invention provides an amount of metabisulfite in an amount of 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. In other embodiments, 1.4 equivalents of metabisulfite or 0.7 equivalents of metabisulfite are used for every 1 equivalent of the imine-containing cytotoxic agent. In other embodiments, 1.2 equivalents of metabisulfite or 0.6 equivalents of metabisulfite are used for every 1 equivalent of the imine-containing cytotoxic agent. As used herein, a specific equivalent refers to a specific value ± 0.05.

[0508] In a fourth aspect, for the above methods, the reaction of 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 of 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 of 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 of 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 other embodiments, the reaction of 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.

[0509] In the fifth aspect, for the methods described above or in aspects 1, 2, 3 or 4, the reaction of 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, dichloromethane, 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 of 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) can be present in an amount of 1%-99%, 1%-95%, 10%-80%, 20%-70%, 30%-70%, 1%-60%, 5%-60%, 10%-60%, 20%-60%, 30%-60%, 40%-60%, 45%-55%, 10%-50%, or 20%-40%, based on the total volume of water and the organic solvent. In some embodiments, the reaction of step (a) is carried out in a mixture of DMA and water, wherein the volume ratio of DMA to water is 1:1.

[0510] In the sixth aspect, for the methods described above or in aspects 1, 2, 3, 4 or 5, the reaction of 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 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 other embodiments, the reaction can be carried out at 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 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.

[0511] In a seventh aspect, for the methods described above or in aspects 1, 2, 3, 4, 5, or 6, the reaction of 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 carried out for 4 to 6 hours. In other embodiments, the reaction is carried out 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 carried out for 4 hours. In other embodiments, the reaction is carried out for 2 hours.

[0512] In an eighth aspect, for the method described above or in aspect 1, 2, 3, 4, 5, 6, or 7, the reaction of step (b) is carried out at a pH of 4 to 9. In some embodiments, the reaction of 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 of 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.

[0513] In some embodiments, for the methods described above or in aspects 1, 2, 3, 4, 5, 6, 7, or 8, the reaction of step (b) is carried out in an aqueous solution comprising a mixture of water and an organic solvent. Any suitable organic solvent described above can be used. More specifically, the organic solvent is DMA. In some embodiments, the aqueous solution comprises less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, less than 2%, or less than 1% organic solvent (e.g., DMA) by volume.

[0514] In some embodiments, for the methods described above or in aspects 1, 2, 3, 4, 5, 6, 7, or 8, the bisulfite is sodium bisulfite or potassium bisulfite, and the metabisulfite is sodium or potassium metabisulfite. In specific embodiments, the bisulfite is sodium bisulfite, and the metabisulfite is sodium metabisulfite.

[0515] In some embodiments, for the methods disclosed herein or in aspects 1, 2, 3, 4, 5, 6, 7, or 8, the modified cytotoxic agent is not purified prior to reacting with the cell-binding agent in step (b). Alternatively, the modified cytotoxic agent is purified prior to reacting with the cell-binding agent in step (b). Any suitable method disclosed herein can be used to purify the modified cytotoxic agent.

[0516] In some embodiments, for the above methods, the reaction of step (a) does not result in substantial sulfonation of the maleimide groups. 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 maleimide sulfonation is equal to the total amount of maleimide-sulfonated cytotoxic agent (a cytotoxic agent having sulfonation only on the maleimide) and disulfonated cytotoxic agent (a cytotoxic agent having sulfonation on both the maleimide and imine moieties) divided by the starting amount of the imine-containing cytotoxic agent prior to reaction with bisulfite or metabisulfite.

[0517] In some embodiments, the immunoconjugates prepared by any of the above methods are subjected to a purification step. In this regard, tangential flow filtration (TFF), non-adsorptive chromatography, adsorption chromatography, adsorptive filtration, selective precipitation or any other suitable purification method and combinations thereof can be used to purify the immunoconjugates from the other components of the mixture.

[0518] In some embodiments, a single purification step (e.g., TFF) is used to purify the immunoconjugate. In some embodiments, a single purification step (e.g., TFF) is used to purify the conjugate and exchange it into an appropriate formulation. In other embodiments, two consecutive purification steps are used to purify the immunoconjugate. For example, the immunoconjugate can first be purified by selective precipitation, adsorption filtration, absorption chromatography or non-absorption chromatography, and then purified with TFF. It will be understood by those of ordinary skill in the art that the purification of the immunoconjugate enables the separation of a stable conjugate comprising a cell binding agent chemically coupled to a cytotoxic agent.

[0519] Any suitable TFF system can be used for purification, including Pellicon-type systems (Millipore, Billerica, Mass.), Sartocon Cassette systems (Sartorius AG, Edgewood, NY), and Centrasette-type systems (Pall Corp., East Hills, NY).

[0520] 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, reversed phase chromatography and combinations thereof. The example of suitable hydroxyapatite resin includes ceramic hydroxyapatite (CHT I type and II type, Bio-Rad Laboratories, Hercules, Calif.), HA Ultrogel hydroxyapatite (Pall Corp., East Hills, NY) and ceramic fluorapatite (CFT I type and II type, Bio-Rad Laboratories, Hercules, Calif.). The example of suitable HCIC resin is MEP Hypercel resin (Pall Corp., East Hills, NY). Examples of suitable HIC resins include butyl-agarose, hexyl-agarose, phenyl-agarose, and octyl agarose resins (all from GE Healthcare, Piscataway, NJ) and Macro-prep methyl and Macro-Prep tert-butyl resins (Biorad Laboratories, Hercules, Calif.). Examples of suitable ion exchange resins include SP-agarose, CM-agarose, and Q-agarose resins (all from GE Healthcare, Piscataway, NJ) and Unosphere S resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable mixed-mode ion exchangers include Bakerbond CBAx resin (JT Baker, Phillipsburg NJ). Examples of suitable IMAC resins include chelating agarose resin (GE Healthcare, Piscataway, NJ) and Profinity IMAC resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable dye-ligand resins include blue agarose resin (GE Healthcare, Piscataway, NJ) and Affi-gel blue resin (Bio-Rad Laboratories, Hercules, Calif.).Examples of suitable affinity resins include protein A agarose resin (e.g., MabSelect, GE Healthcare, Piscataway, NJ), in which the cell binding agent is an antibody; and lectin affinity resins, e.g., Lentil lectin agarose resin (GE Healthcare, Piscataway, NJ), in which the cell binding agent carries an appropriate lectin binding site. Alternatively, an antibody specific for the cell binding agent can be used. Such an antibody can be immobilized on, for example, Sepharose 4 fast flow resin (GE Healthcare, Piscataway, NJ). Examples of suitable reverse phase resins include C4, C8, and C18 resins (Grace Vydac, Hesperia, Calif.).

[0521] Any suitable non-adsorptive chromatographic resin can be used for purification. Examples of suitable non-adsorptive chromatographic resins include, but are not limited to, SEPHADEX TM G-25, G-50, G-100, SEPHACRYL TM Resins (such as S-200 and S-300), SUPERDEX TM Resins (e.g., SUPERDEX TM 75 and Superdex TM 200), Resins (e.g., P-6, P-10, P-30, P-60, and P-100), as well as others known to those skilled in the art.

[0522] Immunoconjugates comprising EpBA covalently linked to a maytansinoid compound as described in the third embodiment herein can be prepared according to any suitable method known in the art.

