Conditionally active anti-EpCAM antibodies, antibody fragments, and constructs incorporating the same

Abstract

A conditionally active bispecific antibody comprising: an IgG antibody or antibody fragment that binds to human EpCAM protein, the IgG antibody or antibody fragment comprising a light chain variable region having three complementarity-determining regions L1, L2, and L3, and a heavy chain variable region having three complementarity-determining regions H1, H2, and H3; and at least one scFv antibody fragment that binds to a T lymphocyte protein linked to the C-terminus of at least one light chain of the IgG antibody or antibody fragment.

Description

[Technical Field]

[0001] The present disclosure relates to anti-EpCAM antibodies, antibody fragments, and constructs incorporating anti-EpCAM antibodies and antibody fragments, such as multispecific antibodies and conjugates. Diagnostic and therapeutic uses of these antibodies, antibody fragments, and constructs are also disclosed. [Background technology]

[0002] Epithelial cell adhesion / activation molecule (EpCAM, also known as CD326, HEA125, MK-1, EGP-2, EGP34, GA733-2, KSA, TROP-1, KS1 / 4, and ESA) (Figure 1) is one of the first and most important immunotherapeutic targets in cancer therapy because it is expressed at high levels and with high frequency in most carcinomas of different origins (Herlyn et al., Proc Natl Acad Sci USA, 76:1438-1442, 1979; Went et al., Hum Pathol, 35:122-128, 2004). This molecule is a relatively small, 314 amino acid (aa) type I transmembrane glycoprotein that is highly conserved throughout evolution. It has been reported to mediate calcium-independent homotypic cell-cell adhesion (Litvinov et al., J Cell Biology, 125:437-446, 1994). This molecule consists of a short 26-aa intracellular domain containing two binding sites for α-actinin for interaction with the actin cytoskeleton (Balzar et al., Mol Cell Biol., 18(8):4833-4843, 1998), a 23-aa transmembrane region, a 242-aa extracellular domain (ECD), and a 23-aa signal peptide that is cleaved from its mature form. The extracellular domain of EpCAM contains three N-linked glycosylation sites. It has been reported that glycosylation status differs between normal and malignant tissues in certain types of cancer (Pauli et al., Cancer Lett., 193:25-32, 2003).

[0003] The extracellular domain contains three domains. The first two are thought to resemble epidermal growth factor (EGF)-like repeats, containing 12 cysteine ​​residues (Balzar et al., Mol Cell Biol, 21:2570-2580, 2001). However, some studies suggest that the second EGF-like repeat of EpCAM is actually a thyroglobulin (TY) domain (Linnenbach et al., Proc Natl Acad Sci USA, 86:27-31, 1989; Chong and Speicher, J Biol Chem, 276:5804-5813, 2001). The third domain is a unique cysteine-poor region (CPR), unrelated to any known molecules (Baeuerle and Gires, Br J Cancer, 96:417-423, 2007). EpCAM plays an important role in cell-cell adhesion, cell signaling, migration, proliferation, and prevention of differentiation (Figure 1).

[0004] EpCAM expression in humans is epithelial-specific. Except for squamous epithelia and some specific epithelial cell types such as epidermal keratinocytes, hepatocytes, gastric parietal cells, and myoepithelial cells, the majority of human epithelial cells express EpCAM (Balzar et al., J Mol Med, 77:699-712, 1999; Momburg et al., Cancer Res, 47:2883-2891, 1987). Higher expression levels are generally observed in tumors of epithelial origin (Balzar et al., J Mol Med, 77:699-712, 1999; Winter et al., Am J Pathol, 163:2139-2148, 2003; Went et al., Hum Pathol, 35:122-128, 2004; Went et al., Br J Cancer, 94:128-135, 2006). For example, the EpCAM protein has been found to be expressed in a wide variety of human adenocarcinomas and squamous cell carcinomas (Went et al., Hum Pathol, 35:122-128, 2004). Recent studies using immunohistochemical (IHC) staining in conjunction with microarray technology have found EpCAM expression in a significant number of samples from patients with breast, ovarian, renal, esophageal, colon, gastric, prostate, and lung cancer (Spizzo et al., Breast Cancer Res Treat, 86:207-213, 2004; Spizzo et al., Gynecol Oncol, 103:483-488, 2006; Stoecklein et al., BMC Cancer, 6:165, 2006; Kimura et al., Int J Oncol, 30:171-179, 2007; Went et al., Am J Surg Pathol, 29:83-88, 2005; Went et al., Br J Cancer, 94:128-135, 2006). These data highlight the potential utility of EpCAM as an immunotherapeutic target for the treatment of human cancers.

[0005] Epithelial cells are known to be the most important cell type in the development of human malignancies, and since more than 90% of all malignancies are of epithelial origin (Birchmeiera et al., Acta Anatomica, 156(3):217-226, 1996), EpCAM is currently considered one of the most frequently and centrally expressed tumor-associated antigens. This molecule has been independently discovered multiple times as an immunogenic tumor-associated antigen for monoclonal antibody development (Gottlinger et al., Int J Cancer, 38:47-53, 1986; Edwards et al., Cancer Res, 46:1306-1317, 1986; Spurr et al., Int J Cancer, 38:631-636, 1986; Momburg et al., Cancer Res, 47:2883-2891, 1987; Schoen et al., J Investig Dermatol, 102:987-991, 1994; Bumol et al., Hybridoma, 7:407-415, 1988; Quak et al., Hybridoma, 9:377-387, 1990).

[0006] Indeed, the first monoclonal antibody applied to human cancer therapy was a murine IgG2a antibody called mAb17-1A (later named edrecolomab and Panorexs) that targets EpCAM (Sears et al., Lancet, 1(8275):762-765, 1982; Sears et al., J Biol Response Mod, 3(2):138-150, 1984). Since then, edrecolomab and other EpCAM-specific murine, chimeric, and humanized monoclonal antibodies have also been subjected to preclinical and clinical trials for cancer therapy, either in the form of native (naked) antibodies, hybrid bispecific (trifunctional) antibodies, or as conjugates with toxins, radioisotopes, or cytokines (IL-2 or GM-CSF) (Velders et al., Cancer Res, 54(7):1753-1759, 1994; Raum et al., Cancer Immunol Immunother, 50(3):141-150, 2001; Elias et al., Am J Respir Crit Care Med, 150:1114-1122, 1994; Di Paolo et al., Clin Cancer Res, 9:2837-2848, 2003; Andratschke et al., Anticancer Res, 27(1A):431-436, 2007; Xiang et al., Cancer Res, 57(21):4948-4955, 1997; Schanzer et al., J Immunother, 29(5):477-488, 2006; Wimberger et al., Int J Cancer, 105(2):241-248, 2003; Amann et al., Cancer Res, 68(1):143-151, 2008). Currently, a number of different immunotherapeutic approaches targeting EpCAM are still in clinical trials (Baeuerle and Gires, Br J Cancer, 96:417-423, 2007).Data from clinical trials suggest that naked anti-EpCAM antibodies such as edrecolomab (17-1A; Panorexs) and adecatumab (MT201) exhibit only limited antitumor activity (Punt et al., Lancet, 360:671-677, 2002), likely due to activation of the complement system (CDC) and antibody-dependent cellular cytotoxicity (ADCC) effects (Schwartzberg, Crit Rev Oncol Hematol, 40(1):17-24, 2001; Naundorf et al., Int J Cancer, 100(1):101-110, 2002; Prang et al., BrJ Cancer 92(2):342-349, 2005; Oberneder et al., Eur J Cancer, 42(15):2530-2538, 2006). Antibodies conjugated with highly potent effector mechanisms, such as IL-2, PE toxin, or anti-CD3 antibodies, appear to exhibit superior antitumor efficacy, but several adverse effects limit the systemic administration of these anti-EpCAM antibodies (Baeuerle and Gires, Br J Cancer, 96:417-423, 2007).

[0007] ING-1 is a high-affinity human recombinant monoclonal antibody that targets EpCAM-positive cells. It is being used in a phase I clinical trial in patients with advanced adenocarcinoma refractory to standard therapy. Data from this study suggest that high-affinity antibodies against EpCAM, while more cytotoxic to tumor cells, can also induce rapid toxic pancreatic damage, limiting their therapeutic window for systemic administration (De Bono et al., Clin Cancer Res, 10(22):7555-65, 2004). Potential systemic toxicity associated with the therapeutic use of high-affinity anti-EpCAM antibodies can be mitigated by a pretargeting strategy that includes a tracking step to clear circulating antibodies at a given time. Alternatively, the use of high-affinity anti-EpCAM antibodies may be limited to locoregional treatment.

[0008] The side effects of known anti-EpCAM antibodies are related to the presence of EpCAM on normal epithelial cells, although at a lower expression density than on tumor cells (Kim et al., Clin Cancer Res, 10:5464-5471, 2004; Osta et al., Cancer Res, 64:5818-5824, 2004). Therefore, increasing the affinity or specificity of an anti-EpCAM antibody does not reduce the effect of the anti-EpCAM antibody on normal tissues that express EpCAM, resulting in side effects.

[0009] The present disclosure aims to provide anti-EpCAM antibodies or antibody fragments that are suitable for therapeutic and diagnostic applications, particularly cancer diagnosis and treatment, and that have reduced or minimized side effects. Some of these anti-EpCAM antibodies or antibody fragments may have higher binding affinity for EpCAM in tumors compared to EpCAM present in normal tissues. These anti-EpCAM antibodies or antibody fragments typically have efficacy at least equivalent to that of known anti-EpCAM antibodies. In addition, the present anti-EpCAM antibodies or antibody fragments may exhibit reduced side effects compared to monoclonal anti-EpCAM antibodies known in the art to have relatively low binding affinity for EpCAM in normal tissues. These advantages may result in more selective targeting of EpCAM with respect to tumors, and because the antibodies are selective for EpCAM present in tumors, higher doses of these anti-EpCAM antibodies or antibody fragments may be used, thereby achieving more effective therapeutic treatment without increasing the corresponding undesirable side effects.

[0010] Antibodies have become a major class of therapeutic proteins. Traditional antibodies usually bind to a single epitope on an antigen. Novel antibody constructs, called multispecific antibodies, have been developed to bind to two or more antigens or two or more epitopes on the same antigen. Multispecific antibodies can be, for example, bispecific, trispecific, or tetraspecific antibodies. Multispecific antibodies have shown potential in a wide range of clinical and diagnostic applications. Two bispecific antibody drugs (Catumaxomab™ and Blinatumab™) have been approved for the treatment of oncological diseases in the European Union and the United States. Their unique characteristics make multispecific antibodies attractive for next-generation antibody therapeutics.

[0011] U.S. Patent Application Publication No. 2013 / 0017200 discloses a method for synthesizing multispecific antibodies. A first antibody fragment is obtained from a first parent antibody having a first monospecificity. The first antibody fragment has a free sulfhydryl group, which can be reacted with a thio-reactive crosslinker to generate an antibody fragment-crosslinker moiety. This antibody fragment-crosslinker moiety is then pairwise reacted with two or more additional antibody fragments, each of which has a free sulfhydryl group, obtained from other parent antibodies having a different monospecificity from the first antibody fragment to generate a multispecific antibody. This multispecific antibody may be suitable as a novel therapeutic or diagnostic agent.

[0012] Brinkmann and Kontermann ("The Making of Bispecific Antibodies," MABS, 2017, Vol. 9, pp. 182-212, 2017) explore bispecific antibody formats, including small molecules consisting of only the antigen-binding sites of two antibodies, molecules with IgG structure, and large composite molecules consisting of different antigen-binding moieties, often combined with a dimerization module. Depending on the application, bispecific antibodies can vary in the size, arrangement, valency, mobility, and geometry of their binding modules, as well as their distribution and pharmacokinetic properties. Overall, bispecific formats increase the diversity of antibodies that can be applied to the development of therapeutics for various indications. Examples of specific bispecific formats are shown in Figures 2 and 3.

[0013] It is also desirable to generate useful antibodies with conditional activity, for example, antibodies that are effectively inactive under normal physiological conditions and have significantly increased activity outside of normal physiological conditions (e.g., abnormal conditions), antibodies that are activated or inactivated in certain microenvironments (e.g., activated in the tumor microenvironment), or antibodies that are activated or inactivated over time. In addition to temperature, other trigger conditions under which antibodies may be evolved or optimized include pH, osmolality, osmolality, oxidative stress, and electrolyte concentration. In addition to activity, other desirable properties of antibodies that may be optimized during evolution include stability, half-life, chemical resistance, and resistance to proteolysis.

[0014] Many strategies have been published for evolving or engineering parent antibodies into mutant antibodies with desired properties. However, to engineer or evolve a parent antibody that is inactive or virtually inactive (less than 10% activity, and particularly less than 5% activity) under normal physiological conditions but has substantial activity under abnormal conditions, one or more destabilizing mutations must coexist with activity-increasing mutations that do not counteract the destabilizing effect. Destabilizing mutations are expected to reduce the activity of the antibody by an order of magnitude greater than predicted by standard rules, such as the Q10 rule. Thus, the ability to evolve a protein that is substantially inactive under its normal functional conditions but functions efficiently (has higher activity) under specific abnormal conditions, such as lower temperature or pH, creates a surprising new class of antibodies called conditionally active antibodies.

[0015] Embodiments disclosed herein include a novel class of multispecific antibodies that are conditionally active for binding to the EpCAM antigen. This novel class of multispecific antibodies can harness the flexibility and versatility of traditional multispecific antibodies while simultaneously directing their activity, affinity, and / or avidity to the site, tissue, or organ where the activity is desired. In this way, these multispecific antibodies avoid or greatly reduce the deleterious effects associated with binding to normal cells, including, but not limited to, avoidance of excessive cytokine production. Summary of the Invention [Means for solving the problem]

[0016] Provided herein are conditionally active antibodies or antibody fragments that bind to human EpCAM protein or an epitope of the EpCAM protein, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises three complementarity-determining regions L1, L2, and L3, and the heavy chain variable region comprises three complementarity-determining regions H1, H2, and H3. The complementarity-determining regions of various embodiments of the conditionally active anti-EpCAM antibodies and antibody fragments disclosed herein are provided in Table 1.

[0017] [Table 1] JPEG2026507181000003.jpg88170

[0018] In some additional embodiments, the conditionally active anti-EpCAM antibodies and antibody fragments comprise a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 53, a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 54, a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 55, a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 56, a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 56, a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 57, a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 58, a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 59 ... The heavy chain variable region of SEQ ID NO: 52 and the light chain variable region of SEQ ID NO: 60, the heavy chain variable region of SEQ ID NO: 52 and the light chain variable region of SEQ ID NO: 61, the heavy chain variable region of SEQ ID NO: 52 and the light chain variable region of SEQ ID NO: 62, the heavy chain variable region of SEQ ID NO: 52 and the light chain variable region of SEQ ID NO: 63, the heavy chain variable region of SEQ ID NO: 52 and the light chain variable region of SEQ ID NO: 64, the heavy chain variable region of SEQ ID NO: 52 and the light chain variable region of SEQ ID NO: 65, the heavy chain variable region of SEQ ID NO: 52 and the light chain variable region of SEQ ID NO: 66, the heavy chain variable region of SEQ ID NO: 52 and the light chain variable region of SEQ ID NO: 67, the heavy chain variable region of SEQ ID NO: 52 and the light chain variable region of SEQ ID NO: 68, and the heavy chain variable region of SEQ ID NO: 52 and the light chain variable region of SEQ ID NO: 69.

[0019] In further embodiments, the conditionally active anti-EpCAM antibodies and antibody fragments comprise a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 70, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 71, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 72, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 73, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 74, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 75, Light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 76, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 77, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 78, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 79, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 80, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 81, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 82, SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 83, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 84, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 85, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 86, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 87, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 88, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 89, a light chain variable region of SEQ ID NO: 51 The light chain variable region of SEQ ID NO: 51 and the heavy chain variable region of SEQ ID NO: 92; the light chain variable region of SEQ ID NO: 51 and the heavy chain variable region of SEQ ID NO: 93; the light chain variable region of SEQ ID NO: 51 and the heavy chain variable region of SEQ ID NO: 94; the light chain variable region of SEQ ID NO: 51 and the heavy chain variable region of SEQ ID NO: 95; and the light chain variable region of SEQ ID NO: 51 and the heavy chain variable region of SEQ ID NO: 96.

[0020] The conditionally active antibody or antibody fragment of the present disclosure exhibits increased binding affinity for EpCAM protein or an epitope of EpCAM protein under abnormal conditions different from normal physiological conditions, and decreased binding affinity for EpCAM protein or an epitope of EpCAM protein under normal physiological conditions. In one embodiment, the abnormal conditions are conditions within a tumor microenvironment, and the normal physiological conditions are conditions within a non-tumor microenvironment. In a further embodiment, the condition is pH. In a specific embodiment, the abnormal conditions are a pH of 5.0 to 6.9 within a tumor microenvironment, and the normal physiological conditions are a pH of 7.0 to 7.6 within a non-tumor microenvironment. In yet another specific embodiment, the conditionally active anti-EpCAM antibody or antibody fragment of the present disclosure has increased binding affinity for EpCAM protein or an epitope of EpCAM protein at a pH of 6.0 compared to the binding affinity of the same conditionally active anti-EpCAM antibody or antibody fragment at a pH of 7.4.

[0021] In one embodiment, the conditionally active anti-EpCAM antibody or antibody fragment disclosed herein is derived from a non-conditionally active anti-EpCAM parent antibody. In a specific embodiment, the non-conditionally active anti-EpCAM parent antibody has a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 52. In this embodiment, the conditionally active anti-EpCAM antibody or antibody fragment disclosed herein has increased binding affinity for the EpCAM protein or an epitope of the EpCAM protein at a tumor microenvironment pH of 5.0 to 6.9 compared to the binding affinity of the same conditionally active anti-EpCAM antibody or antibody fragment at a non-tumor microenvironment pH of 7.0 to 7.6, and decreased binding affinity for the EpCAM protein or an epitope of the EpCAM protein at pH 7.0 to 7.6 compared to the binding affinity of the non-conditionally active parent anti-EpCAM antibody or antibody fragment for the EpCAM protein or epitope of the EpCAM protein at pH 7.0 to 7.6. In certain embodiments, the conditionally active anti-EpCAM antibodies disclosed herein have increased binding affinity for the EpCAM protein or an epitope of the EpCAM protein at a pH of 6.0 compared to the binding affinity of the same conditionally active anti-EpCAM antibody at a pH of 7.4, and decreased binding affinity for the EpCAM protein or an epitope of the EpCAM protein at pH 7.4 compared to the binding affinity of a non-conditionally active parent anti-EpCAM antibody or antibody fragment for the EpCAM protein or an epitope of the EpCAM protein at pH 7.4.

[0022] In one embodiment, the conditionally active anti-EpCAM antibody or antibody fragment disclosed herein has a ratio of binding affinity for the EpCAM protein or an epitope of the EpCAM protein at a pH of 6.0 to binding affinity for the EpCAM protein or an epitope of the EpCAM protein at a pH of 7.4 of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 15:1, or at least about 20:1.

[0023] Also provided herein are multispecific antibodies comprising a conditionally active anti-EpCAM antibody or antibody fragment and at least one scFv antibody fragment that binds to a T lymphocyte antigen linked to the C-terminus of at least one light chain or at least one heavy chain of the conditionally active anti-EpCAM antibody or antibody fragment.

[0024] In one embodiment, the multispecific anti-EpCAM antibody or antibody fragment comprises a light chain variable region having three complementarity determining regions L1, L2, and L3, and a heavy chain variable region having three complementarity determining regions H1, H2, and H3. The light and heavy chain complementarity determining regions of the multispecific conditionally active anti-EpCAM antibody or antibody fragment can be obtained from the various embodiments disclosed in Table 1 above.

[0025] In some additional embodiments, the conditionally active anti-EpCAM antibody or antibody fragment of the multispecific antibody comprises a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 53, a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 54, a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 55, a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 56 ...7, a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO: 58, a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of SEQ ID NO:59, a heavy chain variable region of SEQ ID NO:52 and a light chain variable region of SEQ ID NO:60, a heavy chain variable region of SEQ ID NO:52 and a light chain variable region of SEQ ID NO:61, a heavy chain variable region of SEQ ID NO:52 and a light chain variable region of SEQ ID NO:62, a heavy chain variable region of SEQ ID NO:52 and a light chain variable region of SEQ ID NO:63, a heavy chain variable region of SEQ ID NO:52 and a light chain variable region of SEQ ID NO:64, a heavy chain variable region of SEQ ID NO:52 and a light chain variable region of SEQ ID NO:65, a heavy chain variable region of SEQ ID NO:52 and a light chain variable region of SEQ ID NO:66, a heavy chain variable region of SEQ ID NO:52 and a light chain variable region of SEQ ID NO:67, a heavy chain variable region of SEQ ID NO:52 and a light chain variable region of SEQ ID NO:68, and a heavy chain variable region of SEQ ID NO:52 and a light chain variable region of SEQ ID NO:69.

[0026] In further embodiments, the conditionally active anti-EpCAM antibody or antibody fragment of the multispecific antibody comprises a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 70, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 71, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 72, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 73, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 74, a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 75 Heavy chain variable region, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 76, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 77, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 78, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 79, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 80, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 81, light chain variable region of SEQ ID NO: 51 and heavy chain variable region of SEQ ID NO: 82 a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 83; a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 84; a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 85; a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 86; a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 87; a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 88; a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 89; The combinations include the light chain variable region of SEQ ID NO: 51 and the heavy chain variable region of SEQ ID NO: 90, the light chain variable region of SEQ ID NO: 51 and the heavy chain variable region of SEQ ID NO: 91, the light chain variable region of SEQ ID NO: 51 and the heavy chain variable region of SEQ ID NO: 92, the light chain variable region of SEQ ID NO: 51 and the heavy chain variable region of SEQ ID NO: 93, the light chain variable region of SEQ ID NO: 51 and the heavy chain variable region of SEQ ID NO: 94, the light chain variable region of SEQ ID NO: 51 and the heavy chain variable region of SEQ ID NO: 95, and the light chain variable region of SEQ ID NO: 51 and the heavy chain variable region of SEQ ID NO: 96.