[0523] In some embodiments, the immunoconjugates prepared by any of the above methods are subjected to a purification step. In this regard, tangential flow filtration (TFF), non-adsorptive chromatography, adsorption chromatography, adsorptive filtration, selective precipitation or any other suitable purification method and combinations thereof can be used to purify the immunoconjugates from the other components of the mixture.

[0524] In some embodiments, a single purification step (e.g., TFF) is used to purify the immunoconjugate. In some embodiments, a single purification step (e.g., TFF) is used to purify the conjugate and exchange it into an appropriate preparation. In other embodiments of the present invention, two continuous purification steps are used to purify the immunoconjugate. For example, the immunoconjugate can first be purified by selective precipitation, adsorption filtration, absorption chromatography or non-absorption chromatography, and then purified with TFF. It will be understood by those of ordinary skill in the art that the purification of the immunoconjugate enables the separation of a stable conjugate comprising a cell binding agent chemically coupled to a cytotoxic agent.

[0525] Any suitable TFF system can be used for purification, including Pellicon-type systems (Millipore, Billerica, Mass.), Sartocon Cassette systems (Sartorius AG, Edgewood, NY), and Centrasette-type systems (Pall Corp., East Hills, NY).

[0526] 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, reversed phase chromatography and combinations thereof. The example of suitable hydroxyapatite resin includes ceramic hydroxyapatite (CHT I type and II type, Bio-Rad Laboratories, Hercules, Calif.), HA Ultrogel hydroxyapatite (Pall Corp., East Hills, NY) and ceramic fluorapatite (CFT I type and II type, Bio-Rad Laboratories, Hercules, Calif.). The example of suitable HCIC resin is MEP Hypercel resin (Pall Corp., East Hills, NY). Examples of suitable HIC resins include butyl-agarose, hexyl-agarose, phenyl-agarose, and octyl agarose resins (all from GE Healthcare, Piscataway, NJ) and Macro-prep methyl and Macro-Prep tert-butyl resins (Biorad Laboratories, Hercules, Calif.). Examples of suitable ion exchange resins include SP-agarose, CM-agarose, and Q-agarose resins (all from GE Healthcare, Piscataway, NJ) and Unosphere S resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable mixed-mode ion exchangers include Bakerbond ABx resin (JT Baker, Phillipsburg NJ). Examples of suitable IMAC resins include chelating agarose resin (GE Healthcare, Piscataway, NJ) and Profinity IMAC resin (Bio-Rad Laboratories, Hercules, Calif.). Examples of suitable dye-ligand resins include blue agarose resin (GE Healthcare, Piscataway, NJ) and Affi-gel blue resin (Bio-Rad Laboratories, Hercules, Calif.).Examples of suitable affinity resins include protein A agarose resin (e.g., MabSelect, GE Healthcare, Piscataway, NJ), in which the cell binding agent is an antibody; and lectin affinity resins, such as Lentil lectin agarose resin (GE Healthcare, Piscataway, NJ), in which the cell binding agent carries an appropriate lectin binding site. Alternatively, an antibody specific for the cell binding agent can be used. Such an antibody can be immobilized on, for example, Sepharose 4 fast flow resin (GE Healthcare, Piscataway, NJ). Examples of suitable reverse phase resins include C4, C8, and C18 resins (Grace Vydac, Hesperia, Calif.).

[0527] Any suitable non-adsorptive chromatographic resin can be used for purification. Examples of suitable non-adsorptive chromatographic resins include, but are not limited to, 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), Resins (e.g., P-6, P-10, P-30, P-60, and P-100), and others known to those of ordinary skill in the art.

[0528] 6.6.1 Immunoconjugates Produced by the Provided Methods

[0529] Also provided herein are immunoconjugates produced by the methods described herein.

[0530] In some embodiments, provided herein is an immunoconjugate obtainable by the following method, the method comprising: (a) mixing an activatable EpCAM antibody composed of a heterotetramer of two heavy chains having an amino acid sequence of SEQ ID NO: 103 and two light chains having an amino acid sequence of SEQ ID NO: 179 with a mixture of EPPS buffer and EDTA solution to form an activatable EpCAM reaction mixture; (b) adding a TCEP solution to the activatable EpCAM reaction mixture to form a reduced activatable EpCAM reaction mixture; (c) mixing the reduced activatable EpCAM reaction mixture with a camptothecin linker-toxin stock solution comprising a linker-toxin having the structure

[0531] and (d) terminating the conjugation process. In some embodiments, the method further comprises preparing a camptothecin linker-toxin stock solution by mixing the linker-toxin with anhydrous DMA. In certain embodiments, the linker-toxin and DMA are vortexed until all solids are dissolved.

[0532] In some embodiments, provided herein are immunoconjugates obtainable by the methods described in Example 7.4.1.

[0533] 6.7 Therapeutic Applications

[0534] Also included are methods for inhibiting the growth of cells expressing EpCAM (e.g., human EpCAM or cynomolgus monkey EpCAM). As provided herein, the disclosed EpCAM immunoconjugates have the ability to bind to EpCAM present on the surface of cells (e.g., human cells or cynomolgus monkey cells) and mediate cell killing. In certain embodiments, the immunoconjugate comprises a cytotoxic payload, such as a camptothecin derivative linker-payload that is internalized and mediates cell killing via the activity of the cytotoxic payload. This cell killing activity can be enhanced by immunoconjugate-induced antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC).

[0535] As used herein, the terms "inhibit" and "inhibit" include any inhibitory effect on cell growth, including cell death. Inhibitory effects include temporary effects, sustained effects, and permanent effects.

[0536] The therapeutic applications provided herein include methods for treating subjects suffering from a disease. Diseases treated using the provided methods are those characterized by EpCAM expression (e.g., EpCAM overexpression). Such diseases include, for example, breast cancer, lung cancer, non-small cell lung cancer, gastric cancer, prostate cancer, ovarian cancer, colorectal cancer, colon cancer, esophageal cancer, tracheal cancer, gastric cancer, bladder cancer, uterine cancer, rectal cancer or small intestinal cancer, 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. Those skilled in the art will appreciate that the methods disclosed herein can also be used to treat other diseases not yet described but characterized by EpCAM expression (e.g., overexpression).

[0537] The disclosed EpCAM immunoconjugates can be used to treat cancers that express EpCAM. In some embodiments, the cancer is an epithelial or squamous cell carcinoma. In some embodiments, the cancer is breast cancer, lung cancer, non-small cell lung cancer, gastric cancer, prostate cancer, ovarian cancer, colorectal cancer, colon cancer, esophageal cancer, tracheal cancer, stomach cancer, bladder cancer, uterine cancer, rectal cancer or small intestine cancer, pancreatic cancer, head and neck cancer, endometrial cancer, or metastases thereof.

[0538] The therapeutic applications provided herein can also be practiced in vitro and ex vivo.

[0539] The present disclosure also provides therapeutic applications of the disclosed EpCAM immunoconjugates, wherein the antibodies, antibody fragments, activatable antibodies, or conjugates are administered to a subject in a pharmaceutically acceptable dosage form. They can be administered intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, subcutaneous, parenteral, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. They can also be administered by intratumoral, peritumoral, intralesional, or perilesional routes to exert local as well as systemic therapeutic effects.