[0027] In one embodiment, the conditionally active anti-EpCAM antibody or antibody fragment of the multispecific antibody disclosed herein is derived from a non-conditionally active anti-EpCAM parent antibody. In a specific embodiment, the non-conditionally active anti-EpCAM parent antibody has a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 52. In this embodiment, the conditionally active anti-EpCAM antibody or antibody fragment disclosed herein has increased binding affinity for the EpCAM protein or an epitope of the EpCAM protein at a tumor microenvironment pH of 5.0 to 6.9 compared to the binding affinity of the same conditionally active anti-EpCAM antibody or antibody fragment at a non-tumor microenvironment pH of 7.0 to 7.6, and decreased binding affinity for the EpCAM protein or an epitope of the EpCAM protein at pH 7.0 to 7.6 compared to the binding affinity of the non-conditionally active parent anti-EpCAM antibody or antibody fragment for the EpCAM protein or epitope of the EpCAM protein at pH 7.0 to 7.6. In certain embodiments, the conditionally active anti-EpCAM antibodies disclosed herein have increased binding affinity for the EpCAM protein or an epitope of the EpCAM protein at a pH of 6.0 compared to the binding affinity of the same conditionally active anti-EpCAM antibody at a pH of 7.4, and decreased binding affinity for the EpCAM protein or an epitope of the EpCAM protein at pH 7.4 compared to the binding affinity of a non-conditionally active parent anti-EpCAM antibody or antibody fragment for the EpCAM protein or an epitope of the EpCAM protein at pH 7.4.

[0028] In one embodiment, a conditionally active anti-EpCAM antibody or antibody fragment of a multispecific antibody disclosed herein has a ratio of binding affinity for EpCAM protein or an epitope on the EpCAM protein at a pH of 6.0 to binding affinity for EpCAM protein or an epitope on the EpCAM protein at a pH of 7.4 of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 15:1, or at least about 20:1. In a specific embodiment, a conditionally active anti-EpCAM antibody or antibody fragment of a multispecific antibody disclosed herein has a ratio of binding affinity for EpCAM protein or an epitope on the EpCAM protein at a pH of 6.0 to binding affinity for EpCAM protein or an epitope on the EpCAM protein at a pH of 7.4 of at least about 6:1. In one embodiment, a conditionally active anti-EpCAM antibody or antibody fragment of a multispecific antibody disclosed herein has a ratio of binding affinity for the EpCAM protein or an epitope of the EpCAM protein at a pH of 6.0 to binding affinity for the EpCAM protein or an epitope of the EpCAM protein at a pH of 7.4 of at least about 8:1.

[0029] In one embodiment, the conditionally active anti-EpCAM antibody or antibody fragment of the multispecific antibody is an IgG antibody or antibody fragment. In another embodiment, the conditionally active anti-EpCAM IgG antibody or antibody fragment is part of a bispecific antibody. In a further embodiment, an anti-lymphocyte antigen scFv antibody or antibody fragment is attached to the C-terminus of the light chain of the conditionally active anti-EpCAM IgG antibody or antibody fragment.

[0030] In certain embodiments, the scFv anti-lymphocyte antigen antibody or antibody fragment is an anti-CD3 scFv antibody. In certain embodiments, the anti-CD3 scFv antibody or antibody fragment is a conditionally active antibody or antibody fragment, while in other embodiments, the anti-CD3 scFv is a non-conditionally active antibody or antibody fragment. In embodiments in which the anti-CD3 scFv antibody or antibody fragment is conditionally active, the anti-CD3 scFv antibody has a higher binding affinity for CD3 antigen at a tumor microenvironment pH of 5.0 to 6.9 compared to the binding affinity of the same conditionally active anti-CD3 scFv antibody or antibody fragment at a non-tumor microenvironment pH of 7.0 to 7.6. In a specific embodiment, the anti-CD3 scFv antibody or antibody fragment comprises a light chain variable region of SEQ ID NO: 101 and a heavy chain variable region of SEQ ID NO: 100. In another embodiment, the anti-CD3 scFv antibody comprises SEQ ID NO: 97. In a specific embodiment, the conditionally active anti-EpCAM / anti-CD3 multispecific antibody comprises a light chain of SEQ ID NO:98 and a heavy chain of SEQ ID NO:99.

[0031] In another embodiment, the conditionally active anti-EpCAM antibody or antibody fragment of the multispecific antibody binds to cynomolgus monkey EpCAM protein in addition to human EpCAM protein and has a ratio of binding affinity for cynomolgus monkey EpCAM protein at a pH of 6.0 to binding affinity for cynomolgus monkey EpCAM protein at a pH of 7.4 of at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 15:1, or at least about 20:1. In a specific embodiment, the anti-EpCAM antibody or antibody fragment of the multispecific antibody has light chain variable region CDRs 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, respectively, and heavy chain variable region CDRs 1, 2, and 3 of SEQ ID NOs: 4, 5, and 45, respectively. In another embodiment, the anti-EpCAM antibody or antibody fragment of the multispecific antibody has a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 91. In any of the foregoing embodiments, the multispecific antibody may be a bispecific antibody. In certain embodiments, the bispecific antibody is an anti-EpCAM / anti-CD3 antibody. In these embodiments, the anti-CD3 antibody is an scFv antibody having a light chain variable region of SEQ ID NO: 101 and a heavy chain variable region of SEQ ID NO: 100. In more specific embodiments, the anti-CD3 scFv antibody has the amino acid sequence of SEQ ID NO: 97. In yet another embodiment, the anti-EpCAM / anti-CD3 bispecific antibody has the light chain sequence of SEQ ID NO: 98 and the heavy chain sequence of SEQ ID NO: 99. [Brief explanation of the drawings]

[0032] [Figure 1] 1 shows a schematic diagram of the function of EpCAM in cancer metastasis and progression. [Figure 2] 1 shows the schematic structure of a bivalent multispecific antibody that is a heterodimer with one arm for binding to EpCAM (antigen) and the other arm for binding to CD3. [Figure 3] 1 shows a schematic structure of a tetravalent multispecific antibody, which is a homodimer in which each arm has an antigen (Ag)-binding site and a CD3-binding site. [Figure 4A-4B]1 shows a sequence alignment of exemplary light chain variable regions of anti-EpCAM antibodies of the present disclosure. [Figures 5A-5C] 1 shows a sequence alignment of exemplary heavy chain variable regions of anti-EpCAM antibodies of the present disclosure. [Figure 6A] Figure 1 shows the binding affinity of BA3182 to human CD3 / human EpCAM at pH 6.0 and pH 7.4. Binding of BA3182 to human CD3 and human EpCAM at pH 6.0 and pH 7.4 was measured by ELISA. The average OD values ​​from two replicates at pH 6.0 and pH 7.4 were plotted against various concentrations of BA3182. This figure shows the dose-response binding curve of BA3182 to human CD3 / human EpCAM antigen at different pH values. Data are representative of three experiments. Y-axis: OD 450nm. X-axis: concentration of BA3182. [Figure 6B] Figure 1 shows the binding affinity of BA3182 to human CD3 / cyno EpCAM at pH 6.0 and pH 7.4. BA3182 binding to human CD3 and cyno EpCAM at pH 6.0 and pH 7.4 was measured by ELISA. The average OD values ​​from two replicates at pH 6.0 and pH 7.4 were plotted against various concentrations of BA3182. This figure shows the dose-response binding curve of BA3182 to human CD3 / cyno EpCAM antigen at different pH values. Data are representative of three experiments. Y-axis: OD 450nm. X-axis: BA3182 concentration. [Figure 7] Figure 1 shows the binding of BA3182 bispecific antibody to human CD3 and human EpCAM at different pH values ​​tested by sandwich affinity ELISA. The mean OD values ​​from two replicates for each pH value are shown. Y-axis: OD450nm. X-axis: pH value. [Figure 8] Figure 1 shows the binding of BA3182 to human CD3 and human, cyno, rat and mouse EpCAM extracellular domains at pH 6.0 and pH 7.4. [Figure 9A-9B]Figure 1 shows the binding of BA3182 to human CD3 / human EpCAM and human CD3 / human Trop2 extracellular domains at pH 6.0 and pH 7.4, respectively. Binding of BA3182 to recombinant human CD3 and (A) human EpCAM, or (B) human Trop2 ECD at pH 6.0 and pH 7.4, was measured by sandwich ELISA. Results of one representative experiment are shown. Black bars: BA3182 bispecific antibody; white bars: anti-Trop2 bispecific antibody. [Figures 10A-10B] Figure 1 shows the binding of BA3182 to human CD3 / human EpCAM and human CD3 / unrelated human antigen extracellular domain at pH 6.0 and pH 7.4, respectively. Binding of BA3182 to recombinant human CD3 and (A) human EpCAM, or (B) unrelated human antigen ECD at pH 6.0 and pH 7.4 was measured by sandwich ELISA. Results of one representative experiment are shown. Black bars: BA3182 bispecific antibody; white bars: positive control bispecific antibody targeting an unrelated antigen not disclosed. [Figures 11A-11B] Figure 11 shows the binding of BA3182 to human EpCAM and human Trop2 extracellular domains at pH 6.0 and pH 7.4, respectively. Binding of BA3182 to human EpCAM (Figure 11A) and human Trop2 ECD (Figure 11B) at pH 6.0 and pH 7.4 was measured by affinity ELISA. Results of a representative experiment are shown. Black bars: BA3182 bispecific antibody; patterned bars: anti-Trop2 antibody. [Figures 12A-12C] Figure 12 shows a representative gating strategy for FACS analysis of EpCAM-positive cells. FACS analysis of CHO-hEpCAM cells stained only with the secondary antibody goat anti-human IgG1 AF488. Figure 12A shows a density plot of the viable cell population by forward scatter (FSC) vs. side scatter (SSC). Figure 12B shows the single cell population as determined by forward scatter height (FSC-H) vs. forward scatter area (FSC-A) under the viable cell gate. Figure 12C is a histogram plot showing the cutoff gate for determining AF488-positive single cells. [Figures 13A-13C]Binding analysis of BA3182 to EpCAM-expressing cells at pH 6.0 and pH 7.4 is shown. Binding analysis of BA3182 to the cell surface-expressed EpCAM antigen is shown in Figure 13A, to CHO hEpCAM cells and CHO cynoEpCAM cells in Figure 13B, and to HCT116 cells in Figure 13C. Cells were stained with BA3182 at pH 6.0 and pH 7.4. Data shown are representative of three independent experiments. Y-axis: median fluorescence intensity (MFI). X-axis: antibody concentration. The starting concentration of BA3182 for staining CHO hEpCAM and HCT116 cells was 500 nM. For staining CHO cynoEpCAM cells, the starting concentration of BA3182 was 1500 nM. [Figures 14A-14C] Figure 14 shows the binding analysis of BA3182 to CD3-expressing cells at pH 6.0 and pH 7.4. Binding analysis of BA3182 to the CD3 antigen expressed on the cell surface is shown in Figure 14A for human PBMC, Figure 14B for Cyno PBMC, and Figure 14C for Jurkat cells. Cells were stained with BA3182 at pH 6.0 and pH 7.4. Y-axis: median fluorescence intensity (MFI) value. X-axis: antibody concentration. The starting concentration of BA3182 for CD3-expressing cell staining was 2500 nM. [Figure 15A] A standard curve for PE beads is shown. [Figure 15B] Figure 1 shows the expression levels of the EpCAM antigen on the surface of CHO hEpCAM, CHO cynoEpCAM, and HCT116 cells. The EPCAM expression levels on CHO-hEpCAM, CHO-cynoEpCAM, and HCT116 cells were estimated using the QantiBrite™ PE quantification kit from BD, which contains a mixture of beads loaded with known amounts of phycoerythrin (PE) molecules (high, medium, and low). A standard curve was constructed using the log-geometric mean of PE fluorescence intensity from the beads and the number of PE molecules per bead provided by the vendor. The number of PE molecules on EpCAM-expressing cells stained with anti-hEpCAM PE-conjugated antibodies was calculated by extrapolation from the bead standard curve using the log-geometric mean of PE fluorescence on the stained cells. The anti-hEpCAM antibody bound well to both human and cyno EpCAM-expressing cells. [Figures 16A-16C] Figure 16 shows the in vitro functional activity of BA3182 at pH 6.0 and pH 7.4 against EpCAM-expressing target cells. BA3182 mediated T cell activation at pH 6.0 and pH 7.4. (Figure 16A) CHO hEpCAM cells. (Figure 16B) CHO cynoEpCAM cells, and (Figure 16C) HCT116 cells. Average RLU values ​​from two replicates are shown. Y-axis: relative luminescence intensity (RLU). X-axis: log antibody concentration (nM). Red circles: pH 6.0. Blue squares: pH 7.4. Data are representative of three experiments. [Figures 17A-17C] Figure 17 shows the in vitro cytotoxic activity of BA3182 at pH 6.0 and pH 7.4 against HCT116 cells using human PBMCs and against cynoEpCAM-expressing CHO cells using cynoPBMCs. Cytolysis of EpCAM-expressing target cells mediated by PBMCs activated with the BA3182 antibody at pH 6.5 and pH 7.4. Figure 17A shows HCT116 cells / human PBMCs at pH 6.5, Figure 17B shows HCT116 cells / human PBMCs at pH 7.4, and Figure 17C shows CHO cynoEpCAM / cynoPBMCs. X-axis: Log concentration of BA3182 (pM); Y-axis: % cytolysis. *N=10 for pH 6.5, N=6 for pH 7.4. Four PBMC lots did not yield sufficient cell numbers for testing at either pH value. *N=10 (pH 6.5), N=6 (pH 7.4). For four PBMC lots, there were not enough cells to test at both pH values. [Figures 18A-18B] Figure 18A shows the induction of IL-2 by stimulation of human PBMCs with BA3182 (Figure 18A) and an isotype control antibody (Figure 18B) in the presence of HCT116 cancer cells. Cultures were maintained at 37°C, 5% CO2 for 48 hours, and supernatants were harvested before measuring IL-2 cytokine using the Human IL-2 Quantikine™ ELISA assay (R&D Systems). PBMCs from nine human subjects were tested. Y-axis: IL-2 concentration (pg / mL). X-axis: log concentration of BA3182 or isotype control antibody. [Figures 19A-19B]Figure 19A shows the induction of IFNγ upon stimulation of human PBMCs with BA3182 (Figure 19A) and an isotype control antibody (Figure 19B) in the presence of HCT116 cancer cells. Cultures were maintained at 37°C, 5% CO2 for 48 hours, and supernatants were harvested before measuring IL-2 cytokine using the Human IFNγ Quantikine™ ELISA assay (R&D Systems). PBMCs from nine human subjects were tested. Y-axis: IFNγ concentration (pg / mL). X-axis: log concentration of BA3182 or isotype control antibody. [Figures 20A-20B] Figure 20A shows the induction of IL-6 upon stimulation of human PBMCs with BA3182 (Figure 20A) and an isotype control antibody (Figure 20B) in the presence of HCT116 cancer cells. Cultures were maintained at 37°C and 5% CO2 for 48 hours, and supernatants were harvested before measuring IL-2 cytokine using the Human IL-6 Quantikine™ ELISA assay (R&D Systems). PBMCs from nine human subjects were tested. Y-axis: IL-6 concentration (pg / mL). X-axis: log concentration of BA3182 or isotype control antibody. [Figures 21A-21B] Figure 21A shows the induction of IL-10 by stimulation of human PBMCs with BA3182 (Figure 21A) and an isotype control antibody (Figure 21B) in the presence of HCT116 cancer cells. Cultures were maintained at 37°C, 5% CO2 for 48 hours, and supernatants were harvested before measuring IL-2 cytokine using the Human IL-10 Quantikine™ ELISA assay (R&D Systems). PBMCs from nine human subjects were tested. Y-axis: IL-10 concentration (pg / mL). X-axis: log concentration of BA3182 or isotype control antibody. [Figures 22A-22B]Figure 1 shows the induction of TNFα upon stimulation of human PBMCs with BA3182 and an isotype control antibody in the presence of HCT116 cancer cells. Cultures were maintained at 37°C and 5% CO2 for 48 hours, and supernatants were harvested before measuring IL-2 cytokine using the Human TNFα Quantikine™ ELISA assay (R&D Systems). PBMCs from nine human subjects were tested. Y-axis: TNFα concentration (pg / mL). X-axis: log concentration of BA3182 or isotype control antibody. [Figure 23] Figure 1 shows an ELISA assay demonstrating binding of human C1q protein to BA3182 and B12 antibodies. B12, a human IgG1,k antibody against HIV viral envelope protein 120 (gp120), was used as a positive control for this assay. The mean OD values ​​from two replicates are shown. Data are representative of two experiments. Y-axis: OD 450 nm. X-axis: C1q concentration (nM). [Figure 24] Figure 1 shows SPR sensorgrams of BA3182 binding to the extracellular domain of human and cyno EpCAM at various pH values. Binding of BA3182 to human and cyno EpCAM was measured by SPR at pH 6.0, pH 6.5, and pH 7.4. The binding curves of one representative experiment at each pH value are shown. Data were fitted with a 1:1 Langmuir binding model. The maximum binding signal decreased from pH 6.0 to pH 7.4, which is due to the engineered pH-dependent binding of BA3182. Left column: Sensorgrams of BA3182 binding to human EpCAM at pH 6.0 (top), pH 6.5 (middle), and pH 7.4 (bottom). Right column: Sensorgrams of BA3182 binding to cyno EpCAM at pH 6.0 (top), pH 6.5 (middle), and pH 7.4 (bottom). [Figure 25]Figure 1 shows SPR sensorgrams of BA3182 binding to human and cyno CD3 extracellular domains at various pH values. BA3182 binding to human and cyno CD3 was measured by SPR at pH 6.0, pH 6.5, and pH 7.4. The binding curves of one representative experiment at each pH value are shown. Data were fitted with a 1:1 Langmuir binding model. The maximum binding signal decreased from pH 6.0 to pH 7.4, which is due to the engineered pH-dependent binding of BA3182. Left column: Sensorgrams of BA3182 binding to human CD3 at pH 6.0 (top), pH 6.5 (middle), and pH 7.4 (bottom). Right column: Sensorgrams of BA3182 binding to cyno CD3 at pH 6.0 (top), pH 6.5 (middle), and pH 7.4 (bottom). [Figure 26] Figure 1 shows the analysis of the binding kinetics of BA3182 to human EpCAM at various pH values ​​using SPR simulation software. The simulated sensorgrams (right) were generated using SPR simulation software as described in 3.6. From the simulations, it is clear that the decrease in maximum signal from pH 6.0 (top) to pH 6.5 (middle) and pH 7.4 (bottom) can best be simulated by a decrease in active ligand on the sensor surface. [Figure 27] Figure 1 shows the analysis of the binding kinetics of BA3182 to human CD3 at various pH values ​​using SPR simulation software. The simulated sensorgrams (right) were generated using SPR simulation software as described in 3.6. From the simulations, it is clear that the decrease in maximum signal from pH 6.0 (top) to pH 6.5 (middle) and pH 7.4 (bottom) can be best simulated by a decrease in active ligand on the sensor surface. [Figure 28]Figure 1 shows the binding of BA3182 to FcγRI (CD64). Sensorgrams for BA3182 (top left) and IgG1-control mAb (top right) are shown on the same scale. Bottom: Kinetic fitting of BA3182 binding to FcγRI using a steady-state model. Sensorgrams for the reference spot and buffer-only injection were subtracted from the test sensorgrams. The experimental data for the IgG1-control mAb were fitted with a 1:1 binding model, and the data for BA3182 were fitted with a steady-state model (blue: IgG1-control mAb, red: BA3182). Molar concentrations were calculated using molecular weights of 150 kDa for the IgG1-control mAb and 200 kDa for BA3182. The IgG1-control antibody, which has a wild-type human IgG1 Fc domain, binds to FcγRI with an affinity of 1.18 nM. The binding signal for BA3182 was much lower and was only detected at the highest three concentrations tested. Because all binding curves reached saturation, the data were analyzed using a steady-state model. However, the signal intensities and differences between concentrations were too small to calculate meaningful dissociation constants (KD). The data indicate that BA3182 does not bind to FcγRI. [Figure 29] Figure 1 shows the binding of BA3182 to FcγRIIa (CD32a). Sensorgrams for BA3182 (top left) and IgG1-control mAb (top right) are shown on the same scale. Bottom: Kinetic fitting of experimental data for FcγRIIa using a steady-state model (blue: IgG1-control mAb, red: BA3182). Sensorgrams for reference spots and buffer-only injections were subtracted from the test sensorgrams. Molar concentrations were calculated using molecular weights of 15 kDa for the IgG1-control mAb and 200 kDa for BA3182. The IgG1-control antibody, which has a wild-type human IgG1 Fc domain, binds to FcγRIIa with an affinity of 0.82 μM. In the case of BA3182, no binding to FcγRIIa was detected, indicating that BA3182 does not interact with FcγRIIa. [Figure 30]Figure 1 shows the binding of BA3182 to FcγRIIb / c (CD32b / c). Sensorgrams for BA3182 (top left) and IgG1-control mAb (top right) are shown on the same scale. Bottom: Kinetic fitting of experimental data for FcγRIIb / c using a steady-state model (blue: IgG1-control mAb, red: BA3182). Sensorgrams for reference spots and buffer-only injections were subtracted from the test sensorgrams. Molar concentrations were calculated using molecular weights of 150 kDa for the IgG1-control mAb and 200 kDa for BA3182. The IgG1-control antibody, which has a wild-type human IgG1 Fc domain, binds to FcγRIIb / c with an affinity of 4.2 μM. In the case of BA3182, no binding to FcγRIIb / c was detected, indicating that BA3182 does not interact with FcγRIIb / c. [Figure 31] Figure 1 shows the binding of BA3182 to FcγIIIa CD16a (F158). Sensorgrams for BA3182 (top left) and IgG1-control mAb (top right) are shown on the same scale. Bottom: Kinetic fitting of experimental data to FcγRIIIa using a steady-state model (blue: IgG1-control mAb, red: BA3182). Sensorgrams for reference spots and buffer-only injections were subtracted from the test sensorgrams. Molar concentrations were calculated using molecular weights of 150 kDa for the IgG1-control mAb and 200 kDa for BA3182. The IgG1-control antibody, which has a wild-type human IgG1 Fc domain, binds to FcγRIIIa with an affinity of 0.86 μM. In the case of BA3182, no binding to FcγRIIIa was detected, indicating that BA3182 does not interact with FcγRIIIa. [Figure 32]Figure 1 shows the binding of BA3182 to FcγRIIIb (CD16b). Sensorgrams for BA3182 (top left) and IgG1-control mAb (top right) are shown on the same scale. Bottom: Kinetic fitting of experimental data to FcγRIIIb using a steady-state model (blue: IgG1-control mAb, red: BA3182). Sensorgrams for reference spots and buffer-only injections were subtracted from the test sensorgrams. Molar concentrations were calculated using a molecular weight of 150 kDa for the IgG1-control mAb and 200 kDa for BA3182. The IgG1-control antibody, which has a wild-type human IgG1 Fc domain, binds to FcγRIIIa with an affinity of 6.5 μM. In the case of BA3182, no binding to FcγRIIIa was detected, indicating that BA3182 does not interact with FcγRIIIa. [Figure 33] Figure 1 shows the binding of BA3182 to FcRn. Binding of BA3182 to FcRn. Top: Sensorgrams of BA3182 (left) and IgG1-control mAb (right) are shown on the same scale. Bottom: Kinetic fitting of experimental data to FcRn using a steady-state model (blue: BA3182, red: IgG1-control mAb). Sensorgrams for reference spots and buffer-only injections were subtracted from the test sensorgrams. The molar concentration of FcRn was calculated using a molecular weight of 61.91 kDa. An IgG1-control antibody with a wild-type human IgG1 Fc domain binds to FcRn with an affinity of 366 nM. Similarly, BA3182 showed a binding affinity of 347 nM to FcRn. [Figures 34A-34H]Figure 34 shows cytokine release in human PBMC cultures stimulated with soluble BA3182 or CD3 / CD28 Dynabeads at pH 6.5. Cultures of human PBMCs were stimulated with soluble BA3182 or CD3 / CD28 Dynabeads in medium at pH 6.5 for 48 hours. Supernatants were collected, and cytokine concentrations were measured using a Luminex multiplex cytokine assay. Data show the levels (pg / mL) of various cytokines: IFNγ (Figure 34A), IL1β (Figure 34B), IL2 (Figure 34C), IL4 (Figure 34D), IL6 (Figure 34E), IL10 (Figure 34F), IL17 (Figure 34G), and TNFα (Figure 34H) in cultures of human PBMCs from nine different donors with CD3 / CD28 Dynabeads, but none were detected after soluble BA3182 treatment. [Figures 35A-35H] Figure 35 shows cytokine release in human PBMC cultures stimulated with soluble BA3182 or CD3 / CD28 Dynabeads at pH 7.4. Cultures of human PBMCs were stimulated with soluble BA3182 or CD3 / CD28 Dynabeads in medium at pH 7.4 for 48 hours. Supernatants were collected, and cytokine concentrations were measured using a Luminex multiplex cytokine assay. Data show the levels (pg / mL) of various cytokines: IFNγ (Figure 35A), IL1β (Figure 35B), IL2 (Figure 35C), IL4 (Figure 35D), IL6 (Figure 35E), IL10 (Figure 35F), IL17 (Figure 35G), and TNFα (Figure 35H) in cultures of human PBMCs from nine different donors with CD3 / CD28 Dynabeads, but none were detected after soluble BA3182 treatment. [Figures 36A-36H]Figure 36 shows cytokine production in human PBMC cultures stimulated with immobilized BA3182 and anti-CD3 antibody clone OKT3 at pH 6.5. Cultures of human PBMCs were stimulated with immobilized BA3182 or OKT3 for 48 hours in medium at pH 6.5. Supernatants were collected, and cytokine concentrations were measured using a Luminex multiplex cytokine assay. Data show the levels (pg / mL) of various cytokines: IFNγ (Figure 36A), IL1β (Figure 36B), IL2 (Figure 36C), IL4 (Figure 36D), IL6 (Figure 36E), IL10 (Figure 36F), IL17 (Figure 36G), and TNFα (Figure 36H) in cultures of human PBMCs from nine donors. [Figures 37A-37H] Figure 37 shows cytokine production in human PBMC cultures stimulated with immobilized BA3182 and anti-CD3 antibody clone OKT3 at pH 7.4. Cultures of human PBMCs were stimulated with immobilized BA3182 or OKT3 for 48 hours in medium at pH 7.4. Supernatants were collected, and cytokine concentrations were measured using a Luminex multiplex cytokine assay. Data show the levels (pg / mL) of various cytokines: IFNγ (Figure 37A), IL1β (Figure 37B), IL2 (Figure 37C), IL4 (Figure 37D), IL6 (Figure 37E), IL10 (Figure 37F), IL17 (Figure 37G), and TNFα (Figure 37H) in cultures of human PBMCs from nine donors. [Figure 38] Figure 1 shows the dose-response binding curve of BAP150.31-BF45 to the human EpCAM antigen as measured by affinity ELISA. The average OD values ​​from two replicates were plotted against various concentrations of BAP150.31-BF45. X-axis: Log concentration of BAP150.31-BF45 (ng / mL), Y-axis: OD value at 450 nm. [Figure 39] 1 shows the serum concentration-time profile after a single IV dose of BAP150.31-BF45 at 1 mg / kg in mice. Using PK Solver 2.0, the mean serum concentrations of BAP150.31-BF45 after a single IV dose of BAP150.31-BF45 at 1 mg / kg were plotted against the various serum collection time points. [Figure 40]1 shows the serum concentration-time profile after a single IV dose of BAP150.31-BF45 at 10 mg / kg in mice. Using PK Solver 2.0, the mean serum concentrations of BAP150.31-BF45 after a single IV dose of BAP150.31-BF45 at 10 mg / kg were plotted against the various sample collection time points. DETAILED DESCRIPTION OF THE INVENTION