[0540] Also provided are methods for treating, preventing, and / or delaying the onset or progression of, or alleviating symptoms associated with aberrant expression and / or activity of EpCAM in a subject using EpCAM immunoconjugates that bind to EpCAM and neutralize or otherwise inhibit at least one biological activity of EpCAM and / or EpCAM-mediated signaling.

[0541] In some embodiments, the present disclosure provides methods for treating, preventing, and / or delaying the onset or progression of symptoms associated with the presence, growth, proliferation, metastasis, and / or activity of cells expressing or aberrantly expressing EpCAM in a subject using EpCAM immunoconjugates that bind to, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells expressing or aberrantly expressing EpCAM. The present disclosure also provides methods for treating, preventing, and / or delaying the onset or progression of symptoms associated with the presence, growth, proliferation, metastasis, and / or activity of cells expressing EpCAM in a subject using EpCAM immunoconjugates that bind to, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells expressing EpCAM.

[0542] The present disclosure also provides methods for treating, preventing, and / or delaying the onset or progression of, or alleviating symptoms associated with the presence, growth, proliferation, metastasis, and / or activity of cells that aberrantly express EpCAM in a subject using EpCAM immunoconjugates that bind to, target, neutralize, kill, or otherwise inhibit at least one biological activity of cells that aberrantly express EpCAM.

[0543] The present disclosure provides methods of preventing, delaying progression of, treating, alleviating symptoms of, or otherwise ameliorating 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.

[0544] The present disclosure also provides methods of preventing cancer (e.g., epithelial cancer and its metastasis), delaying the progression of cancer, treating cancer, alleviating the symptoms of cancer, or otherwise ameliorating cancer 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 a variety of cancers, including most cancers of epithelial origin (and metastases).

[0545] In some embodiments, the cancer is epithelial or squamous carcinoma.

[0546] In some embodiments, the cancer is head and neck cancer, breast cancer, lung cancer, non-small cell lung cancer, gastric cancer, prostate cancer, ovarian cancer, colorectal cancer, colon cancer, esophageal cancer, tracheal cancer, stomach cancer, bladder cancer, uterine cancer, endometrial cancer, rectal cancer, pancreatic cancer, or small intestine cancer.

[0547] In some embodiments, the cancer is breast cancer, lung cancer, gastric cancer, colorectal cancer, colon cancer, rectal cancer, small intestine cancer, ovarian cancer, stomach cancer, or esophageal cancer.

[0548] In some embodiments, the cancer is ovarian cancer, uterine cancer, gastric cancer, pancreatic cancer, or colorectal cancer.

[0549] In some embodiments, the cancer is ovarian cancer.

[0550] In some embodiments, the cancer is uterine cancer.

[0551] In some embodiments, the cancer is gastric cancer.

[0552] In some embodiments, the cancer is pancreatic cancer.

[0553] In some embodiments, the cancer is colorectal cancer.

[0554] In some embodiments, the cancer is head and neck cancer.

[0555] In some embodiments, the cancer is breast cancer. In certain embodiments, the cancer is triple-negative breast cancer.

[0556] 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.

[0557] The EpCAM immunoconjugates used in any of the embodiments of these methods and uses can be administered at any stage of the disease. For example, such EpCAM immunoconjugates can be administered to patients with cancer at any stage from early to metastatic. The terms subject and patient are used interchangeably herein.

[0558] In some embodiments, the subject is a mammal, such as a human, non-human primate, companion animal (e.g., cat, dog, horse), farm animal, 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 veterinary care.

[0559] EpCAM immunoconjugates and therapeutic formulations thereof are administered to subjects suffering from or susceptible to a disease or condition associated with aberrant EpCAM expression and / or activity (e.g., cancer). Subjects suffering from or susceptible to a disease or condition associated with aberrant 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 any of a variety of clinical and / or laboratory tests (e.g., physical examination and analysis of blood, urine, and / or stool to assess health). For example, subjects suffering from inflammation and / or inflammatory conditions are identified using any of a variety of clinical and / or laboratory tests (e.g., physical examination and / or analysis of body fluids, such as blood, urine, and / or stool to assess health).

[0560] Administration of an EpCAM immunoconjugate to a patient with a disease or condition associated with aberrant EpCAM expression and / or activity (e.g., a cancer, such as 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 with a disease or condition associated with aberrant EpCAM expression and / or activity is considered successful if one or more symptoms associated with the disease or condition are alleviated, reduced, suppressed, or do not progress to a further (i.e., worse) state. Administration of an EpCAM immunoconjugate to a patient with a disease or condition associated with aberrant EpCAM expression and / or activity is considered successful if the disease or condition enters remission or does not progress to a further (i.e., worse) state.

[0561] In some embodiments, EpCAM immunoconjugates and therapeutic formulations thereof are administered to a subject suffering from or susceptible to a disease or condition, such as a subject suffering from cancer or other neoplastic conditions, wherein the subject's 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. Any of a variety of methods known in the art can be used to identify subjects suffering from or susceptible to a disease or condition in which the subject's diseased cells express EpCAM. For example, any of a variety of clinical and / or laboratory tests (such as physical examination and blood, urine, and / or stool analysis to assess health status) can be used to identify subjects suffering from cancer or other neoplastic conditions. For example, any of a variety of clinical and / or laboratory tests (such as physical examination and / or analysis of body fluids, such as blood, urine, and / or stool analysis to assess health status) can be used to identify subjects suffering from inflammation and / or inflammatory conditions.

[0562] In some embodiments, EpCAM immunoconjugates and therapeutic formulations thereof are administered to subjects suffering from or susceptible to a disease or condition associated with cells expressing EpCAM or the presence, growth, proliferation, metastasis, and / or activity of such cells, such as 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. Any of a variety of methods known in the art is used to identify subjects suffering from or susceptible to a disease or condition associated with cells expressing EpCAM. For example, any of a variety of clinical and / or laboratory tests (such as physical examination and blood, urine, and / or stool analysis to assess health status) is used to identify subjects suffering from cancer or other neoplastic conditions. For example, any of a variety of clinical and / or laboratory tests (such as physical examination and / or analysis of body fluids, such as blood, urine, and / or stool analysis to assess health status) is used to identify subjects suffering from inflammation and / or inflammatory conditions.

[0563] Administration of an EpCAM immunoconjugate to a patient suffering from a disease or condition associated with EpCAM-expressing cells (e.g., a cancer, such as carcinoma) is considered successful if any of various laboratory or clinical objectives are achieved. For example, administration of an EpCAM immunoconjugate to a patient suffering from a disease or condition associated with EpCAM-expressing cells is considered successful if one or more symptoms associated with the disease or condition are alleviated, reduced, suppressed, or do not progress to a further (i.e., worse) state. Administration of an EpCAM immunoconjugate to a patient suffering from a disease or condition associated with EpCAM-expressing cells is considered successful if the disease or condition enters remission or does not progress to a further (i.e., worse) state.

[0564] The present disclosure provides conjugated EpCAM antibodies that can be used in methods for treating, preventing, delaying the progression of, or ameliorating and / or alleviating the symptoms of diseases or conditions associated with aberrant EpCAM expression and / or activity. For example, conjugated EpCAM activatable antibodies can be used in methods for treating, preventing, delaying the progression of, or ameliorating and / or alleviating the symptoms of cancer or other neoplastic conditions.

[0565] The present disclosure provides conjugated EpCAM antibodies that can be used in methods for treating, preventing, delaying the progression of, ameliorating, and / or alleviating the symptoms of diseases or conditions associated with cells expressing 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 for treating, preventing, delaying the progression of, ameliorating, and / or alleviating the symptoms of cancer or other neoplastic conditions.

[0566] The present disclosure provides EpCAM antibodies and antibody fragments and / or conjugated EpCAM activatable antibodies, which can be used in methods for treating, preventing, delaying the progression of, improving and / or alleviating the symptoms of diseases or conditions 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 for treating, preventing, delaying the progression of, improving and / or alleviating the symptoms of cancer or other neoplastic conditions.

[0567] In some embodiments, the conjugated EpCAM antibodies or activatable antibodies provided herein have excellent therapeutic indexes. 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 relative to the therapeutic index of other conjugated EpCAM antibodies or activatable antibodies. For example, huEpCAM23Gv4.2-GMBS-DM21L (see, e.g., Example 7.4.5) has a therapeutic index of about 1.

[0568] The EpCAM antibodies or activatable antibodies provided herein have relatively low systemic toxicity. In cynomolgus monkeys, for example, EpCAM-CPT66 tolerates up to 60 mg / kg (see Example 7.7). This is in stark contrast to EpCAM-DM21, which is an immunoconjugate comprising the same EpCAM activatable antibody as EpCAM-CPT66 but with an auristatin-based DM21 linker-payload, which only tolerates up to 6 mg / kg. Compared to fam-trastuzumab delutec-nxki, the tolerability of EpCAM-CPT66 is also favorable, where HNSTD is determined to be 30 mg / kg in cynomolgus monkeys.

[0569] 6.7.1 Pharmaceutical Compositions

[0570] The provided compositions include bulk pharmaceutical compositions (e.g., impure or non-sterile compositions) that can be used to manufacture pharmaceutical compositions and pharmaceutical compositions that can be used in the form of unit dosage forms (i.e., compositions suitable for administration to a subject or patient). Such compositions contain a prophylactically or therapeutically effective amount of a provided immunoconjugate and a pharmaceutically acceptable carrier.

[0571] In some embodiments, provided compositions comprise a prophylactically or therapeutically effective amount of a disclosed immunoconjugate and a pharmaceutically acceptable carrier.

[0572] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic agent is administered. In general, the ingredients of the compositions provided herein are provided separately or mixed together in unit dosage form (e.g., as a dry lyophilized powder or water-free concentrate) in a sealed container (e.g., an ampoule or sachet) indicating the amount of active agent. When the composition is to be administered by infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0573] The present disclosure also provides a pharmaceutical package or kit comprising one or more containers filled with an immunoconjugate provided herein, alone or in combination with a pharmaceutically acceptable carrier. The present disclosure also provides a pharmaceutical package or kit comprising one or more containers filled with one or more ingredients of a pharmaceutical composition provided herein. Optionally, associated with such one or more containers may be a notice in a form prescribed by a government agency governing the manufacture, use, or sale of drugs or biological products, the notice reflecting the agency's permission for manufacture, use, or sale for human administration.

[0574] The present disclosure provides kits that can be used in the above methods. The kits can contain any of the immunoconjugates disclosed herein.

[0575] 6.7.2 Application method

[0576] By administering a therapeutically effective amount of the immunoconjugates provided herein to a subject, the disclosed compositions can be provided for treating, preventing, and improving one or more symptoms associated with a disease or illness. In particular aspects, such compositions are substantially purified (i.e., substantially free of substances that limit their effects or produce undesirable side effects). In particular embodiments, the subject is an animal, in some cases a mammal, such as a non-primate (e.g., cattle, horses, cats, dogs, rodents, etc.) or a primate (e.g., monkeys such as cynomolgus monkeys, people, etc.). In specific embodiments, the subject is a person.

[0577] Methods of administering 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., intranasal and oral routes) administration. In specific embodiments, the immunoconjugates provided herein are administered intramuscularly, intravenously, or subcutaneously. The compositions can be administered by any convenient route, such as by infusion or bolus injection, and can be administered together with other bioactive agents. Administration can be systemic or local.

[0578] The present disclosure also provides for packaging formulations of the disclosed immunoconjugates in airtight containers, such as ampoules or sachets indicating the amount of the molecule. In one embodiment, such molecules can be provided as dry, sterile, lyophilized powders or water-free concentrates in an airtight container and can be reconstituted, for example, with water or saline to an appropriate concentration for administration to a subject. In some embodiments, the immunoconjugates are provided as dry, sterile, lyophilized powders in an airtight container.

[0579] Lyophilized formulations of the immunoconjugates provided herein should be stored in their original containers at between 2°C and 8°C, and the molecules should be administered within 12 hours, in some cases 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 an airtight sealed container indicating the amount and concentration of the molecule, fusion protein, or conjugated molecule. In some embodiments, when provided in liquid form, such immunoconjugates are provided in an airtight sealed container.

[0580] As used herein, a "therapeutically effective amount" of a pharmaceutical composition is an amount sufficient to produce beneficial or desired results, including but not limited to clinical results, such as reducing symptoms of cancer (e.g., proliferation of cancer cells, presence of tumors, tumor metastasis, etc.), thereby improving the quality of life of patients suffering from the disease, reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug (e.g., by targeting and / or internalization), delaying the progression of the disease and / or prolonging the survival of an individual.

[0581] A therapeutically effective amount can be administered in one or more administrations.For the purposes of this disclosure, a therapeutically effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to reduce the proliferation (or effects) of a viral presence and / or reduce and / or delay the development (directly or indirectly) of a viral disease.

[0582] 6.8 Diagnostic and research applications

[0583] 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 EpCAM immunoconjugates provided can be used, for example, for purification, detection, and targeting of EpCAM included in in vitro and in vivo diagnostic methods. For example, antibodies and / or fragments 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, for example, Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd edition 1988), the entire contents of which are incorporated herein by reference.

[0584] 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)).

[0585] Detectably labeling the EpCAM immunoconjugate can be accomplished by linking to an enzyme for enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA). The linked enzyme reacts with the exposed substrate to produce a chemical moiety that can be detected, for example, by spectrophotometry, fluorescence, or by visual means. Enzymes that can be used to detectably label, for example, the disclosed EpCAM immunoconjugates 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.

[0586] By radiolabeling the EpCAM immunoconjugate, it is possible to detect EpCAM by using a radioimmunoassay (RIA) (see, e.g., Work et al., Laboratory Technology and Biochemistry in Molecular Biology, North Holland Publishing Company, NY (1978)). The radioisotope can be detected by means such as using a gamma counter or scintillation counter or by autoradiography. Particularly useful isotopes for the purposes of the present disclosure are: 3 H. 125 I.131 I. 35 S. 14 C, and in some cases 125 I.

[0587] It is also possible to label EpCAM immunoconjugates with fluorescent compounds. When the fluorescently labeled antibody, antibody fragment, or activatable antibody is exposed to light of the appropriate wavelength, its presence can then be detected due to fluorescence. The most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

[0588] EpCAM immunoconjugates can also be detectably labeled with fluorescent metals such as 125Eu or other metals from the lanthanide series. These metals can be linked to EpCAM antibodies, EpCAM-binding antibody fragments thereof, and EpCAM-activatable antibodies using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0589] In another embodiment, the EpCAM immunoconjugate is detectably labeled by coupling with a chemiluminescent compound. The presence of the chemiluminescent-labeled antibody or antibody fragment is then determined by detecting the presence of luminescence generated during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters. Similarly, bioluminescent compounds can be used to label EpCAM antibodies, EpCAM-binding antibody fragments thereof, EpCAM-activatable antibodies, or derivatives thereof. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds used for labeling purposes are luciferin, luciferase, and aequorin.