[0033] definition To facilitate understanding of the examples described herein, certain frequently occurring terms are defined below.

[0034] As used herein, the term "about" in reference to a measured quantity refers to the normal variation in the measured quantity that would be expected by one of ordinary skill in the art making the measurement and exercising a level of care commensurate with the purpose of the measurement and the precision of the measuring device used. Unless otherwise specified, "about" refers to a ±10% variation of the provided value.

[0035] The term "abnormal conditions," as used herein, refers to conditions that deviate from the normal acceptable range for that condition in a subject. The term "normal physiological conditions," as used herein, refers to conditions that are considered to be within the normal range in a subject for a tissue or organ at a location or site of action in a subject, such as the site of administration.

[0036] The term "affinity," as used herein, refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity," as used herein, refers to the intrinsic binding affinity, which represents a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Specific explanations and exemplary embodiments for measuring binding affinity are described herein.

[0037] As used herein, the term "affinity matured" antibody refers to an antibody that has one or more modifications in one or more heavy or light chain variable regions, compared to a parent antibody that does not contain the modifications, which modifications increase the affinity of the antibody for antigen.

[0038] As used herein, the term "amino acid" refers to any organic compound that contains an amino group (--NH2) and a carboxyl group (--COOH), preferably as a free group or as part of a peptide bond after condensation. "Twenty naturally encoded polypeptide-forming α-amino acids" are understood in the art and refer to the following: alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine ​​(cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).

[0039] As used herein, the term "antibody" refers to intact immunoglobulin molecules and fragments of immunoglobulin molecules, such as Fab, Fab', (Fab')2, Fv, and SCA fragments, that are capable of binding to an epitope of an antigen. These antibody fragments retain some ability to selectively bind to the antigen (e.g., polypeptide antigen) of the antibody from which they are derived and can be produced using methods well known in the art, such as those described herein. Unless otherwise specified, use of the term antibody or antibodies includes functional fragments of an antibody or antibodies. Antibodies can be used to separate aliquots of antigens by immunoaffinity chromatography. Various other uses of such antibodies include the diagnosis and / or staging of disease (e.g., neoplasms) and therapeutic uses for treating disease (e.g., neoplasms, autoimmune diseases, AIDS, cardiovascular diseases, infectious diseases, etc.). Chimeric, human-like, humanized, or fully human antibodies are particularly useful for administration to human patients. The antibodies and antibody fragments of the present disclosure can be obtained by evolution or mutation of a parent antibody or antibody fragment that has the same type of activity, for example, binding activity or affinity for the EpCAM protein.

[0040] A Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule and can be produced by digestion of whole antibody molecules with the enzyme papain to yield a fragment consisting of an intact light chain and a portion of the heavy chain.

[0041] Fab' fragments of antibody molecules can be obtained by treating whole antibody molecules with pepsin, followed by reduction, to yield molecules consisting of an intact light chain and a portion of the heavy chain, with two Fab' fragments being obtained for each antibody molecule so treated.

[0042] The (Fab')2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0043] An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.

[0044] The term "antibody fragment" as used herein refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0045] As used herein, the terms "anti-EpCAM antibody," "EpCAM antibody," and "antibody that binds to EpCAM" refer to an antibody that is capable of binding to EpCAM protein or an epitope of EpCAM protein with sufficient affinity such that it is useful as a diagnostic and / or therapeutic agent in targeting EpCAM. In one embodiment, the extent to which an anti-EpCAM antibody binds to an unrelated, non-EpCAM protein is less than about 10% of the extent to which the antibody binds to EpCAM, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to EpCAM has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 M). In certain embodiments, the anti-EpCAM antibody binds to an epitope of EpCAM that is conserved among EpCAMs from different species, such as the extracellular domain of EpCAM.

[0046] The term "antigen" or "Ag," as used herein, is defined as a molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immunocompetent cells, or both. Those skilled in the art will understand that virtually any macromolecule, including all proteins or peptides, as well as polysaccharides, nucleic acids, or lipids, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Thus, those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen may not be encoded by a "gene" at all. It is readily apparent that antigens can be generated, synthesized, or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0047] The term "binding" as used herein refers to the interaction of an antibody variable region or Fv with an antigen, where the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody variable region or Fv recognizes and binds to a specific protein structure, rather than proteins in general. As used herein, the terms "specifically bind to" or "specifically binds to" mean that an antibody variable region or Fv binds or associates with a particular antigen more frequently, more rapidly, for a longer period of time, and / or with higher affinity than with other proteins. For example, an antibody variable region or Fv specifically binds to an antigen with higher affinity, avidity, more readily, and / or for a longer period of time than it binds to other antigens. As another example, an antibody variable region or Fv binds to a cell surface protein (antigen) with substantially higher affinity than antigens generally recognized by related proteins or other cell surface proteins or polyreactive natural antibodies (i.e., natural antibodies known to bind to a variety of antigens naturally present in humans). However, "specifically binds" does not necessarily require exclusive binding or binding below the limits of detection of other antigens, which is what is meant by the term "selective binding." As an example, "specific binding" of an antibody variable region or Fv (or other binding region) to an antigen means that the antibody variable region or Fv binds to the antigen with an equilibrium constant (KD) of 100 nM or less, e.g., 50 nM or less, e.g., 20 nM or less, e.g., 15 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less.

[0048] As used herein, the terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various head and neck cancers.

[0049] The term "cellular antigen" or "cell-associated antigen," as used herein, refers to any protein, carbohydrate, or other moiety derived from or expressed by a cell that has the ability to elicit an immune response. For example, the cell can be any cell of a subject, particularly cancer cells and senescent cells. A cellular antigen can be an antigen that is on the surface of a cell or inside a cell. This definition is intended to include, but is not limited to, proteins purified from the cell surface or membrane of a cell or unique carbohydrate moieties associated with the cell surface of a cell. This definition also includes antigens from the surface of a cell, which require special treatment of the cell in order to be reached by the antibodies of the present disclosure.

[0050] As used herein, the terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder involving some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0051] As used herein, the term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remaining portion of the heavy and / or light chain is derived from another source or species.

[0052] As used herein, the term "conditionally active antibody" refers to an anti-EpCAM antibody or antibody fragment that is more active under conditions in a tumor microenvironment than under conditions in a non-tumor microenvironment. Conditions in a tumor microenvironment include lower pH, higher lactate and pyruvate concentrations, hypoxia, lower glucose concentrations, and slightly higher temperatures than in a non-tumor microenvironment. For example, a conditionally active antibody is substantially inactive at normal body temperature but becomes active at the higher temperatures in the tumor microenvironment. In yet another aspect, a conditionally active antibody has low activity in normal oxygenated blood but becomes more active in the hypoxic environment present in a tumor. In yet another aspect, a conditionally active antibody has low activity at normal physiological pH (7.0-7.6 or 7.2-7.6) but becomes more active at an acidic pH of 5.0-6.9 or 6.0-6.8 present in a tumor microenvironment. Other conditions in the tumor microenvironment known to those skilled in the art may also be used in the present disclosure as conditions under which an anti-EpCAM antibody has a different binding affinity for EpCAM.

[0053] As used herein, the term "diabody" refers to small antibody fragments with two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L ), the light chain variable region (V L ) linked to a heavy chain variable region (V H ). If a linker is used that is too short to allow pairing between the two domains on the same chain, the domains will be forced to pair with the complementary domains of another chain and create two antigen-binding sites.

[0054] As used herein, the term "detectably labeled" refers to any substance whose direct or indirect detection or measurement by physical or chemical means indicates the presence of an antigen in a sample. Representative examples of useful detectable labels include, but are not limited to, molecules or ions that are directly or indirectly detectable based on light absorption, fluorescence, reflectance, light scattering, phosphorescence, or luminescence properties; molecules or ions that are detectable by radioactive properties; and molecules or ions that are detectable by nuclear magnetic resonance or paramagnetic properties. For example, the group of molecules that are indirectly detectable based on light absorption or fluorescence includes various enzymes that convert an appropriate substrate, for example, from a non-light-absorbing molecule to a light-absorbing molecule or from a non-fluorescent molecule to a fluorescent molecule.

[0055] As used herein, the term "diagnosis" refers to determining a subject's susceptibility to a disease or disorder, determining whether a subject currently has a disease or disorder, prognosis of a subject with a disease or disorder (e.g., identifying pre-metastatic or metastatic cancer status, the stage of the cancer, or the responsiveness of the cancer to treatment), and thermetrics (e.g., monitoring a subject's condition to provide information regarding the efficacy or effectiveness of treatment). In some embodiments, the diagnostic methods of the present disclosure are useful for detecting cancer, particularly in early stages.

[0056] As used herein, the term "effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors) and B cell activation.

[0057] As used herein, the term "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0058] The term "epitope" or "antigenic determinant," as used herein, refers to a site on an antigen to which an antibody binds. Epitopes can be formed either by contiguous amino acids (linear epitopes) or by non-contiguous amino acids juxtaposed by tertiary folding of a protein (conformational epitopes). Epitopes formed by contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include three or more amino acids. Typically, an epitope consists of at least 5-7 amino acids (e.g., 5, 6, or 7 amino acids in the epitope), or at least 8-11 amino acids (e.g., 8, 9, 10, or 11 amino acids in the epitope), or more than 11 amino acids (e.g., 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in the epitope), or more than 20 amino acids (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in the epitope), and less frequently, epitopes can contain 31-40 amino acids. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). A preferred method for mapping epitopes on antigens is surface plasmon resonance.

[0059] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of a constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0060] As used herein, the term "framework" or "FR" refers to variable region residues other than the complementarity determining regions (CDRs or H1-3 in the heavy chain and L1-3 in the light chain). The FR of a variable region generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, V H (or V L ), the CDR and FR sequences usually appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0061] The terms "full-length antibody," "intact antibody," or "whole antibody" refer to an antibody that contains an antigen-binding variable region (V H or V L ) and the light chain constant domain (C L) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Full-length antibodies can be assigned to different "classes" depending on the amino acid sequence of the constant domain of their heavy chain. There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different antibody classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0062] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell or an antibody from a non-human source that utilizes sequences encoding a human antibody repertoire or other human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0063] As used herein, the term "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable regions, all or nearly all of which CDRs correspond to those of a non-human antibody, and all or substantially all of which FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0064] An "individual," "patient," or "subject" is a human or an animal. For example, the subject can be a mammal selected from domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0065] As used herein, an "isolated" antibody refers to an antibody that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase high performance liquid chromatography (HPLC)). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B, vol. 848, pp. 79-87, 2007.

[0066] The term "metastasis," as used herein, refers to any process in which EpCAM plays a role in supporting cancer cells to disperse from a primary tumor, invade lymphatic and / or blood vessels, circulate via the bloodstream, and grow at distant foci in normal tissues elsewhere in the body (metastasis). In particular, the term refers to cellular events such as tumor cell proliferation, migration, adhesion independence, apoptosis evasion, or secretion of angiogenic factors that underlie metastasis and are stimulated or mediated by EpCAM.

[0067] As used herein, the term "microenvironment" refers to any part or region of a tissue or body that has steady or transient physical or chemical differences from other regions of the tissue or body. With respect to tumors, the term "tumor microenvironment" refers to the environment in which the tumor resides, i.e., the acellular regions within the tumor and the region immediately outside the tumor tissue, but not the intracellular compartments of the cancer cells themselves. Tumors and the tumor microenvironment are closely associated and constantly interact. Tumors can alter their microenvironment, which influences how tumors grow and spread. Typically, tumor microenvironments have a low pH, ranging from 5.0 to 7.0, or from 5.0 to 6.9, or from 5.8 to 6.8, or from 6.2 to 6.8. Normal physiological pH, on the other hand, ranges from 7.0 to 7.6 or from 7.2 to 7.8. The tumor microenvironment is known to have lower concentrations of glucose and other nutrients, but higher concentrations of lactate, compared to plasma. Furthermore, the tumor microenvironment may have a temperature 0.3-1°C higher than normal physiological temperature. The tumor microenvironment is discussed in Gillies et al., "MRI of the Tumor Microenvironment," Journal of Magnetic Resonance Imaging, vol. 16, pp. 430-450, 2002, which is incorporated herein by reference in its entirety. The term "non-tumor microenvironment" refers to the microenvironment at a site other than a tumor.

[0068] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, excluding variant antibodies (e.g., those containing naturally occurring mutations or those arising during the production of a monoclonal antibody preparation), which are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present disclosure may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0069] The term "multispecific antibody," as used herein, refers to a full-length antibody, antibody fragment, or construct comprising one or more full-length antibodies and antibody fragments, having at least two different binding sites, each capable of binding to an epitope on the same or different antigens. Engineered antibody constructs having two, three, or more (e.g., four, five, six, or seven) functional antigen-binding sites are within the scope of multispecific antibodies (see, e.g., U.S. Patent Application Publication No. 2002 / 0004587 A1 and Brinkman and Kontermann, MAbs, vol. 9, pp. 182-212, 2017).

[0070] As used herein, the term "package insert" refers to instructions typically included in commercial packaging for therapeutic products, which contain information regarding instructions, usage, dosage, administration, combination therapy, contraindications and / or warnings regarding the use of such therapeutic products.

[0071] As used herein, "percent amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of one in the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0072] As used herein, the term "pharmaceutical formulation" refers to a formulation that is in a form that allows the biological activity of the active ingredients contained therein to be effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to which it is administered.

[0073] As used herein, the term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation other than an active ingredient, which is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0074] As used herein, the terms "purified" and "isolated," with respect to an antibody or nucleotide sequence of the present disclosure, refer to a molecule that is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" means that the molecule is present in the substantial absence of other biological macromolecules of the same type, preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free from other nucleic acid molecules that do not encode that polypeptide, although the molecule may contain additional bases or moieties that do not adversely affect the essential characteristics of the composition.

[0075] As used herein, the term "recombinant antibody" refers to an antibody (e.g., a chimeric, humanized, or human antibody, or an antigen-binding fragment thereof) expressed by a recombinant host cell containing nucleic acid encoding the antibody. Examples of "host cells" for producing recombinant antibodies include: (1) mammalian cells, such as Chinese hamster ovary cells (CHO), COS cells, myeloma cells (such as Y0 cells and NS0 cells), baby hamster kidney cells (BHK), HeLa cells, and Vero cells; (2) insect cells, such as sf9 cells, sf21 cells, and Tn5 cells; (3) plant cells, such as plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, such as cells belonging to the genus Saccharomyces, e.g., Saccharomyces cerevisiae, or Aspergillus (e.g., Aspergillus niger); (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells. subtilis) cells, etc.

[0076] As used herein, the term "single-chain Fv" ("scFv") refers to a covalently linked V H ::V LA heterodimer, which is typically a V linked by a peptide-encoding linker. H and V L It is expressed from a gene fusion containing the encoding gene. "dsFv" is a V stabilized by a disulfide bond. H ::V L Heterodimers. Divalent and multivalent antibody fragments can form spontaneously by the association of monovalent scFvs or can be generated by coupling monovalent scFvs with peptide linkers, such as divalent sc(Fv)2.

[0077] A "therapeutically effective amount" of an antibody of the present disclosure refers to a sufficient amount of antibody to treat said cancer at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the antibodies and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose for a particular patient will vary depending on a variety of factors, including: the disorder being treated and the severity of the disorder; the activity of the specific antibody used; the specific composition used, the age, weight, health, sex, and diet of the patient; the time of administration, route of administration, and excretion rate of the specific antibody used; the duration of treatment; drugs used in combination with or simultaneously with the specific antibody used; and similar factors well known in the medical arts. For example, it is well known to those skilled in the art to start administering a compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0078] As used herein, the terms "treatment," "treat," or "treating" refer to a clinical intervention that seeks to alter the natural course of the individual being treated, and can be performed either for prophylactic purposes or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of disease, prevention of metastasis, slowing the rate of disease progression, improvement or mitigation of disease state, and remission or improved prognosis. In some embodiments, the antibodies of the present disclosure are used to delay disease onset or slow the progression of disease.