[0590] EpCAM immunoconjugates can be used for in vivo imaging, wherein an EpCAM immunoconjugate labeled with a detectable moiety, such as a radiopaque agent or a radioisotope, is administered to a subject, in some cases into the bloodstream, and the presence and location of the labeled antibody or antibody fragment in the host is determined. This imaging technique can be used in the staging and treatment of malignant tumors. The EpCAM immunoconjugate can be labeled with any moiety that is detectable in the host (whether by nuclear magnetic resonance, radiology, or other detection means known in the art).

[0591] The label used according to the disclosed methods can be any detectable moiety that is capable of directly or indirectly producing a detectable signal. For example, the label can be a biotin label, an enzyme label (e.g., luciferase, alkaline phosphatase, β-galactosidase, and horseradish peroxidase), a radioactive label (e.g.,3 H. 14 C. 32 P. 35 S and 125 I), fluorophores such as fluorescent or chemiluminescent compounds (e.g., fluorescein isothiocyanate, rhodamine), imaging agents (e.g., Tc-m 99 and indium ( 111 In)) and metal ions (e.g., gallium and europium).

[0592] Any method known in the art for conjugating EpCAM immunoconjugates to labels can be used, including those exemplary methods 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).

[0593] 7 Examples

[0594] 7.1 Example 1. EpCAM Antibodies and Fragments

[0595] The generation and selection of monoclonal antibodies against human and cynomolgus monkey (cyno) EpCAM are described in detail in WO 2020 / 086665, the disclosure of which is hereby incorporated by reference in its entirety for all purposes. The generation, selection and humanization of the EpCAM antibodies and fragments thereof of the present disclosure are summarized below.

[0596] Three different immunization protocols were used to generate monoclonal antibodies. In the first immunization protocol, wild-type BALB / c female mice were subcutaneously injected with the 300-19 cell line expressing cynomolgus monkey-EpCAM (which is a BALB / c-derived pre-B cell) three times, followed by injection of the 300-19 cell line expressing human EpCAM twice. In the second immunization protocol, wild-type BALB / c female mice were subcutaneously injected with the NSCLC cell line H1568 four times, followed by injection of cynomolgus monkey primary renal epithelial cells four times. In the third immunization protocol, FcγR2b ko / ko BALB / c female mice (model #579, Taconic) were subcutaneously injected with 300-19 cells expressing human EpCAM three times, followed by injection of 300-19 cells expressing cynomolgus monkey-EpCAM twice. In all three protocols, cells were prepared in PBS and cultured at 5×10 6The mice were injected with a dose of 10 cells / mouse / injection, with a two-week interval between injections. To boost the immune response, an anti-GITR Ab (clone DTA-1) was injected one week after the first immunization. Three days before sacrifice for hybridoma generation, the immunized mice received another intraperitoneal injection of 300-19 cells expressing human EpCAM. Hybridoma clones were prepared for antibody screening.

[0597] Other immunization and hybridoma generation 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).

[0598] Hybridoma screening was performed using a flow cytometric binding assay using 300-19 cells expressing human EpCAM and wild-type 300-19 cells. Hybridomas that showed positive binding to human and cynomolgus monkey EpCAM antigens but negative binding to wild-type 300-19 cells were further subcloned by limiting dilution. One subclone from each hybridoma that showed specific binding to human and cynomolgus monkey EpCAM antigens was selected for subsequent analysis.

[0599] During this investigation, 20 fusions were performed. Sixty-three hybridomas specific for human and cynomolgus macaque EpCAM antigens were generated, and 29 hybridomas were subcloned. Stable subclones were cultured, and the monoclonal antibody isotypes were identified using a commercially available mouse IgG isotyping reagent.

[0600] Murine antibodies were purified and binding affinity was determined using purified antibodies to mEpCAM23 by flow cytometry binding assays performed with HSC2 cells. d In 3.8x10 -10 to 7.9x10 -10 The sequence of mEpCAM23 was identified, and mEpCAM23 clones were selected for chimerization, humanization, and further evaluation.

[0601] The VL and VH chains of the EpCAM hybridoma were cloned and sequenced, and then the amino acid sequence of the variable region of the murine EpCAM antibody was 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.

[0602] Antibody humanization was performed using a complementarity determining region (CDR) grafting procedure essentially as described in Jones et al., Nature 321: 604-608 (1986); Verhoeyen et al., Science 239: 1534-1536 (1988); U.S. Patent Nos. 5,225,539 and 5,585,089. CDR grafting typically involves replacing the Fv framework region (FR) of a mouse antibody with a human antibody Fv framework region while retaining the mouse CDR residues that are critical for the specific antigen binding properties of the parent antibody. Exemplary CDRs of murine EpCAM-23 antibodies that follow the Kabat CDR definition are indicated in Table 13 below.

[0603] Table 13.

[0604]

[0605]

[0606] The initial CDR grafting of muEpCAM-23 was generated by grafting 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. L There are 12 framework residues substituted in V H In addition, variants containing one or more back mutations of Vernier region residues were prepared and subsequently evaluated for EpCAM binding.

[0607] Several humanized versions of EpCAM23 retained binding to human HSC2 cells. However, only the Gv2.2 versions (VLGv2 and VH Gv2) and the Gv4.2 versions (VL Gv4 and VH Gv2) also retained binding to primary cynomolgus monkey kidney epithelial cells. Affinity variants of the humanized antibody Gv4.2 were generated. Variants were created by mutagenesis of solvent-exposed CDR residues or VH / VL framework residues that are rarely present in the mouse / human natural repertoire (frequency <5%).

[0608] An initial panel of humanized anti-EpCAM23Gv4.2 affinity variants was tested in an enzyme-linked immunosorbent assay (ELISA) using mFc-tagged huEpCAM or cynoEpCAM proteins. Two affinity variants, huEpCAM23Gv4.2-1361-H and huEpCAM23Gv4.2-1565-Y, were selected for further evaluation. In particular, these variants were selected for their slightly lower binding affinity relative to the parent antibody on HSC2 cells (i.e., Kd was reduced by approximately 3-fold compared to the parent antibody on HSC2 cells) and their different mutation sites.

[0609] 7.2 Example 2. Mask Discovery

[0610] The identification and characterization of masking moieties (MMs or "masks") for use in the activatable anti-EpCAM antibodies and fragments thereof ("activatable antibodies") of the present disclosure are described in detail in WO 2020 / 086665 and are described below.

[0611] A humanized anti-human EpCAM monoclonal antibody (EpCAM23Gv4.2) that cross-reacts with both human and cynomolgus monkey EpCAM was used to screen random X-ray diffraction patterns using a method similar to that described in PCT International Publication No. WO 2010 / 081173, published on July 15, 2010, the disclosure of which is incorporated herein by reference in its entirety. 15 Peptide library, where X is any amino acid. Screening included one round of MACS and three rounds of FACS sorting. Initial MACS sorting was performed using Protein-A Dynabeads (Invitrogen) with anti-EpCAM antibody. For MACS, anti-EpCAM antibody was conjugated to DyLight-488 (ThermoFisher), EpCAM binding activity was confirmed, and anti-EpCAM-488 was used as a fluorescent probe for all FACS rounds. Individual peptide clones were identified by sequence analysis from each FACS round.