[0079] The term "tumor," as used herein, refers to any neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor," as used herein, are not mutually exclusive.

[0080] As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (V, respectively) of a native antibody H and V L ) generally have a similar structure, with each domain consisting of four conserved framework regions (FR) and three complementarity-determining regions (CDR) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single V domain is responsible for conferring antigen-binding specificity. H or V L Furthermore, an antibody that binds to a particular antigen may be able to differentiate between V domains from the antibody that binds to that antigen. H or V L Complementation using domain V H or V L Domains can be isolated by screening libraries of each domain (see, for example, Portolano et al., J. Immunol., vol. 150, pp. 880-887, 1993; Clarkson et al., Nature, vol. 352, pp. 624-628, 1991).

[0081] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined quantity of a conditionally activated multispecific antibody of the present disclosure calculated as the amount of the multispecific antibody of the present disclosure sufficient to produce the desired therapeutic effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle.

[0082] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Furthermore, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The terms "comprise," "include," "have," and "comprise" can also be used interchangeably.

[0083] Unless otherwise indicated, all numerical values ​​expressing properties such as quantities, molecular weights, percentages, ratios of ingredients, reaction conditions, and the like used in the specification and claims are understood to be modified in each instance by the term "about," whether or not the term "about" is present. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be achieved by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of at least the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in its respective testing measurements.

[0084] It is understood that each component, compound, substituent, or parameter disclosed herein should be construed as disclosed for use alone or in combination with one or more of all other components, compounds, substituents, or parameters disclosed herein.

[0085] It is also to be understood that each amount / value or amount / value range for each component, compound, substituent, or parameter disclosed herein is to be construed as being disclosed in combination with each amount / value or amount / value range disclosed for any other component(s), compound(s), substituent(s), or parameter(s) disclosed herein; and thus, any combination of amounts / values ​​or amount / value ranges for two or more components, compounds, substituents, or parameters disclosed herein are disclosed in combination with each other for purposes of this description.

[0086] It is further understood that each range disclosed herein should be interpreted as a disclosure of each specific value having the same number of significant digits within the disclosed range. Thus, a range of 1 to 4 should be interpreted as an explicit disclosure of the values ​​1, 2, 3, and 4. It is further understood that each lower limit of each range disclosed herein should be interpreted as a disclosure in combination with each upper limit of each range and each specific value within each range for the same component, compound, substituent, or parameter. Thus, the present disclosure should be interpreted as a disclosure of all ranges derived by combining each lower limit of each range with each upper limit of each range or each specific value within each range, and combining each upper limit of each range with each specific value within each range.

[0087] Furthermore, a specific amount / value of a component, compound, substituent, or parameter disclosed in the present description or examples should be construed as disclosing either the lower or upper limit of a range and, therefore, can be combined with any other lower or upper limit of a range or specific amount / value for the same component, compound, substituent, or parameter disclosed elsewhere in this application to form a range for that component, compound, substituent, or parameter.

[0088] In one aspect, the present disclosure provides an isolated antibody that specifically binds to an EpCAM protein, particularly a human EpCAM protein, comprising at least one heavy chain variable region comprising three complementarity-determining regions (H1, H2, and H3) and at least one light chain variable region comprising three complementarity-determining regions (L1, L2, and L3). Specific examples of suitable combinations of heavy and light chain complementarity-determining regions are shown in Table 1.

[0089] Exemplary combinations of light and heavy chain variable regions are also provided, including antibodies comprising the heavy chain variable region of SEQ ID NO: 52 in combination with any one of the light chain variable regions of SEQ ID NOs: 53-69, or the light chain variable region of SEQ ID NO: 51 in combination with any one of the light chain variable regions of SEQ ID NOs: 70-96.

[0090] An alignment of various embodiments of light chain variable regions can be found in Figures 4A-4B, and an alignment of various embodiments of heavy chain variable regions can be found in Figures 5A-5B.

[0091] The heavy and light chain variable regions of the present disclosure were each derived from a parent (wild-type) antibody using the methods disclosed in U.S. Patent No. 8,709,755. This method for producing the heavy and light chain variable regions, as well as the methods for producing the antibodies and antibody fragments disclosed in U.S. Patent No. 8,709,755, are incorporated herein by reference.

[0092] Antibodies and antibody fragments comprising these heavy and light chain variable regions can specifically bind to EpCAM, e.g., human EpCAM. Antibodies or antibody fragments comprising a combination of one of these heavy chain variable regions and one of these light chain variable regions have been found to have higher binding affinity for EpCAM at a pH within a tumor microenvironment (e.g., pH 6.0-6.8) than at a pH within a non-tumor microenvironment (e.g., pH 7.0-7.6). As a result, anti-EpCAM antibodies or antibody fragments have higher binding affinity for EpCAM in the tumor microenvironment compared to their binding affinity within a typical normal tissue microenvironment.

[0093] In another embodiment, the present disclosure provides a multispecific antibody comprising at least one binding site for EpCAM and at least one binding site for a tumor-reactive lymphocyte antigen. The multispecific antibody binds to at least one EpCAM epitope and the tumor-reactive lymphocyte antigen with higher affinity under a first physiological condition than under a second physiological condition. In certain embodiments, the tumor-reactive lymphocyte antigen is CD3. In some embodiments, the first physiological condition is an abnormal condition and the second physiological condition is a normal physiological condition. For example, the abnormal condition can be a condition within a tumor microenvironment. The multispecific antibody of the present disclosure can be referred to as a conditionally active multispecific antibody.

[0094] In some embodiments, a conditionally active multispecific antibody is effectively inactive under normal physiological conditions but active under abnormal conditions, optionally with an activity level that is higher than the activity of the conditionally active multispecific antibody under normal physiological conditions or the activity of the parent antibody from which it is derived under normal physiological conditions. In another embodiment, a conditionally active multispecific antibody is effectively inactive at a pH of 7.0 to 7.6 but active at a lower pH of 5.0 to 6.8. In some cases, a conditionally active multispecific antibody is reversibly inactivated under normal physiological conditions. In another example, a conditionally active multispecific antibody can be more or less active in high-oxygen blood, e.g., after passage through the lungs, or in the low pH environment found in a tumor microenvironment. Conditionally active multispecific antibodies can be used as drugs, therapeutic agents, or diagnostic agents.

[0095] Without wishing to be limited by theory, the conditionally active multispecific antibodies of the present disclosure bind to both target cells and tumor-reactive lymphocytes, thereby bringing the target cells into close proximity with the tumor-reactive lymphocytes. This is thought to promote attack by the tumor-reactive lymphocytes against the target cells, thereby inhibiting, damaging, or destroying the target cells. The conditionally active multispecific antibodies of the present disclosure can be used to guide reactive lymphocytes to tumor cells, thereby inhibiting, destroying, and eliminating tumor cells from a subject, thereby achieving the therapeutic effect of inhibiting or eliminating tumor cells.

[0096] The structure / format of the multispecific antibody can be any one of the structures / formats described in Brinkmann and Kontermann, "The making of bispecific antibodies," MABs, vol. 9, pp. 182-212, 2017. Specifically, Figure 2 of Brinkmann and Kontermann describes 19 different structures / formats of bispecific antibodies. These structures / formats include: (1) bispecific antibody conjugates; (2) hybrid bispecific IgG2; (3) "variable domain only" bispecific antibody molecules; (4) CH1 / CL fusion proteins; (5) Fab fusion proteins; (6) non-immunoglobulin fusion proteins; (7) Fc-modified IgG; (8) adjunct and Fc-modified IgG; (9) modified Fc and CH3 fusion proteins; (10) adjunct IgG-HC fusions; (11) adjunct IgG-LC fusions; (12) adjunct IgG-HC and LC fusions; (13) Fc fusions; (14) CH3 fusions; (15) IgE / IgM CH2 fusions; (16) F(ab')2 fusions; (17) CH1 / CL fusion proteins; (18) modified IgG; and (19) non-immunoglobulin fusions.

[0097] In detailed embodiments, the multispecific antibody can be a bivalent scFv-Fc heterodimer as shown in Figure 2 or a tetravalent homodimeric "butterfly" as shown in Figure 3. In these two structures, the reactive lymphocyte antigen is not limited to CD3, which is depicted merely as a representative example of a tumor-reactive lymphocyte antigen. The multispecific antibody of Figure 2 has a first binding site for EpCAM linked to a first heavy chain constant region (e.g., IgG) and a second binding site for a reactive lymphocyte antigen (e.g., CD3) linked to a second heavy chain constant region (e.g., IgG). The two heavy chains are engineered, for example, using knobs-in-holes technology, to form only heterodimers. The first and second binding sites are scFv antibodies that bind to EpCAM and the reactive lymphocyte antigen, respectively. Either or both of the first and second binding sites have conditionally active binding activity for their respective antigens.

[0098] The conditionally active multispecific antibody of Figure 3 may have a full-length IgG antibody that binds to EpCAM and an scFv antibody that binds to a reactive lymphocyte antigen (e.g., CD3). The scFv antibody is linked to the C-terminus of the light chain of the IgG antibody via a linker. The linker may be a short alanine linker (Ala). n , serine linker (Ser) n , hydrophilic linker, or glycine-serine rich linker. The heavy chain of an IgG antibody pairs with the light chain of an IgG antibody linked to the scFv antibody, thus forming one half of a homodimer. This multispecific antibody has a "butterfly" configuration.

[0099] In some embodiments, the multispecific antibody comprises an IgG antibody or fragment thereof that binds to a tumor-reactive lymphocyte antigen and a single-chain antibody that binds to EpCAM, also forming a "butterfly" configuration as shown in Figure 3. The single-chain antibody may be an scFv antibody. The scFv antibody may be attached at the C-terminus of the IgG antibody via a linker as described herein.

[0100] The binding sites of the multispecific antibodies of the present disclosure each comprise a light chain variable region and a heavy chain variable region. The light chain variable region and heavy chain variable region may be in a single-chain antibody format or in a two-chain format, as formed by pairing of a light chain and a heavy chain (FIGS. 2 and 3). In a conditionally active binding site, one or both of the light and heavy chain variable regions may be conditionally active. An exemplary conditionally active anti-CD3 scFv antibody comprises the light chain variable region of SEQ ID NO: 101 and the heavy chain variable region of SEQ ID NO: 100. Additional sequences of conditionally active and non-conditionally active anti-CD3 antibodies useful in the multispecific antibodies of the present disclosure are described in WO 2019 / 241216, the disclosure of which is incorporated herein by reference.

[0101] Examples of mutant regions of conditionally active anti-EpCAM antibodies that can be used to construct the multispecific antibodies of the present specification include a combination of the heavy chain mutant region of SEQ ID NO: 52 with one of the light chain mutant regions of SEQ ID NOs: 53 to 69, and a combination of the light chain variable region of SEQ ID NO: 51 with one of the heavy chain variable regions of SEQ ID NOs: 70 to 96.

[0102] In some other embodiments, a multispecific antibody can be constructed having two variable regions that form binding sites for EpCAM and two other variable regions that form binding sites for reactive lymphocyte antigens (e.g., CD3), as shown in Figure 2. These variable regions can be selected from light and heavy chain variable regions having the amino acid sequences provided herein. One or both of the binding sites must have conditional activity for its respective antigen. In each binding site with conditional activity, at least one of the light and heavy chain variable regions has increased affinity for its antigen under a first physiological condition (e.g., an abnormal condition) compared to its affinity under a second physiological condition (e.g., normal physiological condition). Thus, one skilled in the art can select appropriate light and heavy chain variable regions to construct a multispecific antibody as shown in Figure 2. The heavy chain fragment in Figure 2 is selected from the constant region of an IgG antibody, including any of the IgG subclasses: IgG1, IgG2, IgG3, and IgG4.

[0103] In some other embodiments, a multispecific antibody can be constructed as shown in Figure 3. Similarly, the light and heavy chain variable regions of an scFv antibody and the light and heavy chain variable regions of a full-length IgG antibody can be selected from light and heavy chain variable regions having the amino acid sequences provided herein. In each binding site with conditional activity, at least one of the light and heavy chain variable regions has increased affinity for its antigen under a first physiological condition (e.g., an abnormal condition) compared to its affinity under a second physiological condition (e.g., a normal physiological condition). In this way, one skilled in the art can select appropriate light and heavy chain variable regions provided herein to construct the multispecific antibody shown in Figure 3. The constant regions in Figure 3 are selected from the constant regions of IgG antibodies, including any of the IgG subclasses: IgG1, IgG2, IgG3, and IgG4.

[0104] The conditionally active anti-EpCAM antibodies or antibody fragments herein are expected to exhibit reduced side effects compared to non-conditionally active anti-EpCAM antibodies due to their reduced binding affinity for EpCAM in normal tissue microenvironments. The anti-EpCAM antibodies or antibody fragments of the present disclosure are also expected to have efficacy comparable to monoclonal anti-EpCAM antibodies known in the art. These combined properties may allow the use of higher doses of these anti-EpCAM antibodies or antibody fragments due to reduced side effects, thereby providing a more effective treatment option.

[0105] In some embodiments, the abnormal conditions are an acidic pH ranging from about 5.0 to about 7.0, or from about 5.2 to about 6.8, or from about 5.4 to about 6.8, or from about 5.6 to about 6.8, or from about 5.8 to about 6.8, or from about 6.0 to about 6.8, or from about 6.2 to about 6.8, or from about 6.4 to about 6.8, or from about 6.6 to about 6.8. In some embodiments, the acidic pH can be in the range of about 6.4 to about 7.0, or from about 6.6 to about 7.0, or from about 6.8 to about 7.0. Normal physiological conditions can be the normal physiological pH of blood, which is well established in the art. In some embodiments, the normal physiological pH of blood can be in the range of 7.0 to about 7.8, or from about 7.1 to about 7.7, or from about 7.2 to about 7.6, or from about 7.2 to about 7.5, or from about 7.2 to about 7.4.

[0106] In certain embodiments, the multispecific antibodies of the present disclosure have a ratio of affinity or avidity for EpCAM and / or tumor-reactive lymphocyte antigens (e.g., CD3) under abnormal conditions to the same affinity or avidity under normal physiological conditions of at least about 1.3:1, or at least about 2:1, or at least about 3:1, or at least about 4:1, or at least about 5:1, or at least about 6:1, or at least about 7:1, or at least about 8:1, or at least about 9:1, or at least about 10:1, or at least about 1.5 ...2:1, or at least about 3:1, or at least about 4:1, or at least about 5:1, or at least about 6:1, or at least about 7:1, or at least about at least about 11:1, or at least about 12:1, or at least about 13:1, or at least about 14:1, or at least about 15:1, or at least about 16:1, or at least about 17:1, or at least about 18:1, or at least about 19:1, or at least about 20:1, or at least about 30:1, or at least about 40:1, or at least about 50:1, or at least about 60:1, or at least about 70:1, or at least about 80:1, or at least about 90:1, or at least about 100:1.

[0107] In some embodiments, the antibody comprises one or more non-naturally occurring amino acids. For example, non-naturally occurring amino acids include carbonyl, acetyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide, or alkyne groups. For example, see U.S. Pat. No. 7,632,924 for suitable non-naturally occurring amino acids. The term "non-naturally occurring amino acid" also includes amino acids that are created by modification (e.g., post-translational modification) of naturally occurring amino acids, but that are not themselves naturally incorporated into a growing polypeptide chain by a living organism's translation complex. Examples of such non-naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0108] In some embodiments, the antibody is in "mimetic" or "peptidomimetic" form, which is either composed entirely of synthetic, non-natural analogues of amino acids, or is a chimeric molecule of some naturally occurring amino acids and some non-natural analogs of amino acids. The mimetic can also incorporate any amount of naturally occurring amino acid conservative substitutions as long as such substitutions also do not substantially alter the structure and / or activity of the antibody.

[0109] Mimetics can include any combination of non-natural structural components. In one aspect, mimetics of the present disclosure include one or all of the following three structural groups: a) residue linking groups other than natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues that induce secondary structure mimicry, i.e., residues for inducing or stabilizing secondary structure, such as β-turn, γ-turn, β-sheet, α-helical conformations, etc. For example, a multispecific antibody can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be alternatives to the traditional amide bond ("peptide bond") linkage include, for example, ketomethylene (e.g., -C(=O)-CH2- for -C(=O)-NH-), aminomethylene (CH2-NH), ethylene, olefin (CH=CH), ether (CH2-O), thioether (CH2-S), tetrazole, thiazole, retroamide, thioamide, and ester (see, for example, Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, vol. 7, pp. 267-357, "Peptide Backbone Modifications," in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, vol. 7, B. Weinstein, ed., New York: Marcell Dekker, pp. 257-267).

[0110] Further examples of non-naturally occurring amino acid residues include D- or L-naphthylalanine; D- or L-phenylglycine; D- or L-2 thienylalanine; D- or L-1, -2, -3, or -4-pyrenylalanine; D- or L-3 thienylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; and D-(trifluoromethyl)-phenylglycine. D-(trifluoromethyl)-phenylalanine; Dp-fluoro-phenylalanine; D- or Lp-biphenylphenylalanine; D- or Lp-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and D- or L-alkylamines (wherein alkyl can be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, sec-isobutyl, isopentyl, or a non-acidic amino acid). Aromatic rings of unnatural amino acids include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

[0111] Acidic unnatural amino acids can be generated by substitution with, for example, noncarboxylic acid amino acids; (phosphono)alanine; sulfated threonine, etc., while maintaining the negative charge. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R'~NC--N--R'), such as 1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl-3(4-azonia-4,4-dimethylpentyl)carbodiimide. Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0112] Basic unnatural amino acids can be generated, for example, by substituting ornithine, citrulline, or (guanidino)-acetic acid or (guanidino)alkyl-acetic acid (where alkyl is as defined above) (in addition to lysine and arginine). Nitrile derivatives (e.g., containing a CN-moiety instead of COOH) can be substituted for asparagine or glutamine. Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues. Arginine residue mimics can be generated by reacting arginyl with, for example, one or more conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, under alkaline conditions. Tyrosine residue mimics can be generated by reacting tyrosyl with, for example, aromatic diazonium compounds or tetranitromethane. N-acetylimidazole and tetranitromethane can be used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Cysteine ​​residue mimetics can be generated by reacting cysteinyl residues with, for example, α-haloacetates, such as 2-chloroacetic acid or chloroacetamide, and corresponding amines to form carboxymethyl or carboxyamidomethyl derivatives. Cysteine ​​residue mimetics can also be generated by reacting cysteinyl residues with, for example, bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4-nitrophenol; or chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can be generated (and amino-terminal residues can be altered) by reacting lysinyl with, for example, succinic acid or other carboxylic acid anhydrides.Lysine and other α-amino-containing residue mimetics can also be generated by reaction with imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transamidase-catalyzed reactions with glyoxylic acid. For example, methionine mimetics can be generated by reaction with methionine sulfoxide. Proline mimetics include, for example, pipecolic acid, thiazolidinecarboxylic acid, 3- or 4-hydroxyproline, dehydroproline, 3- or 4-methylproline, or 3,3-dimethylproline. Histidine residue mimetics can be generated by reacting histidyl with, for example, diethylpyrocarbonate or p-bromophenacyl bromide. Other mimetics include those produced, for example, by hydroxylation of proline and lysine; phosphorylation of the hydroxyl group of seryl or threonyl residues; methylation of the α-amino groups of lysine, arginine, and histidine; acetylation of N-terminal amines; methylation or substitution of backbone amide residues with N-methyl amino acids; or amidation of the C-terminal carboxyl group.

[0113] Antibody mimetics may also contain one or more amino acids of opposite chirality. Thus, any naturally occurring amino acid in the L configuration (which may also be referred to as R or S, depending on the structure of the chemical entity) may be replaced by an amino acid of the same chemical structure type, or peptidomimetic, but of opposite chirality, referred to as a D-amino acid (these forms may also be referred to as R or S).

[0114] Antibody mimetics can be synthesized using any protein chemical synthesis technique. In a typical in vitro protein synthesis process, the length of a peptide is extended by one amino acid at a time through the formation of a peptide bond between the peptide and an amino acid. The peptide bond is formed using a ligation reaction that can use natural or unnatural amino acids. Thus, unnatural amino acids can be introduced into the antibodies of the present disclosure to create mimetics in this manner.

[0115] In some embodiments, the non-naturally occurring amino acid in the antibody allows for conjugation to a macromolecule, such as a polymer, protein, or fatty acid. In some embodiments, the multispecific antibody is linked (e.g., covalently bonded) to a polymer (e.g., a polymer other than a polypeptide). Suitable polymers include, for example, biocompatible polymers, water-soluble biocompatible polymers, synthetic polymers, and naturally occurring polymers. Examples of polymers include substituted or unsubstituted linear or branched polyalkylene, polyalkenylene, and polyoxyalkylene polymers, and branched or unbranched polysaccharides, such as homopolysaccharides or heteropolysaccharides.Further examples of suitable polymers include ethylene vinyl alcohol copolymer (commonly known as EVOH or EVAL); polybutyl methacrylate; poly(hydroxyvalerate); poly(L-lactic acid); polycaprolactone; poly(lactide-co-glycolide); poly(hydroxybutyrate); poly(hydroxybutyrate-co-valerate); polydioxanone; polyorthoesters; polyanhydrides; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolic acid-co-trimethylene carbohydrate); carbonates; polyphosphoesters; polyphosphoesterurethanes; poly(amino acids); cyanoacrylates; poly(trimethylene carbonate); poly(iminocarbonates); copoly(ether-esters) (e.g., poly(ethylene oxide)-poly(lactic acid) (PEO / PLA) copolymers); polyalkylene oxalates; polyphosphazenes; biomolecules, such as fibrin, fibrinogen, cellulose, starch, collagen, and hyaluronic acid; polyurethanes; silicones; polyesters; polyolefins; polyisobutylene and ethylene ethylene-α-olefin copolymers; acrylic polymers and copolymers; vinyl halide polymers and copolymers such as polyvinyl chloride; polyvinyl ethers such as polyvinyl methyl ether; polyvinylidene halides such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketone; polyvinyl aromatics such as polystyrene; polyvinyl esters such as polyvinyl acetate; copolymers of vinyl monomers with each other and with olefins, for example, ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin, and ethylene-vinyl acetate copolymer; polyamides such as nylon 66 and polycaprolactam; alkyd resins; polycarbonate; polyoxymethylene; polyimides; polyethers; epoxy resins; polyurethanes; rayon; rayon-triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; amorphous Teflon™; poly(ethylene glycol); and carboxymethyl cellulose.

[0116] Examples of synthetic polymers include unsubstituted and substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) and derivatives thereof, substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) and derivatives thereof. Suitable naturally occurring polymers include, for example, albumin, amylose, dextran, glycogen and derivatives thereof.

[0117] The linked polymer can have an average molecular weight in the range of 500 Da to 50,000 Da, e.g., 5,000 Da to 40,000 Da or 25,000 to 40,000 Da. For example, in some embodiments, when the multispecific antibody comprises a poly(ethylene glycol) (PEG) or methoxypoly(ethylene glycol) polymer, the PEG or methoxypoly(ethylene glycol) polymer can have a molecular weight in the range of about 0.5 kilodaltons (kDa) to 1 kDa, about 1 kDa to 5 kDa, 5 kDa to 10 kDa, 10 kDa to 25 kDa, 25 kDa to 40 kDa, or 40 kDa to 60 kDa.

[0118] For example, a water-soluble polymer (e.g., PEG) can be linked to an antibody by reacting a water-soluble polymer containing a carbonyl group with an antibody bearing a non-naturally occurring amino acid containing an aminooxy, hydrazine, hydrazide, or semicarbazide group. As another example, an antibody containing an alkyne-containing amino acid can be linked to a water-soluble polymer by reacting the antibody with a water-soluble polymer containing an azide moiety. In some cases, the azide or alkyne group is linked to the PEG molecule through an amide bond.

[0119] In some embodiments, the macromolecule linked to the antibody is albumin. The albumin can be, for example, the albumin of the subject to which the antibody is administered. For example, if the antibody is intended for use in humans, human albumin can be linked to the antibody. If the antibody is intended for use in dogs, canine albumin can be linked to the multispecific antibody. Generally speaking, for antibodies intended for use in a certain species, albumin from that species is linked to the multispecific antibody.

[0120] Examples of linkers for conjugating macromolecules to antibodies include glutaraldehyde, homobifunctional crosslinkers, or heterobifunctional crosslinkers. Glutaraldehyde crosslinks polypeptides via their amino moieties. Homobifunctional crosslinkers (e.g., homobifunctional imidoesters, homobifunctional N-hydroxysuccinimidyl (NHS) esters, or homobifunctional sulfhydryl-reactive crosslinkers) contain two or more identical reactive moieties and can be used in a one-step reaction procedure in which the crosslinker is added to a solution containing a mixture of the macromolecule and antibody to be linked. At slightly alkaline pH, imidoesters react only with primary amines to form imidoamides, and the overall charge of the crosslinked macromolecule and antibody is not affected. Homobifunctional sulfhydryl-reactive crosslinkers include bismaleimidohexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-di-(3',2'-pyridyldithio)propionamidobutane (DPDPB).

[0121] Heterobifunctional crosslinkers have two or more different reactive moieties (e.g., an amine-reactive moiety and a sulfhydryl-reactive moiety) that crosslink one of the macromolecules and antibodies through the amine- or sulfhydryl-reactive moiety and then react with the other of the macromolecule and antibody through the unreacted moiety. Similar to pyridyl disulfide crosslinkers, several heterobifunctional haloacetyl crosslinkers are available. Carbodiimides are a classic example of heterobifunctional crosslinking reagents for coupling carboxyl groups to amines to form amide bonds.

[0122] The antibody may be glycosylated, e.g., covalently linked to a carbohydrate or polysaccharide moiety. Glycosylation of multispecific antibodies is typically N-linked or O-linked.

[0123] N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue in the antibody. The tripeptide sequences "asparagine-X-serine" or "asparagine-X-threonine," where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in an antibody creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine (although 5-hydroxyproline or 5-hydroxylysine can also be used).

[0124] Addition of glycosylation sites to an antibody can be accomplished by altering its amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by adding, or substituting, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). Conversely, removal of glycosylation sites can be achieved by amino acid alterations within the native glycosylation sites of the multispecific antibody.

[0125] Antibodies can be covalently linked to another macromolecule (e.g., lipid, polypeptide, synthetic polymer, carbohydrate, etc.) using a linker selected from glutaraldehyde, a homobifunctional crosslinker, or a heterobifunctional crosslinker. Glutaraldehyde crosslinks multispecific antibodies through their amino moieties. Homobifunctional and heterobifunctional crosslinkers are described herein.

[0126] Linkers used in the construction of multispecific antibodies can be flexible peptides that ensure proper folding of the multispecific antibody. Exemplary linkers include (Ser)n, (Ser-Ala)n, and (Ala)n.

[0127] In certain embodiments, a conditionally active anti-EpCAM antibody of the present disclosure binds to EpCAM of a non-human primate, such as a cynomolgus monkey (Macaca fascicularis), in addition to binding to human EpCAM. The ability to bind to both human and non-human primate EpCAM proteins is advantageous for safety and efficacy testing, as it allows for earlier testing in non-human primates rather than human subjects. In certain embodiments, anti-EpCAM antibodies, including bispecific antibodies, bind to human and cynomolgus EpCAM proteins, respectively, with at least five-fold, at least four-fold, or at least three-fold higher affinity than the same antibody that binds to rat or mouse EpCAM protein. In another embodiment, a conditionally active bispecific antibody of the present disclosure binds to human and cynomolgus EpCAM proteins, respectively, with at least five-fold higher affinity than the same antibody that binds to mouse or rat EpCAM protein. In further embodiments, these antibodies bind to cynomolgus EpCAM protein with an affinity that is at least 45% or at least 50% of the binding affinity of the same antibody to human EpCAM protein.

[0128] In each of the embodiments disclosed herein, the antibody or antibody fragment may have higher antigen-binding activity for the EpCAM protein at a certain value of a condition in a tumor microenvironment compared to a different value of the same condition occurring in a non-tumor microenvironment. In one embodiment, the condition is pH.

[0129] A conditionally active anti-EpCAM antibody or antibody fragment also has at least 70% of the antigen-binding activity at pH 6.0 compared to the same antigen-binding activity at pH 6.0 of the parent antibody or antibody fragment from which it is derived, and the antibody or antibody fragment may have less than 50%, or less than 40%, or less than 30%, or less than 20%, or less than 10% of the antigen-binding activity at pH 7.4 compared to the same antigen-binding activity at pH 7.4 of the parent antibody or antibody fragment from which it is derived. The antigen-binding activity may be, for example, binding to the EpCAM protein or binding to CD3.

[0130] In each of the foregoing embodiments, antigen binding activity may be measured by ELISA.

[0131] The processes described herein serve as a guide in deriving these variants. Variants of the heavy and light chain variable regions can be prepared by introducing appropriate modifications into the nucleotide sequences encoding the heavy and light chain variable regions or by peptide synthesis. Such modifications include, for example, deletion of, and / or insertion and / or substitution of, residues from the amino acid sequences of the heavy and light chain variable regions. Any combination of deletion, insertion, and substitution can be made to obtain an antibody or antibody fragment of the invention, provided that it possesses the desired properties, such as antigen binding to human EpCAM and / or conditional activity.

[0132] In certain embodiments, antibody or antibody fragment variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include the CDRs and framework regions (FRs). Conservative substitutions are shown in Table 2 under the heading of "Preferred Substitutions." More substantial changes are shown in Table 2 under the heading of "Exemplary Substitutions," and are further described below with respect to amino acid side chain classifications. Amino acid substitutions are introduced into the antibody or antibody fragment of interest, and the resulting products are screened for a desired activity, e.g., retained / improved antigen binding or reduced immunogenicity.

[0133] [Table 2]

[0134] Amino acids can be classified based on common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0135] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0136] One type of substitutional variant involves substituting one or more complementarity-determining region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have altered (e.g., improved) specific biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or may substantially retain specific biological properties of the parent antibody. Exemplary substitutional variants include affinity-matured antibodies, which may be conveniently generated using, for example, phage-display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies are displayed on phage and then screened for a specific biological activity (e.g., binding affinity).

[0137] For example, modifications (e.g., substitutions) can be made to the CDRs to improve antibody affinity. Such modifications can be made to CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during somatic maturation (e.g., Chowdhury, Methods Mol. Biol., vol. 207, pp. 179-196, 2008), and / or SDRs (a-CDRs), and the resulting mutant V H or VL The binding affinity of the antibody is evaluated. Affinity maturation by construction and reselection of a secondary library is described, for example, in Hoogenboom et al. in Methods in Molecular Biology, vol. 178, pp. 1-37, 2001. In one embodiment of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, oligonucleotide-directed mutagenesis). A secondary library is then created. This library is screened to identify any antibody variants with the desired affinity. Another method for introducing diversity is the CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be identified using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3, in particular, are often targeted.

[0138] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such modifications do not substantially reduce the ability of the antibody or antibody fragment to bind to the antigen. For example, conservative substitutions (e.g., conservative substitutions described herein) that do not substantially reduce binding affinity may be made in the CDRs. Such substitutions may occur in CDR "hot spots" or outside of the SDRs. The aforementioned mutant V H and V L In particular embodiments of the sequences, each CDR is unaltered or contains no more than one, two or three amino acid substitutions.

[0139] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells, Science, vol. 44, pp. 1081-1085, 1989. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody or antibody fragment with the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody or antibody fragment and the antigen. Such contact residues and neighboring residues can be targeted or removed as candidates for substitution. The mutants can be screened to determine whether they have the desired properties.

[0140] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibodies include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide which extends the serum half-life of the antibody.

[0141] Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. H and V L Only the CDRs of the V H and V L It is known that when a humanized antibody is produced by simply grafting the V of a non-human antibody onto the V of a non-human animal, the antigen-binding activity is reduced compared to that of the original antibody derived from a non-human animal. H and V LSome amino acid residues in the CDRs as well as in the FRs are thought to be directly or indirectly involved in antigen-binding activity. H and V L It is thought that substitution of different amino acid residues from the FR of the V of a human antibody will reduce binding activity. H and V L It is necessary to attempt to identify amino acid residues from the amino acid sequence of the FR that are directly involved in binding to the antibody, that interact with amino acid residues in the CDR, or that maintain the three-dimensional structure of the antibody and are directly involved in binding to the antigen. The reduced antigen-binding activity can be increased by substituting the identified amino acids with amino acid residues of the original antibody derived from a non-human animal.

[0142] Modifications and changes can be made to the structure of the antibodies of the present disclosure and the DNA sequences that encode them, thereby obtaining a functional molecule that still encodes an antibody with desired properties.

[0143] When making changes to the amino acid sequence, the hydrophobicity index of the amino acid may be taken into consideration. The importance of the hydrophobicity index in conferring interactive biological function to a protein is generally understood in the art. The relative hydrophobicity characteristics of amino acids are recognized to contribute to the secondary structure of the resulting protein, which determines the interaction of the protein with other molecules such as enzymes, substrates, receptors, DNA, antibodies, and antigens. Each amino acid has been assigned a hydrophobicity index based on its hydrophobicity and charge characteristics as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0144] Another object of the present disclosure also includes variants that retain the function of the antibodies of the present disclosure.

[0145] Two amino acid sequences are "substantially homologous" or "substantially similar" if greater than 80%, preferably greater than 85%, and more preferably greater than 90%, of the amino acids are identical or more than about 90%, preferably greater than 95%, similar (functionally identical) over the entire length of the shorter sequence. Preferably, similar or homologous sequences are identified by alignment using, for example, the pileup program of GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) or any sequence comparison algorithm such as BLAST or FASTA.

[0146] For example, certain amino acids in a protein structure can be substituted with other amino acids without a significant loss of activity. Because the interaction capabilities and properties of a protein determine its biological functional activity, certain amino acid substitutions can be made in the protein sequence, and of course, in its DNA coding sequence, while still obtaining a protein with similar properties. Thus, it is believed that various changes can be made to the sequences of the antibodies or antibody fragments of the present disclosure, or the corresponding DNA sequences encoding said antibodies or antibody fragments, without significantly impairing their biological activity.

[0147] It is known in the art that certain amino acids can be substituted with other amino acids having a similar hydrophobicity index or score to obtain proteins with similar biological activity, i.e., proteins with equivalent biological function.

[0148] As outlined herein, amino acid substitutions are generally made based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions taking into account the various characteristics listed above are well known to those of skill in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0149] In certain embodiments, the anti-EpCAM antibodies or antibody fragments provided herein may be modified to increase or decrease the degree of glycosylation of the antibody or antibody fragment. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0150] If the antibody contains an Fc region, the carbohydrate attached thereto can vary. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, generally N-linked to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH, vol. 15, pp. 26-32, 1997. The oligosaccharides can contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and can contain fucose attached to GlcNAc in the "stem" portion of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present disclosure can be performed to generate antibody variants with specific improved properties.

[0151] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the total amount of all glycostructures (e.g., complex structures, hybrid structures, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (EU numbering of Fc region residues) in the Fc region. However, due to minor sequence variations in antibodies, Asn297 can also be located approximately three amino acids upstream or downstream from position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo). Examples of publications related to "defucosylated" or "fucose-lacking" antibody variants include: U.S. Patent Application Publication Nos. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication Nos. 2003 / 0115614; 2002 / 0164328; 2004 / 0093621; and 2004 / 0132140. No. 2004 / 0110704; No. 2004 / 0110282; No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al. J. Mol. Biol., vol. 336, pp. 1239-1249, 2004; Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004.Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys., vol. 249, pp. 533-545, 1986; U.S. Patent Application Publication No. 2003 / 0157108A; and WO 2004 / 056312A1, particularly Example 11), as well as knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng., vol. 87, pp. 614-622, 2004; Kanda, Y. et al. al., Biotechnol. Bioeng., vol. 94, pp. 680-688, 2006 and WO 2003 / 085107).

[0152] Antibody variants are further provided with branched oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are branched by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878; U.S. Pat. No. 6,602,684; and WO 2005 / 0123546. Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0153] In certain embodiments, Fc region variants can be generated by introducing one or more amino acid modifications into the Fc region of an anti-EpCAM antibody or antibody fragment provided herein. The Fc region variants can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0154] Certain embodiments contemplate antibody variants that retain some, but not all, effector functions, making them desirable candidates for uses in which in vivo antibody half-life is important but certain effector functions (e.g., ADCC) are unnecessary or deleterious. To confirm reduced / depleted CDC and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / absent CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Mmunol., vol. 9, pp. 457-492, 1991. Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (e.g., Hellstrom et al. Proc. Nat'l Acad. Sci. USA, vol. 83, pp. 7059-7063, 1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA, vol. 82, pp. 1499-1502, 1985; U.S. Pat. No. 5,821,337 (see also Bruggemann et al., J. Exp. Med., vol. 166, pp. 1351-1361, 1987). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.) and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. SciUSA, vol. 95, pp. 652-656, 1998. A C1q binding assay can also be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs described in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods, vol. 202, pp. 163-171, 1996; Cragg, MS et al., Blood, vol. 101, pp. 1045-1052, 2003; and Cragg, MS, and MJ Glennie, Blood, vol. 103, pp. 2738-2743, 2004). Measurements of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol., vol. 18, pp. 1759-1769, 2006).

[0155] Antibody or antibody fragment variants with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 have been substituted with alanine (U.S. Pat. No. 7,332,581).

[0156] Certain antibody variants with improved or decreased binding to FcRs have been described in the art (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. vol. 9, pp. 6591-6604, 2001).

[0157] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at Fc region positions: 298, 333 and / or 334 (EU numbering of residues).

[0158] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or attenuated) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol., vol. 164, pp. 4178-4184, 2000.

[0159] Antibodies with extended half-lives and improved binding to the neonatal Fc receptor (FcRn), responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., vol. 117, pp. 587-593, 1976 and Kim et al., J. Immunol., vol. 24, p. 249, 1994), are described in U.S. Patent Application Publication No. 2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Pat. No. 7,371,826). For other examples of Fc region variants, see also Duncan & Winter, Nature, vol. 322, pp. 738-740, 1988; U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94 / 29351.

[0160] In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an anti-EpCAM antibody or antibody fragment are substituted with cysteine ​​residues. In certain embodiments, the substituted residues are located at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are positioned at accessible sites of the antibody that can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further detailed herein. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and 5400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0161] In certain embodiments, the anti-EpCAM antibodies or antibody fragments provided herein may be further modified to contain additional nonproteinaceous moieties known and readily available in the art. Suitable moieties for derivatization of antibodies or antibody fragments include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous for manufacturing because it is stable in water. The polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody or antibody fragment can vary, and when multiple polymers are attached, they can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody or antibody fragment to be improved and whether the derivative will be used in therapy under certain conditions.

[0162] The anti-EpCAM antibody or antibody fragment, or variant thereof, of the present disclosure has a higher binding affinity for EpCAM under conditions in a tumor microenvironment than under conditions in a non-tumor microenvironment. In one embodiment, the conditions in a tumor microenvironment and the conditions in a non-tumor microenvironment are both pH. Thus, in one embodiment, an anti-EpCAM antibody or antibody fragment of the present disclosure selectively binds to EpCAM at a pH of about 5.0 to 6.8, but exhibits reduced binding affinity to EpCAM at a pH of about 7.2 to 7.8, which is found in a normal non-tumor microenvironment. In another embodiment, an anti-EpCAM antibody or antibody fragment of the present disclosure selectively binds to EpCAM at a pH of about 5.0 to 6.9, but exhibits reduced binding affinity to EpCAM at a pH of about 7.0 to 7.6, which is found in a normal non-tumor microenvironment. In yet another embodiment, an anti-EpCAM antibody or antibody fragment of the present disclosure selectively binds to EpCAM at a pH of about 5.0 to 6.9, but exhibits reduced binding affinity to EpCAM at a pH of about 7.0 to 7.8, which is found in a normal non-tumor microenvironment. In yet another embodiment, the anti-EpCAM antibody or antibody fragment of the present disclosure selectively binds to EpCAM at a pH of about 5.0 to 6.8, but exhibits reduced binding affinity for EpCAM at a pH of about 7.2 to 7.6, which is found in normal, non-tumor microenvironments. In one embodiment, the anti-EpCAM antibody or antibody fragment exhibits higher binding affinity for EpCAM at pH 6.0 than at pH 7.4 in screening assays such as those described herein.

[0163] In certain embodiments, the anti-EpCAM antibody or antibody fragment of the present disclosure has a dissociation constant (Kd) with EpCAM under conditions within a tumor microenvironment: about ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, or 10 -8 M~10 -13 M or 10 -9 M~10 -13In one embodiment, the ratio of the Kd of the antibody or antibody fragment for EpCAM under conditions in a tumor microenvironment to the Kd under the same conditions in a non-tumor microenvironment is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1. In another embodiment, the ratio of the Kd of an antibody or antibody fragment for EpCAM under conditions in a tumor microenvironment to the Kd under the same conditions in a non-tumor microenvironment is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 15:1, or at least about 20:1.

[0164] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed on the Fab version of the antibody of interest and its antigen using the following assay: The solution binding affinity of the Fab for the antigen is measured in the presence of a titration series of unlabeled antigen at the lowest concentration ( 125 I) After equilibration of Fab with labeled antigen, bound antigen is measured by capturing the antigen on an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125[I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, and incubation can be continued for a longer period (e.g., about 65 hours) until equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20®; Packard) is added and counted for 10 minutes in a TOPCOUNT® gamma counter (Packard). For use in competitive binding assays, a concentration of each Fab that results in 20% or less of maximum binding is selected.

[0165] According to another embodiment, Kd is measured by surface plasmon resonance assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with approximately 10 response units (RU) of immobilized antigen on a CM5 chip. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μL / min to achieve approximately 10 response units (RU) of bound protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 μL / min. on ) and dissociation rate (k offThe equilibrium dissociation constant (Kd) is calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. off / k on See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on rate is 10 6 M -1 s -1 If the on-rate exceeds 100 kJ / s, the on-rate can be determined using a fluorescence quenching method, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen at 25°C, as measured in a spectrophotometer, for example, a stop-flow equipped spectrophotometer (Aviv Instruments) or an 8000 Series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0166] The anti-EpCAM antibody of the present disclosure may be a chimeric, humanized, or human antibody. In one embodiment, anti-EpCAM antibody fragments are used, such as Fv, Fab, Fab', Fab'-SH, scFv, diabody, triabody, tetrabody, or F(ab')2 fragments, and multispecific antibodies formed from antibody fragments. In another embodiment, the antibody is a full-length antibody, such as an intact IgG antibody, or other antibody class or isotype as defined herein. For a review of specific antibody fragments, see Hudson et al. Nat. Med., vol. 9, pp. 129-134, 2003. For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have extended in vivo half-lives, see U.S. Pat. No. 5,869,046.

[0167] Diabodies of the present disclosure can be bivalent or bispecific. For examples of diabodies, see, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993. Examples of triabodies and tetrabodies are also described in Hudson et al., Nat. Med., vol. 9, pp. 129-134, 2003.

[0168] In some embodiments, the present disclosure provides single-domain antibody fragments comprising all or a portion of the heavy chain variable region or all or a portion of the light chain variable region of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

[0169] Antibody fragments can be produced by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage).

[0170] In some embodiments, the anti-EpCAM antibody of the present disclosure may be a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp. 6851-6855, 1984. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass of the antibody is changed relative to the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0171] In certain embodiments, the chimeric antibody of the present disclosure is a humanized antibody. Typically, such non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody may optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0172] Humanized antibodies and methods for their production are reviewed, for example, by Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008, and further described, for example, in Riechmann et al., Nature, vol. 332, pp. 323-329, 1988; Queen et al., Proc. Nat'l Acad. Sci. USA, vol. 86, pp. 10029-10033, 1989; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al. al., Methods, vol. 36, pp. 25-34, 2005 (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol., vol. 28, pp. 489-498, 1991 (describing "resurfacing"); Dall'Acqua et al., Methods, vol. 36, pp. 43-60, 2005 (describing "FR shuffling"); and Osbourn et al., Methods, vol. 36, pp. 61-68, 2005 and Klimka et al., Br. J. Cancer, vol 83, pp. 252-260, 2000 (describing "guided selection" approach for FR shuffling).