[0612] Two heavy chain variants of humanized anti-human EpCAM monoclonal antibodies (EpCAM23(1361-H) and EpCAM23(1565-Y)) were generated and used to screen random X-linked proteins using a method similar to that described in PCT International Publication No. WO 2010 / 081173 published on July 15, 2010. 15Peptide library where X is any amino acid. Masks EP101 to EP104 were identified using the EpCAM23 (1565-Y) heavy chain variant. Masks EP105 to EP110 were identified using the EpCAM23 (1361-H) heavy chain variant. Mutations of the lysine residue in the Ep107 mask were also generated.

[0613] Table 14 lists the sequences of selected anti-EpCAM masking moieties.

[0614] Table 14. Masking moieties of EpCAM-activatable antibodies.

[0615]

[0616]

[0617] These masking peptides were used to generate the anti-EpCAM activatable antibodies of the present disclosure.The amino acid sequences of some of these anti-EpCAM activatable antibodies are shown in Table 15 below. In some embodiments, the 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), ISSGLL SGRSANP (SEQ ID NO: 317), ISSGLLSGRSANI (SEQ ID NO: 318), ISSGLLSGRSDNI (SEQ ID NO: 169), AVGLLAPPGGLSGRS DIH (SEQ ID NO: 319), AVGLLAPPGGLSGRSDQH (SEQ ID NO: 320), AVGLLAPPGGLSGRSDNP (SEQ ID NO: 321), AVGLLAPPGGLSGRSANP (SEQ ID NO: 322), AVGLLAPPGGLSGRSANI (SEQ ID NO: 323), NO:323) or AVGLLAPPGGLSGRSDNI (SEQ ID NO:168). In some embodiments, certain anti-EpCAM activatable antibodies of the present disclosure include a non-cleavable portion "NSUB," which is not expected to be cleaved.

[0618] Although certain light chain sequences of the activatable antibodies of the present disclosure shown below comprise an N-terminal spacer sequence of QGQSGQG (SEQ ID NO: 324), one of ordinary skill in the art will appreciate that the activatable anti-EpCAM antibodies of the present disclosure may comprise any suitable spacer sequence, such as, for example, 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 antibodies of the present disclosure may not have a spacer sequence attached to the N-terminus.

[0619] Table 15. Variable light chain amino acid sequences of the activatable antibodies of the present disclosure.

[0620]

[0621]

[0622]

[0623]

[0624]

[0625] Exemplary anti-EpCAM activatable antibodies having the following heavy and light chains are shown in Table 16 below.

[0626] Table 16. Anti-EpCAM activatable antibodies.

[0627]

[0628]

[0629] A solid phase binding assay was used to demonstrate the binding of the anti-human EpCAM antibodies disclosed herein. Briefly, recombinant human EpCAM-mFC protein (Immunogen) was coated on an ELISA plate (50 μL of 1 μg / mL) and then incubated with serially diluted anti-EpCAM antibodies (starting at 62.5 nM) or activatable anti-EpCAM antibodies (starting at 1 μM), wherein the activatable antibodies were assayed in their uncleaved form. Anti-human IgG (anti-Fab) conjugated to horseradish peroxidase (Sigma) was used to detect the amount of bound antibody with Ultra TMB-ELISA reagent (Thermo Fisher Scientific), and the OD was measured at 450 nM. The K values ​​of each antibody and activatable antibody were measured. D , and the ELISA masking efficiency (ME) of each activatable antibody relative to the unmasked antibody was calculated, and exemplary results are shown in Tables 17 and 18. Overall, these data indicate that the anti-human EpCAM activatable antibodies of the present disclosure exhibit altered binding affinity to recombinant EpCAM protein compared to the parent anti-EpCAM antibodies of the present disclosure.

[0630] Table 17. Anti-EpCAM activatable antibody masking efficiency.

[0631]

[0632]

[0633] Table 18. Anti-EpCAM activatable antibody masking efficiency.

[0634]

[0635]

[0636] As depicted in Tables 17 and 18, these exemplary results demonstrate that anti-human EpCAM activatable antibodies exhibit a range of masking efficiencies relative to unmasked anti-EpCAM antibodies.

[0637] 7.3 Example 3. Epitope Mapping

[0638] The human CD326 antigen EpCAM (epithelial cell adhesion molecule) consists of 314 amino acids, an extracellular domain containing 265 amino acids, a transmembrane domain of 23 amino acids, and a cytoplasmic tail of 26 amino acids. 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 contain a first domain comprising a region from glutamine at position 24 of the mature protein (i.e., before signal peptide cleavage) to cysteine ​​at position 59; and a second domain comprising a region from cysteine ​​at position 66 to cysteine ​​at position 135. Then, in series with the first two domains, there is also a third domain (D3) that does not contain cysteine, which contains amino acid residues 136-243.

[0639] Table 19. EpCAM domains.

[0640]

[0641]

[0642] The epitope of the humanized EpCAM antibody Gv4.2 was mapped by engineering a chimeric protein using a combination of the extracellular domains of human and mouse EpCAM. Because human and mouse EpCAM share over 82% amino acid sequence identity and 85% similarity, the topology of the chimeric protein should remain intact.

[0643] The extracellular region of human EpCAM (residues 1-265) was codon-optimized using the HindIII and BamHI restriction sites at Genscript, synthesized, and in-frame cloned into a vector (pGSmuFc2ANL) containing the mouse IgG2a Fc region. Similarly, other expression vectors containing various chimeric variants of the extracellular domain of human / mouse EpCAM were synthesized by replacing residues corresponding to human EpCAM domain D1 (24-59), domain D2 (66-135), domain D3 (136-265), or their combinations 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 transfections were incubated for one week, harvested, and the filtered supernatant purified using a combination of protein A and CHT chromatography.

[0644] The binding of humanized EpCAM antibody huEpCAM23Gv4.2 to the above-mentioned EpCAM protein was tested in an enzyme-linked immunosorbent assay (ELISA) format. 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 ug / mL in 50 mM sodium bicarbonate buffer at pH 9.6, and 100 μL was added to each well. After incubation at 4°C for 16 hours, the plate was washed with Tris-buffered saline (TBST) containing 0.1% Tween-20 and then blocked with 200 μL blocking buffer (TBS containing 1% BSA). Next, 100 μL of the first 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 plate was then washed 3 times with TBST, and 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 then washed three 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 terminated by adding 100 μL of stop solution and the absorbance at 450 nm was read in a multiwell plate reader. OD450 was plotted against antibody concentration in a semi-logarithmic plot. Dose-response curves were generated by nonlinear regression and the EC for each curve was calculated using GraphPad Prism v6. 50 value.

[0645] The binding of the huEpCAM23Gv4.2 antibody to chimeric EpCAM proteins was evaluated compared to wild-type EpCAM. The huEpCAM23Gv4.2 antibody bound to both EpCAM-D2 (66-135) and EpCAM-D3 (136-265) with similar affinity to wild-type EpCAM. In contrast, the huEpCAM23Gv4.2 antibody did not bind to EpCAM-D1 and D1 / D2 constructs, and binding to the chimeric protein EpCAM-D1 (24-59) construct was almost eliminated. These results indicate that the epitope of the huEpCAM23Gv4.2 antibody is primarily located within the D1 (24-59) domain of EpCAM.