[0173] Human framework regions that can be used for humanization include, but are not limited to, the "best-fit" method (see, e.g., Sims et al. J. Immunol., vol. 151, p. 2296, 1993); framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, vol. 89, p. 4285, 1992; and Presta et al. J. Immunol., vol. 151, p. 2623, 1993); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008); and framework regions obtained from FR library screening (see, e.g., Baca et al. al., J. Biol. Chem., vol. 272, pp. 10678-10684, 1997 and Rosok et al., J. Biol. Chem., vol. 271, pp. 22611-22618, 1996).

[0174] In some embodiments, the anti-EpCAM antibodies of the present disclosure are multispecific, e.g., bispecific, antibodies. Multispecific antibodies are monoclonal antibodies with binding specificities for at least two different sites. In certain embodiments, one binding specificity is for EpCAM and the other is for another antigen, such as CD3. A specific example of such an antibody is a bispecific antibody with binding specificities for EpCAM and CD3+. Conditionally active bispecific antibodies can be single-conditionally active or biconditionally active. Thus, in the case of a single-conditionally active bispecific antibody, one binding site is conditionally active and the other is not, such as a combination of wild-type (WT) EpCAM and conditionally active (CAB) CD3+ or a combination of CAB EpCAM and WT CD3+. In the case of a biconditionally active antibody, both binding sites are conditionally active, such as CAB EpCAM x CAB CD3+.

[0175] Multispecific antibodies can be conditionally active under a single, two, three, etc. condition. Thus, any one or more of the binding regions of a multispecific antibody can be conditionally active.

[0176] In certain embodiments, bispecific antibodies can bind to two different epitopes of EpCAM. Bispecific antibodies can also be used to localize cytotoxic drugs to cells expressing EpCAM. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0177] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, vol. 305, pp. 537-540, 1983), WO 93 / 08829 and Traunecker et al., EMBO J., vol. 10, pp. 3655-3659, 1991), and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies have been developed using a variety of techniques, including the manipulation of electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1), cross-linking of two or more antibodies or fragments thereof (see, e.g., U.S. Pat. No. 4,676,980; Brennan et al., Science, vol. 229, pp. 81-83, 1985); the use of leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., vol. 148, pp. 1547-1553, 1992); "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993); and the use of single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., vol. 90, pp. 6444-6448, 1993). al., J. Immunol., vol. 152, pp. 5368-5374, 1994); as well as the preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol., vol. 147, pp. 60-69, 1991.

[0178] Engineered antibodies with three or more functional antigen binding sites (such as "Octopus antibodies") are also included herein (see, e.g., U.S. Patent Application Publication No. 2006 / 0025576A1).

[0179] The anti-EpCAM antibodies or antibody fragments of the present disclosure can be produced using recombinant methods and compositions, which are described in detail in U.S. Patent Application Publication No. 2016 / 0017040.

[0180] The bispecific antibodies herein comprise an anti-EpCAM antibody disclosed herein in combination with an antibody that binds to a T lymphocyte antigen. In one embodiment, the antibody may comprise an anti-EpCAM portion comprising a combination of complementarity-determining regions as shown in Table 1. In another embodiment, the anti-EpCAM portion of the bispecific antibody comprises a heavy chain variable region of SEQ ID NO: 52 and a light chain variable region of any one of SEQ ID NOs: 53-69. In another embodiment, the anti-EpCAM portion of the bispecific antibody comprises a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of any one of SEQ ID NOs: 70-96. In some embodiments, the antibody that binds to a T lymphocyte antigen is an scFv antibody. In certain embodiments, the T lymphocyte antigen antibody may be an anti-CD3 antibody. In certain embodiments, the anti-CD3 antibody comprises a light chain variable region of SEQ ID NO: 101 and a heavy chain variable region of SEQ ID NO: 100. In a more specific embodiment, the anti-CD3 scFv antibody comprises SEQ ID NO: 99. Additional sequences of anti-CD3 antibodies useful in the multispecific antibodies of the present disclosure are described in WO 2019 / 241216.

[0181] In one embodiment, the bispecific antibody comprises an scFv antibody linked to the C-terminus of the light chain of an IgG molecule or a fragment of an IgG antibody (e.g., an (Fab')2 fragment), as shown in FIG. 3. In one embodiment, the IgG portion of the bispecific antibody can comprise any of the anti-EpCAM antibodies disclosed herein, and the scFv portion can be any of the anti-CD3 antibodies disclosed herein. In another embodiment, the IgG portion of the bispecific antibody can be any of the anti-CD3 antibodies disclosed herein, and the scFv antibody can be any of the anti-EpCAM antibodies disclosed herein. In a specific embodiment, the IgG portion of the bispecific antibody comprises a light chain variable region comprising SEQ ID NOs: 1-3 and a heavy chain variable region comprising SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 45, and the scFv portion comprises a light chain variable region of SEQ ID NO: 101 and a heavy chain variable region of SEQ ID NO: 100. In another embodiment, the IgG portion of the bispecific antibody comprises a light chain variable region of SEQ ID NO: 51 and a heavy chain variable region of SEQ ID NO: 91, and an scFv fragment of SEQ ID NO: 99.

[0182] In certain embodiments, any of the anti-EpCAM antibodies or antibody fragments provided herein can be used to detect the presence of EpCAM in a biological sample, either quantitatively or qualitatively. In certain embodiments, the biological sample comprises cells or tissues, such as breast, pancreatic, esophageal, lung, and / or brain cells or tissues.

[0183] Another aspect of the present disclosure relates to an anti-EpCAM antibody or antibody fragment of the present disclosure for diagnosing and / or monitoring cancer or other diseases in which the expression level of EpCAM is increased or decreased from normal physiological levels in at least one location in the body.

[0184] In one embodiment, an antibody or antibody fragment of the present disclosure can be labeled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule, or other label known in the art. For example, an antibody or antibody fragment of the present disclosure can be labeled with a radioactive molecule. For example, suitable radioactive molecules include, but are not limited to, radioactive atoms used in scintigraphy studies, e.g., 123 I, 124 I, 111In, 186 Re and 188 The antibodies or antibody fragments of the present disclosure may also be labeled with spin labels for nuclear magnetic resonance (NMR) imaging, such as iodine-123, iodine-131, indium II, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. After administration of the antibody, the distribution of the radiolabeled antibody within the patient's body is detected. Any suitable known method can be used. Some non-limiting examples include computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence, and ultrasound.

[0185] The antibodies or antibody fragments of the present disclosure may be useful in the diagnosis and staging of cancers and diseases associated with EpCAM overexpression, including squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, sarcoma, blood cancer (leukemia), astrocytoma, and various types of head and neck cancer, or other hyperproliferative diseases that express or overexpress EpCAM.

[0186] The antibodies or antibody fragments of the present disclosure may be useful for diagnosing diseases other than cancer in which EpCAM expression is increased or decreased. Either soluble or cellular forms of EpCAM can be used for such diagnosis. Typically, such diagnostic methods involve the use of a biological sample taken from a patient. Biological samples encompass a variety of sample types taken from a subject, which can be used in diagnostic or monitoring assays. Biological samples include, but are not limited to, blood and other liquid samples of biological origin, solid tissue samples such as biopsies or tissue cultures or cells derived therefrom, and their progeny. For example, biological samples include tissue samples taken from individuals suspected of having a cancer associated with EpCAM overexpression, and in preferred embodiments, cells obtained from glioblastoma, gastric cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, renal cancer, liver cancer, and endometrial cancer. Biological samples include clinical samples, cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0187] In certain embodiments, methods are provided for diagnosing cancer associated with EpCAM overexpression in a subject by detecting EpCAM on cells from the subject using an antibody of the present disclosure. In particular, the methods include: 1. contacting a biological sample from a subject with an antibody or antibody fragment of the present disclosure under conditions suitable for the antibody or antibody fragment to form a complex with cells expressing EpCAM in the biological sample; 2. Detecting and / or quantifying said complex, whereby detection of said complex indicates the presence of a cancer associated with EpCAM overexpression; may include:

[0188] To monitor the progression of cancer, the method can be repeated at various time points to determine whether binding of the antibody to the sample increases or decreases, which can then determine whether the cancer has progressed, regressed, or stabilized.

[0189] In certain embodiments, the present disclosure provides methods for diagnosing diseases associated with EpCAM expression or overexpression, including cancer, human immune disorders, thrombotic diseases (thrombosis and atherothrombosis), and cardiovascular diseases.

[0190] In one embodiment, an anti-EpCAM antibody or antibody fragment is provided for use in a diagnostic or detection method. In yet another aspect, a method for detecting the presence of EpCAM4 in a biological sample is provided. In yet another aspect, a method for quantifying the amount of EpCAM in a biological sample is provided. In certain embodiments, the method comprises contacting an anti-EpCAM antibody or antibody fragment described herein with a biological sample under conditions that allow the anti-EpCAM antibody or antibody fragment to bind to EpCAM, and detecting whether a complex is formed between the anti-EpCAM antibody or antibody fragment and EpCAM. Such a method can be performed in vitro or in vivo. In one embodiment, an anti-EpCAM4 antibody or antibody fragment is used to select subjects eligible for treatment. In some embodiments, the treatment comprises administering an anti-EpCAM antibody or antibody fragment to a subject.

[0191] In certain embodiments, a labeled anti-EpCAM antibody or antibody fragment is provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels) and moieties that are indirectly detected, such as through enzymatic reactions or molecular interactions (e.g., enzymes or ligands). Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C. 125 I, 3 H, 131Examples of suitable dyes include I, fluorescent dyes such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases (e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456)), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase), and heterocyclic oxidases such as glucose-6-phosphate dehydrogenase, uricase, and xanthine oxidase (coupled with an enzyme that uses hydrogen peroxide to oxidize a dye precursor, such as HRP, lactoperoxidase, or microperoxidase), biotin / avidin, spin labels, bacteriophage labels, and stable free radicals.

[0192] Anti-EpCAM antibodies or antibody fragments, and multispecific antibodies incorporating them, have cell-killing activity. This cell-killing activity extends to multiple different types of cell lines. Therefore, anti-EpCAM antibodies, fragments thereof, or multispecific antibodies may be useful in treating proliferative diseases associated with EpCAM expression. These antibodies, fragments, or multispecific antibodies may be used alone or in combination with any suitable drug or other conventional therapy.

[0193] The anti-EpCAM antibodies, antibody fragments, or multispecific antibodies of the present disclosure can be used to treat diseases associated with EpCAM expression, overexpression, or activation. There are no particular limitations on the types of cancers or tissues that can be treated, other than those requiring EpCAM expression. Examples include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, sarcoma, blood cancer (leukemia), astrocytoma, and various types of head and neck cancer. Specific cancers include glioma, gastric cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, renal cancer, liver cancer, and endometrial cancer.

[0194] The anti-EpCAM antibodies, antibody fragments, or multispecific antibodies of the present disclosure are potent activators of the innate immune response and may therefore be used to treat human immune disorders such as sepsis. The anti-EpCAM antibodies or antibody fragments of the present disclosure may also be used as adjuvants for immunization, such as vaccines, and as anti-infective agents against, for example, bacteria, viruses, and parasites.

[0195] In each of the embodiments of the methods of treatment described herein, the anti-EpCAM antibody, antibody fragment, or multispecific anti-EpCAM antibody or antibody fragment may be administered in a manner consistent with conventional techniques associated with the management of the disease or disorder for which treatment is sought. In accordance with the present disclosure, an effective amount of the antibody, antibody fragment, or multispecific antibody is administered to a subject in need of such treatment for a period of time and under conditions sufficient to prevent or treat the disease or disorder. Thus, one aspect of the present disclosure relates to a method of treating a disease associated with EpCAM expression, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody, antibody fragment, or multispecific antibody of the present disclosure.

[0196] For purposes of administration, the anti-EpCAM antibodies, antibody fragments, or multispecific antibodies of the present disclosure can be formulated as pharmaceutical compositions. Pharmaceutical compositions containing anti-EpCAM antibodies, antibody fragments, or multispecific antibodies can be formulated according to known methods for preparing pharmaceutical compositions. In such methods, the therapeutic molecule is typically combined with a mixture, solution, or composition that includes a pharmaceutically acceptable carrier.

[0197] A pharmaceutically acceptable carrier is a substance that can be tolerated by the recipient patient. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable pharmaceutically acceptable carriers are well known to those skilled in the art (see, for example, Gennaro (ed.), Reminton's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, etc.

[0198] The dosage form, administration route, dosage and regimen of the pharmaceutical composition will naturally depend on the condition to be treated, the severity of the condition, the age, weight and sex of the patient, etc. These considerations can be taken into account by those skilled in the art to formulate a suitable pharmaceutical composition. The pharmaceutical compositions herein can be formulated for topical, oral, parenteral, nasal, intravenous, intramuscular, subcutaneous or intraocular administration, etc.

[0199] Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier for an injectable preparation, in particular an isotonic sterile saline solution (such as mono- or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or a mixture of these salts) or a dry (in particular lyophilized) composition, which can be made up into an injectable solution by addition of, for example, sterile water or saline.

[0200] In some embodiments, a tonicity agent (sometimes known as a "stabilizer") is present to adjust or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules such as proteins and antibodies, these are often referred to as "stabilizers" because they can interact with the charged groups on amino acid side chains to reduce the likelihood of inter- and intra-molecular interactions. The tonicity agent may be present in any amount, for example, from 0.1 to 25% by weight of the pharmaceutical composition, e.g., from 1 to 5%. Tonicity agents include trihydric or higher sugar alcohols such as polyhydric sugar alcohols, e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0201] Other excipients include agents that may function as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. Such excipients include polyhydric sugar alcohols (as described herein); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myo-inisitose, myo-inisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycols, and the like. glycols; sulfur-containing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose); disaccharides (e.g., lactose, maltose, sucrose); trisaccharides such as raffinose; and polysaccharides such as dextrin or dextran.

[0202] Non-ionic surfactants or detergents (also known as "wetting agents") can be used to facilitate dissolution of the therapeutic agent and to protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stresses without denaturing the active therapeutic protein or antibody. Non-ionic surfactants can be present in a range of about 0.05 mg / mL to about 1.0 mg / mL, preferably about 0.07 mg / mL to about 0.2 mg / mL.

[0203] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyol, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyethylene stearate 40, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic surfactants that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic surfactants include benzalkonium chloride or benzethonium chloride.

[0204] The dosage used for administration may be adjusted depending on various parameters and in particular on the method of administration used, the pathology involved or alternatively the desired duration of treatment. To prepare a pharmaceutical composition, an effective amount of the antibody, antibody fragment or multispecific antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0205] Suitable dosage forms for injection include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage, and must be protected against the contaminating action of microorganisms such as bacteria and fungi.

[0206] Solutions of the active compound free base or pharmaceutically acceptable salts can be prepared in water with a suitable mixture of surfactants.Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof, and oils.Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0207] The anti-EpCAM antibodies, antibody fragments, or multispecific antibodies of the present disclosure can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, or procaine.

[0208] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is desirable to include isotonic agents (for example, sugars or sodium chloride). Prolonged absorption of injectable compositions can be achieved by using agents delaying absorption (for example, aluminum monostearate and gelatin) in the compositions.

[0209] Sterile injectable solutions are prepared by incorporating the anti-EpCAM antibody, antibody fragment or multispecific antibody described herein in the required amount in a suitable solvent together with one or more of the other ingredients listed above, and then optionally sterilizing by filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above. For sterile powders to prepare sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which yields a powder of the active ingredient and any other desired ingredients from the solution previously sterile-filtered.

[0210] The preparation of more concentrated solutions for direct injection is also contemplated, in which case dimethyl sulfoxide (DMSO) is used as the solvent, which is expected to result in extremely rapid penetration and delivery of high concentrations of the active ingredient to small tumor areas.

[0211] Upon formulation, solutions containing the anti-EpCAM antibody, antibody fragment, or multispecific antibody may be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. These formulations are readily administered in a variety of dosage forms, including injectable solutions of the type described herein.

[0212] For example, for parenteral administration using an aqueous solution, the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration, among others. In this regard, sterile aqueous vehicles that can be used will be well known to those skilled in the art in light of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of hypodermic infusion fluid or injected directly into the proposed injection site (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pages 1035-1038 and 1570-1580). Dosages will necessarily vary depending on the condition, weight, and / or sex of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for the individual subject.

[0213] The antibodies, antibody fragments, or multispecific antibodies disclosed herein can be formulated in therapeutic mixtures to deliver about 0.0001 to 10.0 milligrams, or about 0.001 to 5 milligrams, or about 0.001 to 1 milligram, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0, or even about 10 milligrams per dose. Multiple doses can also be administered at predetermined time intervals.

[0214] In certain embodiments, the use of liposomes and / or nanoparticles for the introduction of antibodies or antibody fragments into host cells is contemplated. The formation and use of liposomes and / or nanoparticles is well known to those of skill in the art.

[0215] Nanocapsules can generally encapsulate compounds stably and reproducibly. To avoid side effects due to intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) are generally designed using polymers that can be degraded in vivo. The present disclosure contemplates the use of biodegradable polyalkylcyanoacrylate nanoparticles that meet these requirements, and such particles can be easily manufactured.

[0216] Liposomes are formed from phospholipids dispersed in an aqueous medium, which spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs typically have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters between 200 and 500 Å, which contain aqueous solution in their cores. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0217] Pharmaceutical formulations comprising the anti-EpCAM antibodies, antibody fragments, or multispecific antibodies described herein are prepared by mixing such antibodies, antibody fragments, or multispecific antibodies having the desired purity, in the form of a lyophilized formulation or aqueous solution, with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A., ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants (such as ascorbic acid and methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight ( polypeptides (fewer than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0218] Examples of pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use thereof, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycan-degrading enzymes, such as chondroitinase.

[0219] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulation using a histidine acetate buffer.

[0220] The formulations described herein may also contain two or more active ingredients as needed for the particular indication being treated. Preferably, ingredients with complementary activities that do not adversely affect each other can be combined in a single formulation. For example, it may be desirable to provide an EGFR antagonist (e.g., erlotinib), an anti-angiogenic agent (e.g., a VEGF antagonist, which may be an anti-VEGF antibody), or a chemotherapeutic agent (e.g., a taxoid or platinum agent) in addition to the anti-EpCAM antibody, antibody fragment, or multispecific antibody of the present disclosure. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0221] Any of the anti-EpCAM antibodies, antibody fragments, or multispecific antibodies described herein can be used in methods of treatment. In one aspect, an anti-EpCAM antibody, antibody fragment, or multispecific antibody for use as a medicament is provided. In yet another aspect, an anti-EpCAM antibody or antibody fragment is provided for use in treating cancer (e.g., breast cancer, non-small cell lung cancer, pancreatic cancer, brain tumor, renal cancer, ovarian cancer, gastric cancer, leukemia, endometrial cancer, colon cancer, prostate cancer, thyroid cancer, liver cancer, osteosarcoma, and / or melanoma). In certain embodiments, an anti-EpCAM antibody, antibody fragment, or multispecific antibody for use in a method of treatment is provided. In certain embodiments, the present disclosure provides an anti-EpCAM antibody, antibody fragment, or multispecific antibody for use in a method of treating an individual having cancer, the method comprising administering to the individual an effective amount of an anti-EpCAM antibody, antibody fragment, or multispecific antibody. Additionally, the present disclosure provides anti-EpCAM antibodies, antibody fragments, or multispecific antibodies for use in inhibiting angiogenesis, inhibiting cell proliferation, inhibiting tumor vasculature (e.g., intratumoral or tumor-associated vasculature), and / or inhibiting tumor stromal function.

[0222] In yet another aspect, the disclosure provides use of an anti-EpCAM antibody or antibody fragment in the manufacture or preparation of a medicament. In one embodiment, the medicament is used in the treatment of cancer (in some embodiments, breast cancer, non-small cell lung cancer, pancreatic cancer, brain tumor, kidney cancer, ovarian cancer, gastric cancer, leukemia, endometrial cancer, colon cancer, prostate cancer, thyroid cancer, liver cancer, osteosarcoma, and / or melanoma). In another embodiment, the medicament is used in a method for treating cancer, comprising administering an effective amount of the medicament to an individual having cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one other therapeutic agent (e.g., as described herein). In yet another embodiment, the medicament is for inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature), and / or inhibiting tumor stromal function. In yet another embodiment, the pharmaceutical agent is for use in a method of inhibiting angiogenesis, inhibiting cell proliferation, inhibiting tumor vasculature (e.g., intratumoral or tumor-associated vasculature), and / or inhibiting tumor stromal function in an individual, the method comprising administering to the individual an amount of the pharmaceutical agent effective to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), inhibit the development of tumor vasculature (e.g., intratumoral or tumor-associated vasculature), and / or inhibit tumor stromal function. The "individual" in any of the above embodiments may be a human.

[0223] In yet another aspect, the disclosure provides pharmaceutical formulations comprising any of the anti-EpCAM antibodies, antibody fragments, or multispecific antibodies described herein, e.g., for use in any of the aforementioned methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-EpCAM antibodies, antibody fragments, or multispecific antibodies described herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-EpCAM antibodies, antibody fragments, or multispecific antibodies described herein and at least one additional therapeutic agent (e.g., as described below).

[0224] In any of the treatments described herein, the antibodies, antibody fragments, or multispecific antibodies of the present disclosure may be used in therapy alone or in combination with other agents. For example, the antibodies of the present disclosure may be co-administered with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-angiogenic agent. In certain embodiments, the additional therapeutic agent is a VEGF antagonist (in some embodiments, an anti-VEGF antibody, e.g., bevacizumab). In certain embodiments, the additional therapeutic agent is an EGFR antagonist (in some embodiments, erlotinib). In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent and / or a cytostatic agent. In certain embodiments, the additional therapeutic agent is a taxoid (e.g., paclitaxel) and / or a platinum agent (e.g., carboplatin). In certain embodiments, the additional therapeutic agent is an agent that enhances the patient's immunity or immune system.