[0646] 7.4 Example 4. Preparation of EPCAM Antibody Drug Conjugates and Activatable Antibody Drug Conjugates

[0647] 7.4.1 Preparation of HUEPCAM23Gv4.2-LALALALALALA-CPT66

[0648] The following examples provide methods for producing activatable EpCAM antibody drug conjugates according to embodiments of the present disclosure. The activatable EpCAM drug conjugates obtainable by the following methods comprise a heavy chain having an amino acid sequence of SEQ ID NO: 103 and a light chain having an amino acid sequence of SEQ ID NO: 179 and a drug to antibody ratio of 8, wherein the linker payload is

[0649]

[0650] Wherein ** indicates the position of covalent attachment to the activatable EpCAM antibody via one of the eight cysteine ​​residues of the activatable EpCAM antibody that usually participate in the formation of four disulfide bonds.

[0651] Figure 1A Figure 2 shows the camptothecin derivative linker-payload used to prepare EpCAM antibody drug conjugates. Figure 2 As shown, the EpCAM antibody drug conjugate produced comprises an EpCAM antibody, a cleavable substrate linker and a mask. In the uncleaved (inactive) state, the mask inhibits the binding of the EpCAM antibody to EpCAM. The cleavable substrate linker can be cleaved by a protease. After cleavage, the mask is released and the antibody is free to bind to EpCAM. The depicted embodiment includes 8 conjugated camptothecin-derived (CPT66) linkers-payloads. The conjugation of the linker-payload to the activatable EpCAM antibody is random, wherein the conjugation occurs at the interchain cysteine ​​of the antibody.

[0652] Briefly, to prepare a CPT linker-toxin (CPT-LT) stock solution (approximately 40 mM), 9.1 mL of anhydrous DMA was added to a vial containing 336.8 mg of CPT-LT solid and the vial was vortexed until all solids dissolved. The concentration of the stock solution was then determined to be 45.57 mM using UV spectroscopy.

[0653] 2mL (0.05 volume) of EPPS buffer (1M, pH 7.80) was transferred to a 100mL 3-neck round-bottom flask, followed by addition of 0.4mL (0.01 volume) of 0.5M EDTA solution and mixing under stirring. 37.6mL of CX-231 stock solution (24.45g / L; 0.94 volume; previously thawed at 4°C) was then added, and the reaction mixture was stirred at room temperature for 5 minutes to fully mix all starting materials.

[0654] 4.5 molar equivalents of TCEP (2.673 mL of a 10 mM stock solution) were added to the reaction vessel. Immediately after the addition of the TCEP solution, the reaction time was set to T = 0. The reaction mixture was stirred at 500 rpm at room temperature for 3 hours to allow complete reduction of all four interchain disulfide bonds.

[0655] At T = 3 hours, 2.316 mL of DMA and 4.0 molar equivalents of CPT-LT (521 uL of a 45.57 mM stock solution) were added to the reaction vessel using a pipette. Two additional batches of 4.0 molar equivalents of CPT-LT (521 uL of a 45.57 mM stock solution) were then added at T = 4 hours and 5 hours. The total amount of CPT-LT was 12.0 molar equivalents (1.563 mL of a 45.57 mM stock solution). The total volume of DMA (including the DMA in the CPT-LT stock solution and the additional DMA) was 3.879 mL, representing 8.3% of the reaction volume. The total reaction time was 7 hours, including 3 hours for TCEP reduction and 4 hours for CPT-LT conjugation.

[0656] At T = 7 hours, 24 molar equivalents of NAC (2.326 mL of a 10 g / L solution) were added to the reaction and stirred at 500 rpm for 15 minutes at room temperature to terminate the conjugation process.The crude reaction mixture was purified by tangential flow filtration.

[0657] Purified EpCAM-Camp66 immunoconjugate was tested by the following QC assays:

[0658] - Measure concentration and drug to antibody (or activatable antibody) ratio (DAR) by UV-visible spectrophotometry using a SoloVPE instrument;

[0659] - % monomers were measured using SEC-HPLC method;

[0660] -The content of free drug-related impurities (FDRI) was evaluated using HiSep-HPLC method.

[0661] The purity and integrity of the immunoconjugates were confirmed using LC-MS and CE-SDS assays.

[0662] The above conjugation reaction is schematically depicted in Figure 1B As shown, the activatable EpCAM antibody is reduced using TCEP, which exposes reactive cysteines that typically form disulfide bridges between the heavy chains and between the light and heavy chains of the activatable antibody. Importantly, one or more disulfide bonds present within the masked portion of the activatable EpCAM antibody are not disturbed by the reducing conditions. Figure 1A The maleimide functional group of the linker-payload reacts with the thiol group of the exposed reactive cysteine ​​to form a covalent thioester linkage.

[0663] 7.4.2 Preparation of HUEPCAM23GV4.2-MCGGFG-CPT66

[0664] The maleimide GGFG-CPT66 linker toxin was synthesized at Abzena and dissolved in DMSO. The CX-231 activatable antibody buffer was exchanged into 50mM HEPES, 2mM EDTA (pH 7.0) and reduced with 10 molar equivalents of TCEP for 2 hours at room temperature. The reduced activatable antibody was buffer exchanged into 50mM HEPES, 2mM EDTA by a desalting column. 10% DMSO and 12 molar equivalents of maleimide GGFG-CPT66 were added to the reduced activatable antibody and incubated at room temperature for 45 minutes. The reaction was quenched with N-acetylcysteine ​​and then buffer exchanged into 20mM succinate, 6% sucrose (pH 6.5). The purified EpCAM-GGFG-DXd immunoconjugate was tested by the following QC assay:

[0665] - Measure concentration and drug to antibody (or activatable antibody) ratio (DAR) by UV-visible spectrophotometry using a SoloVPE instrument;

[0666] - % monomers were measured using SEC-HPLC method;

[0667] -The content of free drug-related impurities (FDRI) was evaluated using HiSep-HPLC method.

[0668] The purity and integrity of the immunoconjugates were confirmed using LC-MS and CE-SDS assays.

[0669] 7.4.3 Preparation of HUEPCAM23GV4.2-MCGGFG-DXD

[0670] 10mg of maleimide GGFG-DXd linker toxin was purchased from MedChem Express and dissolved in 1mL of DMA to obtain a 9.67mM solution. 5mM EDTA and 12 molar equivalents of TCEP (in 10mM HEPES buffer, pH 7.53) were added to 4ml of CX-231 at a concentration of 25.2mg / mL in 20mM succinate, 4% sucrose (pH 6.5). The reduction reaction was carried out at room temperature for 3 hours. Excess TCEP was removed by a desalting column. 12 molar equivalents of maleimide GGFG-DXd and DMA were added to the reduced CX-231 solution to a final concentration of 10%. The reaction mixture was incubated at room temperature overnight and then exchanged into 20mM succinate, 8% sucrose (pH 6.5) using a desalting column buffer. The purified EpCAM-GGFG-DXd immunoconjugate was tested by the following QC assays:...