[0225] The aforementioned combination therapies include combined administration (where two or more therapeutic agents are in the same or separate formulations) and separate administration, in which case the administration of the antibody, antibody fragment or multispecific antibody may occur before, simultaneously with and / or after the administration of the additional therapeutic agent and / or adjuvant. The antibody or antibody fragment may also be used in combination with radiation therapy and / or surgical intervention.

[0226] Anti-EpCAM antibodies, antibody fragments, or multispecific antibodies may be formulated, dosed, and administered in a manner consistent with the principles of good medical practice. Factors to consider in this regard include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of drug delivery, the method of administration, the administration schedule, and other factors known to physicians. The antibody, antibody fragment, or multispecific antibody may, optionally, but not necessarily, be formulated with one or more agents currently used to treat the disorder. The effective amount of such other agents will depend on the amount of antibody, antibody fragment, or multispecific antibody present in the formulation, the type of disease or treatment, and other factors discussed above. These may generally be used in the same dosages and via any route of administration as described herein, or in any dosage and via any route determined empirically / clinically appropriate.

[0227] The appropriate dosage of an antibody, antibody fragment, or multispecific antibody (used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease will vary depending on the type of disease being treated, the type of antibody, antibody fragment, or multispecific antibody, the severity and course of the disease, whether the antibody or antibody fragment is being administered for prophylactic or therapeutic purposes, previous treatment history, the patient's clinical history and response to the antibody, antibody fragment, or multispecific antibody, and the judgment of the attending physician. The antibody or antibody fragment is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, initial candidate doses for administration to a patient can range from about 1 μg to 40 mg of antibody or antibody fragment per kg of patient body weight, whether by one or more individual administrations or by continuous infusion, for example. For repeated administration over several days or longer, treatment is generally continued until a desired suppression of disease symptoms is achieved, depending on the condition. Such doses can be administered intermittently, for example every week or every three weeks (e.g., whereby the patient receives from about 2 to about 20, or for example about 6, doses of the antibody or antibody fragment). An initial high loading dose followed by one or more lower doses can be administered, although other dosage regimens are also useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0228] Enhancement of the host's immune function to combat tumors is a topic of growing interest. Conventional methods include (i) enhancing APCs, such as (a) injecting DNA encoding foreign MHC alloantigens into tumors, or (b) transfecting biopsied tumor cells with genes that increase the probability of tumor immune antigen recognition (e.g., immunostimulatory cytokines, GM-CSF, costimulatory molecules B7.1, B7.2), and (iii) adoptive cellular immunotherapy or treatment with activated tumor-specific T cells. Adoptive cellular immunotherapy involves isolating tumor-infiltrating host T lymphocytes and expanding the population in vitro, such as through stimulation with IL-2 or tumor, or both. Furthermore, isolated dysfunctional T cells can also be activated by in vitro application of the anti-PD-L1 antibodies of the present disclosure. Such activated T cells can then be readministered to the host. One or more of these methods can be used in combination with the administration of the antibodies, antibody fragments, or multispecific antibodies of the present disclosure.

[0229] Conventional cancer treatments include: (i) radiotherapy (e.g., radiotherapy, X-ray therapy, irradiation) or ionizing radiation, which aims to kill cancer cells and shrink tumors. Radiotherapy can be administered either externally via external beam radiation therapy (EBRT) or internally via brachytherapy; (ii) chemotherapy, i.e., the administration of cytotoxic drugs that act on rapidly dividing cells; (iii) targeted therapy, i.e., drugs that act specifically on dysregulated proteins in cancer cells (e.g., tyrosine kinase inhibitors: imatinib, gefitinib; monoclonal antibodies, photodynamic therapy); (iv) immunotherapy, i.e., enhancing the host's immune response (e.g., vaccines); (v) hormonal therapy, i.e., hormone blockade (e.g., when the tumor is hormone-sensitive); (vi) angiogenesis inhibitors, i.e., inhibiting blood vessel formation and growth; and (vii) palliative care, i.e., treatments aimed at improving the quality of care by reducing pain, nausea, vomiting, diarrhea, and bleeding. Analgesics such as morphine and oxycodone, and antiemetics such as ondansetron and aprepitant allow for more aggressive treatment regimens.

[0230] In treating cancer, any of the conventional therapies described for cancer treatment can be administered before, after, or simultaneously with the administration of the anti-EpCAM antibody, antibody fragment, or multispecific antibody disclosed herein. Furthermore, the anti-EpCAM antibody, antibody fragment, or multispecific antibody can be administered before, after, or simultaneously with conventional cancer therapies, such as administration of a tumor-binding antibody (e.g., a monoclonal antibody, a toxin-conjugated monoclonal antibody) and / or administration of a chemotherapeutic agent.

[0231] Another aspect of the present disclosure provides an article of manufacture containing an anti-EpCAM antibody, antibody fragment, or multispecific antibody and other substances useful for the treatment, prevention, and / or diagnosis of the aforementioned diseases. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV infusion bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition, alone or in combination with another composition, that is effective for the treatment, prevention, and / or diagnosis of a disease, and may have a sterile access port (e.g., the container may be an IV infusion bag or vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the antibody, antibody fragment, or multispecific antibody of the present disclosure. The label or package insert describes use of the composition for treating the selected condition. Additionally, an article of manufacture may include (a) a first container containing a composition comprising an anti-EpCAM antibody, antibody fragment, or multispecific antibody, and (b) a second container containing a composition comprising another cytotoxic or other therapeutic agent. The article of manufacture in this embodiment of the disclosure may further include a package insert indicating that the article may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer (e.g., bacteriostatic water for intravenous injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution). The article of manufacture may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0232] The present disclosure also provides kits comprising at least one antibody, antibody fragment, or multispecific antibody of the present disclosure. Kits comprising the antibody, antibody fragment, or multispecific antibody of the present disclosure are useful for detecting EpCAM expression (increase or decrease) or for therapeutic or diagnostic assays. Kits of the present disclosure may include antibodies bound to a solid support (e.g., tissue culture plate or beads (e.g., Sepharose beads)). Kits may be provided that include antibodies for detecting and quantifying EpCAM in vitro, for example, in ELISA or Western blot. Such antibodies useful for detection may be provided with a label, such as a fluorescent label or a radioactive label.

[0233] The kit may further include instructions for its use. In some embodiments, the instructions include those required for in vitro diagnostic kits by the U.S. Food and Drug Administration (FDA) or other appropriate agencies. In some embodiments, the kit includes one or more antibodies, antibody fragments, or multispecific antibodies. In other embodiments, the kit further includes one or more enzymes, enzyme inhibitors, or enzyme activators. In still other embodiments, the kit further includes one or more chromatography compounds. In yet other embodiments, the kit further includes one or more compounds used in sample preparation for spectrophotometric assays. In still other embodiments, the kit further includes a reference material for interpreting the presence or absence of EpCAM according to the intensity, color spectrum, or other physical property of the indicator.

[0234] The following examples are illustrative, but not limiting, of the anti-EPCAM antibodies of the present disclosure. Other suitable variations and modifications of the variety of conditions and parameters commonly used in the art that are obvious to those skilled in the art are within the scope of the present disclosure. [Example]

[0235] Examples 1 to 15 relating to the production of conditionally active antibodies are described in WO 2017 / 078839.

[0236] Examples 16-28 used an exemplary antibody (BA3182 or BAP150.31-BF45) comprising SEQ ID NOs: 98 and 99.

[0237] [Table 3] JPEG2026507181000006.jpg42170

[0238] Example 16. Affinity ELISA This example demonstrates the binding activity of the BA3182 bispecific antibody to recombinant human CD3 and human EpCAM or cyno EpCAM extracellular domain (ECD) using an enzyme-linked immunosorbent assay (ELISA). The ELISA assay was performed using buffers at pH 6.0 (tumor microenvironment pH) or pH 7.4 (normal physiological pH). Serially diluted BA3182 was added to wells coated with recombinant CD3 epsilon and delta heterodimer extracellular domains. Bound BA3182 was quantified using human or cyno EpCAM extracellular domain fused to mouse Fc and an anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP), which was then reacted with 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric substrate to generate a colored product. The absorbance at 450 nm (OD ) of each well was measured. 450The EC50 values ​​for BA3182 binding to both the human CD3 complex and human or cyno EpCAM were proportional to the amount of BA3182 bound to both the human CD3 complex and human or cyno EpCAM, with a dynamic range of 0.0847 to 50,000 pM. The EC50 values ​​for BA3182 binding to human CD3 and human or cyno EpCAM at different pH values ​​were calculated using a nonlinear fit model (variable slope, 4 parameters) implemented in GraphPad Prism™ software. The EC50 values ​​for BA3182 binding to human CD3 and human EpCAM at pH 6.0 were 297.83 pM and 4155.67 pM at pH 7.4. The EC50 values ​​for BA3182 binding to human CD3 and cyno EpCAM at pH 6.0 were 543.47 pM and 29415.33 pM at pH 7.4. The results demonstrate that the binding activity of BA3182 to CD3 and / or EpCAM at normal physiological pH (pH 7.4) is significantly weaker than the binding at the pH of the tumor microenvironment (pH 6.0).

[0239] material BA3182, 1.03 mg / mL antigen: Recombinant human CD3ε and δ heterodimer, BioVision, catalog #P11830-500; Lot number 7C19P11830. Recombinant human EpCAM fused to mouse Fc (human EpCAM-mFc), Evitria, lot number 12919-SEC. Recombinant cyno EpCAM fused to mouse Fc (cyno EpCAM-mFc), BioAtla, lot no. 20072. antibody: Goat anti-mouse HRP antibody, Promega, catalog #W402B, lot number 0000465785. reagent: Carbonate-bicarbonate buffer capsules, Sigma, catalog #C3041-100CAP, lot number SLBZ3401. 10x PBS, Gibco, catalog #70011-044, lot #2323767. Albumin, bovine (BSA), Sigma Aldrich, catalog #A9647-100G, lot number SLCH8436. Sodium bicarbonate (7.5% solution), Gibco, catalog #25082-094, lot #2336825. Tween-20, Sigma Aldrich, catalog #P9416-50ML, lot number SLCJ0231. TMB Chromogen Solution, Life Technology, Catalog #002023, Lot #05123211-7. 12N HCl:VWR, catalog #87003-251, lot #4118020. ELISA Assay Plates, Thermo Scientific Nunc, Catalog #269787, Lot #1223409. Carbonate-bicarbonate coating buffer: Dissolve the contents of one capsule of carbonate-bicarbonate buffer in 100 mL of sterile water. PBS buffer: Dilute 10x PBS buffer to 1x with distilled water. pH ELISA incubation buffer: Sodium bicarbonate and BSA are added to PBS buffer to contain a final concentration of 2.5 g / L sodium bicarbonate and 1% BSA, and the pH is adjusted to 6.0 or 7.4 using 1N HCl. pH Wash Buffer: Add sodium bicarbonate and Tween-20 to PBS buffer to a final concentration of 2.5 g / L sodium bicarbonate and 0.1% Tween-20, and adjust the pH to 6.0 or 7.4 with 1 N HCl. Stop solution: 1N HCl

[0240] Method A: A three-fold serial dilution of BA3182 was performed in incubation buffer (pH 6.0 and pH 7.4). The starting concentration of BA3182 for the complex hCD3 / hEpCAM-mFc was 50 nM, and the starting concentration of BA3182 for the complex hCD3 / cyno EpCAM-mFc was 150 nM.

[0241] ELISA assay Coat an ELISA plate with 100 μL / well of 1 μg / mL hCD3 antigen in bicarbonate coating buffer. Cover the plate with sealing film and incubate overnight at 4°C. Decant the plate and tap the residual liquid on a stack of paper towels. Wash the wells twice by dispensing 200 μL of pH 6.0 or pH 7.4 incubation buffer into each well and aspirate the contents thoroughly. Add 200 μL of pH 6.0 or pH 7.4 incubation buffer to each well. Cover with sealing film and place the plate on a plate shaker set at 200 rpm for 60 minutes at room temperature. Decant the plate and tap the residual liquid on a stack of paper towels. Prepare 3-fold serial dilutions of BA3182, starting at 50 nM or 150 nM in pH 6.0 or pH 7.4 incubation buffer. Add 100 μL / well of diluted BA3182 to the plate in duplicate. Cover the plate with sealing film and place on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. Decant the plate and gently tap the residual liquid on a stack of paper towels to remove any remaining liquid. Wash the wells three times by dispensing 200 μL of pH 6.0 or pH 7.4 wash buffer into each well and aspirate the contents thoroughly. Dilute human EpCAM-mFc to 1 μg / mL in incubation buffer (pH 6.0 or pH 7.4). Dilute cyno EpCAM-mFc to 2 μg / mL in incubation buffer (pH 6.0 or pH 7.4). Add 100 μL / well of the diluted 1 μg / mL human EpCAM-mFc or 2 μg / mL cyno EpCAM-mFc from above to each well. Cover with sealing film and place on a plate shaker set at 200 rpm at room temperature for 60 minutes. Decant the plate and gently tap the plate on a stack of paper towels to remove any residual liquid. Wash the wells three times by dispensing 200 μL of pH 6.0 or pH 7.4 wash buffer into each well and aspirate the contents thoroughly. Dilute goat anti-mouse HRP secondary antibody 1:2500 in pH 6.0 or pH 7.4 incubation buffer.Add 100 μL of the diluted goat anti-mouse HRP secondary antibody described above to each well. Cover with sealing film and place the plate on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. Decant the plate and gently tap the residual liquid on a stack of paper towels. Wash the wells three times by dispensing 200 μL of pH 6.0 or pH 7.4 wash buffer into each well and aspirate the contents completely. Dispense 50 μL / well of TMB substrate solution into all wells of the plate. Incubate wells containing human EpCAM-mFc for 5 minutes at room temperature. Incubate wells containing cyno EpCAM-mFc for 10 minutes. Add 50 μL / well of 1N HCl to all wells of the plate. Read the plate at 450 nm using a Molecular Devices SpectraMax i3X microplate reader. The mean EC50 values ​​for the binding activity of BA3182 to human CD3 / human EpCAM-mFc or cyno EpCAM-mFc were calculated using a nonlinear fit model (variable slope, 4 parameters) implemented in GraphPad Prism software version 9.2.0.

[0242] Results: A total of three independent ELISA experiments were performed. A summary of the EC50 values ​​of BA3182 for recombinant human CD3 / human EpCAM-mFc and human CD3 / cyno EpCAM-mFc at pH 6.0 and pH 7.4 is shown in Tables 3 and 4. Binding curves from a representative experiment are shown in Figures 6A and 6B. At pH 6.0, the EC50 values ​​of BA3182 measured by ELISA were 297.8 pM for human CD3 / human EpCAM-mFc and 543.37 pM for human CD3 / cyno EpCAM-mFc. At pH 7.4, the EC50 values ​​of BA3182 were 4155.67 pM for human CD3 / human EpCAM-mFc and 29415.33 pM for human CD3 / cyno EpCAM-mFc. Based on these results, it was observed that BA3182 bound to CD3 and EpCAM antigens with high affinity at the pH of the tumor microenvironment (p 6.0), but the affinity was significantly reduced at physiological pH (pH 7.4).

[0243] [Table 4]

[0244] [Table 5]

[0245] Example 17. pH Range ELISA This example demonstrates the binding activity of the BA3182 bispecific antibody to recombinant human CD3 and human EpCAM extracellular domains over a pH range (pH 6.0-pH 7.4) of buffers mimicking the pH of the tumor microenvironment (pH 6.0-pH 6.7) and normal physiological pH (pH 7.4). Binding activity was measured using a sandwich enzyme-linked immunosorbent assay (ELISA). Serially diluted BA3182 was bound to recombinant human CD3ε / δ heterodimer extracellular domains immobilized in wells. The amount of bound BA3182 was quantified using human EpCAM extracellular domain fused to mouse Fc and an anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP), which was then reacted with 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric substrate to generate a colored product. The absorbance at 450 nm (OD ) of each well was then measured. 450 ) is proportional to the amount of BA3182 bound to both the human CD3 complex and human EpCAM in different pH buffers. Data analysis showed that the pH inflection point (= 50% of binding activity compared to pH 6.0) of BA3182 was at pH 6.66, and 90% of binding activity occurred at pH 6.25 (the pH of the tumor microenvironment). At pH 7.4 (normal physiological pH), significantly weaker binding activity of BA3182 was detected.

[0246] material antigen: a) Recombinant human CD3ε and δ heterodimer, BioVision Cat#P1183-500, Lot#7C19P11830. b) Recombinant human EpCAM fused to mouse Fc (EpCAM-mFc), Evitria, lot number 12919-SEC. Antigen coating buffer, distilled water, Millipore. Goat anti-mouse HRP-conjugated antibody, Promega, Cat#W402B, Lot#0000465785. Sodium bicarbonate, Sigma, Cat# S5761-500G, Lot# BCCD6088. PBS (1X): Cellgro, Cat# R21-040-CV, Lot# 17321021. Tween-20: Sigma, Cat# P1379-500ML, Lot# SLBS7482. Albumin, Bovine (BSA), VWR, Cat# 0332, Lot# 20D0656194. TMB Chromogen Solution, Life Technology, Cat# 002023, Lot# 08228211-7. HCl, Titansci, Cat# G81788B, Lot# P1972715. ELISA assay plates, Corning, Cat# 42592, Lot# 00821030. PBS buffer: 0.144g / L KH2PO4, NaCl9g / L, 0.795g / L Na2HPO4pH7.4 pH ELISA incubation buffer, pH 6.0, 6.2, 6.5, 6.7, 7.0, and 7.4: Add 0.1 g sodium bicarbonate and 0.4 g BSA to 40 mL of 1x PBS buffer. Adjust the pH to 6.0, 6.2, 6.5, 6.7, 7.0, and 7.4 with 1N HCl. pH ELISA wash buffer pH 6.0, 6.2, 6.5, 6.7, 7.0, 7.4: To 60 mL of 1x PBS buffer, add 0.15 g sodium bicarbonate, 0.6 g BSA, and 0.1% Tween-20. Adjust the pH to 6.0, 6.2, 6.5, 6.7, 7.0, and 7.4 with 1N HCl. Stop solution: 1N HCl, add 83.3 mL to 1 L of distilled water

[0247] method EA1 was initially diluted to 100 nM and then diluted to 1.5 nM in incubation buffers of various pHs. An ELISA plate was coated with 100 μL of recombinant CD3ε and δ complex antigen at 1 μg / mL in distilled water. The plate was covered with sealing film and incubated overnight at 4°C. The plate was decanted and the residual liquid was removed by tapping on a stack of paper towels. The wells were washed twice by dispensing 200 μL of various pH incubation buffers into the wells and the contents were aspirated completely. 200 μL of various pH incubation buffers were added to the wells, covered with sealing film, and the plate was placed on a plate shaker (set at 200 rpm) for 60 minutes at room temperature. The plate was decanted and the residual liquid was removed by tapping on a stack of paper towels. The test substances were serially diluted to 1.5 nM in the incubation buffers of various pHs. BA3182 was added to the plate at 100 μL / well. Cover with sealing film and place the plate on a plate shaker (set to 200 rpm) for 60 minutes at room temperature. Decant the plate and gently tap the residual liquid on a stack of paper towels. Wash the wells three times by dispensing 200 μL of the various pH incubation buffers into the wells and aspirate the contents completely. Dilute human EpCAM-mFc to 1 μg / mL in the various pH incubation buffers. Add 100 μL / well of 1 μg / mL human EpCAM-mFc diluted in the various pH incubation buffers to each well. Cover with sealing film and place the plate on a plate shaker (set to 200 rpm) for 60 minutes at room temperature. Decant the plate and gently tap the residual liquid on a stack of paper towels. Wash the wells three times by dispensing 200 μL of the various pH incubation buffers into the wells and aspirate the contents completely. Dilute the anti-mouse IgG HRP secondary antibody 1:2500 in various pH incubation buffers. Add 100 μL of the anti-mouse IgG HRP secondary antibody diluted in various pH incubation buffers to each well. Cover with sealing film and place the plate on a plate shaker (set at 200 rpm) at room temperature for 60 minutes.Decant the plate and gently tap the residual liquid on a stack of paper towels. Wash the wells three times by dispensing 200 μL of the various pH incubation buffers into the wells and aspirate the contents completely. Dispense 5 μL of TMB substrate solution into all wells at a rate of 1 μL per well. Incubate at room temperature for 5 minutes. Add 50 μL of 1 N HCl to all wells at a rate of 1 μL per well. Read the plate at 450 nm using a Molecular Devices SpectraMax 190 microplate reader.

[0248] Data analysis: The mean OD values ​​(from two replicates) were plotted against each pH point tested using GraphPad Prism software. EC50 and EC90 were calculated using a four-parameter curve fit with variable slope. The inflection point of the pH curve where 50% binding activity is observed is equal to the EC50 value in the fitting equation. Binding activity at pH 6.0 was set as 100%. The pH value showing 90% binding activity was interpolated from the fitted curve using nonlinear regression to find ECanything with the parameter set to EC90.

[0249] Results: A summary of the binding activity of BA3182 to recombinant human CD3 and human EpCAM in various pH buffers is shown in Table 5. Binding curves from a representative experiment are shown in Figure 7. The pH inflection point for BA3182 binding activity was pH 6.66. The pH inflection point is the pH value at which 50% of the pH-dependent binding of BA3182 was observed. 90% of the binding activity of BA3182 was achieved at pH 6.25 (tumor microenvironment pH). Furthermore, weak binding activity of BA3182 was detected at normal physiological pH (pH 7.4) (Figure 7).