Claims

1. An immunoconjugate, comprising: (a) an activatable antibody comprising a full-length human IgG1 EpCAM antibody having eight cysteines that form four interchain disulfide bonds when in a native conformation, the full-length human IgG1 EpCAM antibody comprising 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) Eight linker-payloads depicted by the following structures: or a pharmaceutically acceptable salt thereof, wherein each of the eight linker-payloads is individually covalently bound to the activatable antibody via one of the eight cysteines.

2. An immunoconjugate, comprising: (a) an activatable antibody, the activatable antibody comprising: (i) an EpCAM antibody or an EpCAM-binding fragment thereof, wherein the EpCAM antibody or the EpCAM-binding fragment thereof comprises: (1) a heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence NYYIH (SEQ ID NO: 13); (2) a heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14); (3) a heavy chain complementarity determining region 3 (CDR3) (VH-CDR3) comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15); (4) a light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO: 42); (5) a light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence QTSNLAS (SEQ ID NO: 40); and (6) a light chain complementary determining region 3 (VL-CDR3) comprising the amino acid sequence AQNLELPNT (SEQ ID NO: 41); (ii) a cleavable portion coupled to the EpCAM antibody or EpCAM-binding fragment thereof, the cleavable portion 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 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, wherein the activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: (masking moiety)-(cleavable moiety)-(antibody or antibody fragment) or (antibody or antibody fragment)-(cleavable moiety)-(masking moiety); and (b) at least one linker-payload depicted by the following structure: or a pharmaceutically acceptable salt thereof.

3. The immunoconjugate of claim 2, wherein the cleavable moiety comprises the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169).

4. The immunoconjugate of claim 2, wherein the cleavable portion comprises the amino acid sequence AVGLLAPPGGLSGRSDNI (SEQ ID NO: 168).

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

6. The immunoconjugate of any one of claims 2 to 5, wherein the activatable antibody is a full-length human IgG1 EpCAM antibody having eight cysteines that form four interchain disulfide bonds when in a native conformation, and wherein each linker-payload is individually covalently bound to the activatable antibody via one of the eight cysteines.

7. An immunoconjugate, comprising: (a) an activatable antibody, the activatable antibody comprising: (i) a full-length human IgG1 EpCAM antibody having eight cysteines that form four interchain disulfide bonds when in a native conformation, the full-length human IgG1 EpCAM antibody comprising: (1) a heavy chain complementary determining region 1 (VH-CDR1) comprising the amino acid sequence NYYIH (SEQ ID NO: 13); (2) a heavy chain complementary determining region 2 (VH-CDR2) comprising the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 14); (3) a heavy chain complementarity determining region 3 (CDR3) (VH-CDR3) comprising the amino acid sequence DGPWFAY (SEQ ID NO: 15); (4) a light chain complementary determining region 1 (VL-CDR1) comprising the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO: 42); (5) a light chain complementary determining region 2 (VL-CDR2) comprising the amino acid sequence QTSNLAS (SEQ ID NO: 40); and (6) a light chain complementary 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 EpCAM-binding fragment thereof, the cleavable moiety comprising the amino acid sequence ISSGLLSGRSDNI (SEQ ID NO: 169); and (iii) a masking moiety coupled to the EpCAM antibody or 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, wherein the activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: (masking moiety)-(cleavable moiety)-(antibody) or (antibody)-(cleavable moiety)-(masking moiety); and (b) Eight linker-payloads, each depicted by the following structure: or a pharmaceutically acceptable salt thereof, wherein each of the eight linker-payloads is individually covalently bound to the activatable antibody via one of the eight cysteines.

8. The immunoconjugate of claim 7, wherein 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. 9 . The immunoconjugate of claim 7 , 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.

10. An immunoconjugate obtainable by the method of embodiment 7.4.

1.

11. An immunoconjugate, which can be obtained by the following method, the method comprising: (a) mixing an activatable EpCAM antibody composed of a heterotetramer of two heavy chains and two light chains with a mixture of EPPS buffer and EDTA aqueous solution to form an activatable EpCAM reaction mixture, wherein the two heavy chains each consist of the amino acid sequence of SEQ ID NO: 103, and the two light chains each consist of the amino acid sequence of SEQ ID NO: 179; (b) mixing an activatable EpCAM antibody composed of a heterotetramer of two heavy chains and two light chains, each consisting of the amino acid sequence of SEQ ID NO: 103, and each consisting of the amino acid sequence of SEQ ID NO: 179, with a mixture of EPPS buffer and an EDTA aqueous solution to form an activatable EpCAM reaction mixture; and (c) mixing the reduced activatable EpCAM reaction mixture with a camptothecin linker-toxin stock solution comprising a linker-toxin represented by the following structure: Thereby forming the immunoconjugate.

12. A nucleic acid comprising one or more coding regions for an activatable antibody of the immunoconjugate of any one of claims 1 to 11. A vector comprising the nucleic acid of claim 12 . The vector of claim 13 , which is a viral vector.

15. A method for producing an immunoconjugate, the method comprising: (a) culturing a host cell comprising the vector of claim 14; (b) isolating the activatable antibody from the host cell; and (c) conjugating at least one linker-payload reactant comprising camptothecin or a derivative thereof to the activatable antibody.

16. The method of claim 15, wherein the linker-payload reactant comprises a compound of formula I: EALD (Formula I), in: EA-Z'-L1-D (Formula I), in: D is represented by the following structural formula: Where R 1 is F and R 2 It 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 of covalent attachment to A; Each R 5 are independently H, methyl or benzyl; Each R 8 are independently H, methyl or benzyl; A is a peptide comprising 2 to 4 amino acids; and E is -C(=O)-(C1-C 10 Alkylene)-X 3 , where X 3 yes 17. The method of claim 15, wherein the linker-payload reactant is: or a pharmaceutically acceptable salt thereof.

18. The method of claim 15, wherein the linker-payload reactant is: or a pharmaceutically acceptable salt thereof.

19. The method of claim 15, wherein the linker-payload reactant comprises exatecan.

20. The method of claim 15, wherein the linker-payload reactant comprises delutecan.

21. A method of producing an immunoconjugate, the method comprising conjugating at least one linker-payload reactant comprising camptothecin or a derivative thereof to the activatable antibody of any one of claims 1 to 11.

22. The method of claim 21, wherein the linker-payload reactant comprises a compound of formula I: E-A-Z’-L 1 -D (Formula I), in: D is represented by the following structural formula: Where R 1 is F and R 2 It 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 of covalent attachment to A; Each R 5 are independently H, methyl or benzyl; Each R 8 are independently H, methyl or benzyl; A is a peptide comprising 2 to 4 amino acids; and E is -C(=O)-(C1-C 10 Alkylene)-X 3 , where X 3 yes 23. The method of claim 21, wherein the linker-payload reactant is: or a pharmaceutically acceptable salt thereof.

24. The method of claim 21, wherein the linker-payload reactant is: or a pharmaceutically acceptable salt thereof.

25. The method of claim 21, wherein the linker-payload reactant comprises exatecan.

26. The method of claim 21, wherein the linker-payload reactant comprises delutecan.

27. A composition comprising: (a) the immunoconjugate of any one of claims 1 to 11; and (b) a pharmaceutically acceptable carrier.

28. The immunoconjugate of any one of claims 1 to 11 or the composition of claim 27 for use in treating a cancer expressing EpCAM.

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