[0250] [Table 6]

[0251] Example 18. Cross-species affinity ELISA This example demonstrates the binding activity of the BA3182 bispecific antibody to recombinant human CD3 and human, cynomolgus monkey (cyno), rat, and mouse EpCAM extracellular domains. The binding activity of BA3182 to human CD3 and EpCAM antigens from different species was assessed using a sandwich enzyme-linked immunosorbent assay (ELISA). The ELISA assay was performed using assay buffers at pH 6.0 (tumor microenvironment pH) and pH 7.4 (normal physiological pH). BA3182 at a concentration of 1.5 nM was added to wells containing immobilized recombinant human CD3ε / δ heterodimer extracellular domains. The amount of bound BA3182 was quantified using human, cyno, rat, or mouse EpCAM extracellular domain fused to mouse Fc (EpCAM-mFc) and an anti-mouse IgG antibody conjugated with horseradish peroxidase (HRP), which was then reacted with TMB colorimetric substrate to generate a colored product. The absorbance at 450 nm (OD ) of each well was measured. 450 ) was proportional to the amount of BA3182 bound to both the human CD3 complex and the EpCAM antigen. BA3182 exhibited strong binding activity to the human CD3 complex and human or cyno EpCAM at pH 6.0, but weak binding activity at pH 7.4. For rat and mouse EpCAM, BA3182 showed very low or no binding activity at any pH.

[0252] material Antibody:BA3182 antigen: a) Recombinant human CD3ε and δ heterodimer, BioVision, Cat#P1183-500, Lot#7C19P11830. b) Recombinant human EpCAM fused to mouse Fc (human EpCAM-mFc), Evitria, lot number 12919-SEC. c) Recombinant cyno EpCAM fused to mouse Fc (cyno EpCAM-mFc), BioAtla, lot number 22007. d) Recombinant rat EpCAM fused to mouse Fc (rat EpCAM-mFc), BioAtla, lot number 22009. e) Mouse EpCAM fused to mouse Fc (mouse EpCAM-mFc), BioAtla, lot number 22011. Antigen coating buffer, distilled water, Millipore. Goat anti-mouse HRP-conjugated antibody, Promega, Cat#W402B, Lot#0000465785. PBS (1X): Cellgro, Cat# R21-040-CV, Lot# 17321021. Albumin, Bovine (BSA), VWR Cat#0332, Lot#20D0656194. Sodium bicarbonate, Sigma Cat#S5761-500G, Lot# BCCD6088. Tween-20, Sigma Cat# P1379-500ML, Lot# SLBS7482. TMB Chromogen Solution, Life Technology Cat#002023, Lot#08228211-7. HCl, Titansci Cat# G81788B, Lot# P1972715. ELISA assay plates, Corning, Cat# 42592, Lot# 01919010. pH meter, Alalis pH400. Shaker, Kylin-Bell TS-2. Plate reader, Thermo Fisher Multiskan™ Sky 51119770DP, SN: 1530-800210C. PBS buffer: 0.144 g / L KH2PO4, 9 g / L NaCl, 0.795 g / L Na2HPO4 pH 7.4. pH ELISA incubation buffer: Add 0.5g sodium bicarbonate and 2g BSA to 200mL of 1x PBS buffer. Adjust the pH to 6.0 or 7.4 with 1N HCl. pH ELISA wash buffer: Add 0.5 g of sodium bicarbonate and 0.1% Tween-20 to 200 mL of 1x PBS buffer. Adjust the pH to 6.0 or 7.4 with 1N HCl. Stop solution: Add 83.3 mL of 1 N HCl to 1 L of distilled water.

[0253] method BA3182 was first diluted to 100 nM in PBS, then to 1.5 nM in the pH 6.0 and pH 7.4 incubation buffers. ELISA plates were coated with 100 μL of 1 μg / mL recombinant CD3ε and δ complex antigen in distilled water. The plates were covered with sealing film and incubated overnight at 4°C. The plates were decanted and tapped on a stack of paper towels to remove residual liquid. The wells were washed twice by dispensing 200 μL of pH 6.0 or pH 7.4 incubation buffer into each well and the contents were aspirated thoroughly. 200 μL of pH 6.0 or pH 7.4 incubation buffer was added to each well, covered with sealing film, and the plates were placed on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. The plates were decanted and tapped on a stack of paper towels to remove residual liquid. Serially dilute the test substance to 1.5 nM in pH 6.0 or pH 7.4 incubation buffer. Add 100 μL / well of the diluted test substance to the plate. Cover with sealing film and place the plate on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. Decant the plate and gently tap the residual liquid on a stack of paper towels. Wash the wells three times by dispensing 200 μL of pH 6.0 or pH 7.4 wash buffer into each well and thoroughly aspirate the contents. Dilute human, cyno, rat, or mouse EpCAM-mFc to 1 μg / mL in pH 6.0 or pH 7.4 incubation buffer. Add 100 μL / well of 1 μg / mL human, cyno, rat, or mouse EpCAM-mFc diluted in pH 6.0 or pH 7.4 incubation buffer to each well. Cover with sealing film and place the plate on a plate shaker (set at 200 rpm) for 60 minutes at room temperature. Decant the plate and gently tap the plate on a stack of paper towels to remove any residual liquid. Wash the wells three times by dispensing 200 μL of pH 6.0 or pH 7.4 wash buffer into each well and aspirate the contents thoroughly. Dilute the anti-mouse IgG HRP secondary antibody 1:2500 in pH 6.0 or pH 7.4 incubation buffer.Add 10 μL of anti-mouse IgG HRP secondary antibody diluted in various pH incubation buffers to each well. Cover with sealing film and place the plate on a plate shaker (set at 200 rpm) for 60 minutes at room temperature. Decant the plate and gently tap the plate on a stack of paper towels to remove any residual liquid. Wash the wells three times by dispensing 200 μL of pH 6.0 or pH 7.4 wash buffer into each well and aspirate the contents thoroughly. Dispense 50 μL of TMB substrate solution into all wells of the plate at a rate of 1 / well. Incubate at room temperature for 5 minutes. Add 50 μL of 1 N HCl into all wells of the plate at a rate of 1 / well. Read the plate at 450 nm using a microplate reader.

[0254] Results: A total of three independent ELISA experiments were performed. Figure 8 shows the results of a representative experiment demonstrating the binding activity of BA3182 to recombinant human CD3 and human, cyno, rat, or mouse EpCAM at pH 6.0 and pH 7.4. BA3182 exhibited strong binding activity to human CD3 and human or cyno EpCAM at the tumor environment pH (pH 6.0), but much lower binding activity at pH 7.4. At both pH values, BA3182 binding to rat and mouse EpCAM was weak or not observed.

[0255] Example 19. Specificity ELISA This example demonstrates the binding activity of the BA3182 bispecific antibody to recombinant human CD3, human EpCAM, human trophoblast cell surface antigen 2 (Trop2), and an unrelated human antigen. Trop2 and EpCAM belong to the tumor-associated calcium signaling gene family. They share high sequence homology in their extracellular and transmembrane domains. The undisclosed unrelated human antigen has no sequence similarity to the human EpCAM molecule. The binding activity of BA3182 to these antigens was assessed by affinity assays and sandwich enzyme-linked immunosorbent assays (ELISAs). Assays were performed using buffers at pH 6.0 (tumor microenvironment pH) or pH 7.4 (normal physiological pH). BA3182 was captured by recombinant human EpCAM or Trop2 (both fused to a His tag) immobilized in the wells, followed by detection with horseradish peroxidase (HRP)-conjugated anti-human IgG antibodies in affinity ELISA assays. In the sandwich ELISA assay, BA3182 was captured by recombinant human CD3ε / δ heterodimer extracellular domain immobilized in the wells, followed by the addition of human EpCAM, human Trop2, or an unrelated antigen (all fused to mouse Fc). Bound complexes were detected with an HRP-conjugated anti-mouse IgG antibody, which reacted with 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric substrate to generate a colored product. The absorbance at 450 nm (OD ) in each well was measured. 450 ) is proportional to the amount of EpCAM, Trop2, or unrelated antigen protein. For human Trop2, little or no binding was observed in either pH 6.0 or pH 7.4 buffer. To confirm binding affinity to the human Trop2 antigen regardless of pH buffer, anti-human Trop2-specific antibodies (affinity ELISA assay) or bispecific antibodies (sandwich ELISA assay) were included as positive controls. Similarly, no binding of BA3182 to unrelated antigens was observed. Thus, the binding activity of the BA3182 bispecific antibody to recombinant human EpCAM was confirmed to be target-specific.

[0256] material Antibody:BA3182 antigen: a) Recombinant human CD3ε and δ heterodimer, BioVision Cat#P1183-500, Lot#7C19P11830. b) Recombinant human EpCAM fused to mouse Fc, Evitria, lot number 12919-SEC. c) Recombinant human Trop2 fused to mouse Fc, BioAtla, lot no. 21042. d) Recombinant unrelated human antigen fused to mouse Fc, BioAtla, lot no. 21044. e) Recombinant human EpCAM fused to a his tag, BioAtla, lot no. 21058. f) Fusion recombinant human Trop2 fused with a his tag, BioAtla, lot no. 19044. Positive control antibody against Trop2, BioAtla, lot number PB01. Positive control bispecific antibody against Trop2, BioAtla, lot number 860650. Positive control bispecific antibody against an unrelated human antigen, BioAtla, lot number 21-10458. Antigen coating buffer, distilled water, Millipore. Carbonate-bicarbonate buffer capsules, Sigma, catalog #C3041-100CAP, lot number SLBZ3401. Goat anti-human IgG HRP-conjugated antibody, Promega, Cat#W403B, Lot#0000423844 Goat anti-mouse IgG HRP antibody, Promega, Cat#W402B, Lot#0000465785. PBS (1x): Cellgro, Cat# R21-040-CV, Lot# 17321021. Albumin, Bovine (BSA), VWR Cat#0332, Lot#20D0656194. Sodium Bicarbonate, Sigma Cat#S5761-500G, Lot#BCCD6088. Tween-20, Sigma Ca#P1379-500ML, Lot#SLBS7482. TMB Chromogen Solution, Life Technology Cat#002023, Lot#08228211-7. HCl, Titansci Cat# G81788B, Lot# P1972715. ELISA Assay Plates, Corning Cat#42592, Lot#01919010. pH meter, Alalis pH400. Shaker, Kylin-Bell TS-2. Plate reader, Thermo Fisher Multiskan™ Sky 51119770DP, SN: 1530-800210C. PBS buffer: 0.144 g / L KH2PO4, 9 g / L NaCl, 0.795 g / L Na2HPO4, pH 7.4. Carbonate-bicarbonate coating buffer: Dissolve the contents of one capsule of carbonate-bicarbonate buffer in 100 mL of sterile water. pH ELISA incubation buffer: Add 0.5g sodium bicarbonate and 2g BSA to 200mL of 1x PBS buffer. Adjust the pH to 6.0 or 7.4 with 1N HCl. pH ELISA Wash Buffer: Add 0.5g sodium bicarbonate and 0.1% Tween-20 to 200mL of 1x PBS buffer. Adjust the pH to 6.0 or 7.4 with 1N HCl. Stop solution: Add 83.3 mL of 1 N HCl to 1 L of distilled water.

[0257] method BA3182 was initially diluted to 100 nM in PBS, then diluted to 2.5 nM for the related family ELISA and 1.5 nM for the unrelated antigen ELISA in pH 6.0 and pH 7.4 incubation buffers. Affinity ELISA plates were coated with 100 μL of 1 μg / mL recombinant human EpCAM or Trop2 antigen in carbonate-bicarbonate coating buffer. Sandwich ELISA plates were coated with 100 μL of 1 μg / mL human recombinant CD3ε and δ complex antigen in distilled water. Plates were covered with sealing film and incubated overnight at 4°C. The plates were decanted and gently tapped on a stack of paper towels to remove residual liquid. Wells were washed twice by dispensing 200 μL of pH 6.0 or pH 7.4 incubation buffer into each well and completely aspirating the contents. Add 200 μL of pH 6.0 or pH 7.4 incubation buffer to each well, cover with sealing film, and place the plate on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. Decant the plate and gently tap the residual liquid on a stack of paper towels. Serially dilute the test substance to 2.5 nM, 1.5 nM, or 0.5 nM in pH 6.0 or pH 7.4 incubation buffer. Add 100 μL / well of diluted BA3182 to the plate. Cover with sealing film and place the plate on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. Decant the plate and gently tap the residual liquid on a stack of paper towels. Wash the wells three times by dispensing 200 μL of pH 6.0 or pH 7.4 wash buffer into each well and completely aspirate the contents. For affinity ELISAs, dilute anti-human IgG HRP secondary antibody 1:2500 in pH 6.0 or pH 7.4 incubation buffer. Add 100 μL of anti-human IgG HRP secondary antibody diluted in various pH incubation buffers to each well. For sandwich ELISAs, dilute human EpCAM-mFc, human Trop2-mFc, or unrelated human antigen-mFc to 1 μg / mL or 2 μg / mL in pH 6.0 or pH 7.4 incubation buffer.Add 100 μL / well of 1 μg / mL human EpCAM-mFc, 2 μg / mL human Trop2-mFc, or unrelated human antigen-mFc diluted as described above. Cover with sealing film and place the plate on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. Decant the plate and gently tap the plate on a stack of paper towels to remove any residual liquid. Wash the wells three times by dispensing 200 μL of pH 6.0 or pH 7.4 wash buffer into each well and aspirate the contents thoroughly. For sandwich ELISA, dilute anti-mouse IgG HRP secondary antibody 1:2500 in pH 6.0 or pH 7.4 incubation buffer. Add 100 μL of anti-mouse IgG HRP secondary antibody diluted in various pH incubation buffers to each well. Cover with sealing film and place the plate on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. Decant the plate and gently tap the remaining liquid on a stack of paper towels. Wash the wells three times by dispensing 200 μL of pH 6.0 or pH 7.4 wash buffer into each well and aspirate the contents completely. Dispense 50 μL of TMB substrate solution into all wells of the plate at a rate of 1 / well. Incubate for 5 minutes at room temperature. Add 50 μL of 1 N HCl to all wells of the plate at a rate of 1 / well. Read the plate at 450 nm using a microplate reader.

[0258] Results: A total of three independent ELISA experiments were performed with duplicate samples. Representative experimental results demonstrating the binding activity of BA3182 to recombinant human CD3 and human EpCAM or human Trop2 ECD at pH 6.0 and pH 7.4 are shown in Figures 9A and 9B. The binding activity of BA3182 to recombinant human CD3 and human EpCAM or an unrelated human antigen at pH 6.0 and pH 7.4 is shown in Figures 10A and 10B. The binding activity of BA3182 to recombinant human EpCAM or human Trop2 ECD at pH 6.0 and pH 7.4 is shown in Figures 11A and 11B.

[0259] BA3182 exhibited high binding activity to human EpCAM (Figures 9A, 10A, and 11A) at pH 6.0 and lower binding at pH 7.4. BA3182 did not exhibit binding to human Trop2 (Figures 9B and 11B) or an unrelated human antigen (Figure 10B) at either pH 6.0 or pH 7.4. This data indicates that BA3182 has high specificity for human EpCAM and CD3.

[0260] Example 20. Binding of BA3182 to EpCAM- or CD3-expressing cells analyzed by FACS. This example demonstrates the binding activity of the BA3182 bispecific antibody to EpCAM- or CD3-expressing cells. The binding activity of BA3182 to EpCAM-expressing cells was evaluated using CHO cells expressing human EpCAM, CHO cells expressing cynoEpCAM, and HCT116 cells. The binding activity of BA3182 to CD3-expressing cells was evaluated using Jurkat T cells and peripheral blood mononuclear cells (PBMCs) (human, cyno, rat, and dog). The above cells were incubated with various concentrations of BA3182 at pH 6.0 (tumor microenvironment pH) and pH 7.4 (normal physiological pH). Cell-bound BA3182 was quantified using an anti-human IgG antibody conjugated to Alexa Fluor 488 (AF488). Stained cells were analyzed by fluorescence-activated cell sorting (FACS) to measure the median fluorescence intensity (MFI), which is proportional to the amount of cell-bound BA3182. At each pH, ​​the MFI values ​​and corresponding BA3182 concentrations were analyzed using GraphPad Prism software. A nonlinear four-parameter curve with variable slope was used to determine the EC50 values ​​for BA3182 binding activity to EpCAM and CD3 molecules expressed on the cell surface under both pH 6.0 and pH 7.4 conditions. The average EC50 values ​​for BA3182 binding to EpCAM- and CD3-expressing cells are shown in Tables 6 and 7. Overall, at pH 6.0, BA3182 bound with high affinity to human and cynoEpCAM and CD3 antigens expressed on the cell surface. At pH 7.4, a decrease in the affinity of BA3182 for these molecules (especially for the CD3 molecule) was observed. No BA3182 binding was observed to rat, mouse, or canine PBMCs. The homology between human and rat, mouse, or canine CD3 molecules is low (less than 60%). This result suggests that the CD3 epitope of BA3182 is not present in rat, mouse, or canine CD3 antigens.

[0261] material BA3182 1.03mg / mL. The isotype control was from Evitria (Zurich, Switzerland), lot number #10229, 3 mg / mL. CHO-hEpCAM: CHO-S cells transfected to stably express human EpCAM (synthesized and constructed at BioAtla), ThermoFisher Cat#R80007, clone #32. CHO-cynoEpCAM: CHO-S cells transfected to stably express cynomolgus monkey EPCAM (synthesized and constructed at BioAtla), ThermoFisher Cat#R80007, clone#3. HCT116: Human colon cancer cells, ATCC, Cat#CCL-247(TM). Jurkat, human T lymphocyte cells, ATCC, Cat#TIB-152, clone E6-1. Human PBMC: Precision for Medicine, Cat#39000, Lot#201013292. Cyno PBMC: Worldwide Primates, Cat#CA-10, Lot#C0767-22. SD rat PBMC: Iqbiosciences, Cat# IQB-RPB101, Lot# P20K0105. Balb / c mouse PBMC: Iqbiosciences, Cat# IQB-MPB101, Lot# P21A0603. Canine (Beagle) PBMC: Iqbiosciences, Cat# IQB-CPB102, Lot# P21C2303. Mouse anti-hEpCAM conjugated to PE: BioLegend, Cat#324206, clone 9C4, lot#B222943. Mouse IgG2b isotype conjugated to PE: BioLegend, Cat#400314, clone MPC-11, lot#B214529. Goat anti-human IgG AF488 antibody: Thermo Fisher, Cat# A11013, Lot# 2110842. PBS: 10x, Thermo Fisher, Cat# 14190-144, Lot# 2152877. Cell culture grade water: Cytiva Cat# SH30529.03, Lot# AH30009593. Trypan blue staining solution: Thermo Fisher, Cat# 15250-061, Lot# 1861515. BSA: Sigma, Cat# A9647, Lot# SLBV4996. TrypLE™ Express Enzyme (1X): Thermo Fisher, Cat# 12605-10, Lot# 2193025 FBS: Thermo Fisher, Cat# 16140-011, Lot# 2372673RP. McCoy's 5A: Thermo Fisher, Cat#16600-082, Lot#232363. DMEM: Thermo Fisher, Cat# 11965-084, Lot# 2346179. RPMI-1640: Thermo Fisher, Cat# 11875-085, Lot# 2463433. 100×NEAA: Thermo Fisher, Cat# 11140-050, Lot 2337217. 100 mM Sodium Pyruvate: Thermo Fisher, Cat# 11360-070, Lot# 2323639. G418: Invivogen, Cat# ant-gn-5, Lot# GNL-38-06A. Sodium Bicarbonate: Thermo Fisher, Cat# 25080-094, Lot# 2039755. 12N HCl:VWR, Cat#87003-251, Lot#4118020. T-150 tissue culture flask: Corning Cat#430825. 50mL conical tubes: BioPioneer Cat# CNT-50, Lot# 10272021. 96-well U-bottom plates: Greiner bio-one, Cat# 650101, Lot# B17033C7. pH meter: ORION STAR A111, Thermo Fisher, serial #J17162 Shaker: MaxQ 2000. Flow cytometer: ACEA NovoCyte 20602. PBS buffer: Add 100 mL of 10x PBS buffer to 900 mL of sterile water. Wash buffer, pH 6.0: Add 10 mL of 75 g / mL sodium bicarbonate solution to 290 mL of 1x PBS buffer. Adjust the pH to 6.0 using 6N or 0.6N HCl. Wash buffer, pH 7.4: Add 10 mL of 75 g / mL sodium bicarbonate solution to 290 mL of 1x PBS buffer. Adjust the pH to 7.4 using 6N or 0.6N HCl. FACS buffer, pH 6.0: Add 1.5 mL of 30% BSA to 43.5 mL of wash buffer. Adjust the pH to 6.0 using 6N or 0.6N HCl. FACS buffer, pH 7.4: Add 1.5 mL of 30% BSA to 43.5 mL of wash buffer. Adjust the pH to 7.4 using 6 N or 0.6 N HCl. CHO-hEpCAM / CHO-cynoEpCAM medium: DMEM + 1x NEAA (0.1 mM non-essential amino acid solution) + 1x sodium pyruvate (1 mM) + 10% FBS + 1 mg / mL G418. HCT116 medium: McCoy's 5A+10%FBS. Jurkat medium: RPMI-1640 + 10% FBS.

[0262] method Cell Culture: CHO-hEpCAM, CHO-cynoEpCAM, HCT116, and Jurkat cells were maintained in the indicated medium and routinely passaged twice weekly. Cells were harvested during exponential growth phase for FACS analysis. Frozen human, cyno, rat, mouse, and canine PBMCs were freshly thawed in RPMI-1640 / 10% FBS medium and analyzed by FACS.

[0263] cell staining On the day of FACS analysis, remove and discard the medium. Wash the cell layer briefly with PBS solution. Add 3 mL of TrypLE™ Express enzyme solution to each T-150 flask. Wait until the cell layer is dispersed. Add 7 mL of medium to the flask and resuspend the cells by gentle pipetting. Pool the cells and transfer the cell suspension to a 50 mL conical tube. Count the cell number using trypan blue staining and then centrifuge at 2000 rpm for 3 minutes at 4°C. Wash the cells once with PBS and then resuspend 3 x 10 5 Transfer cells to an Eppendorf tube. Add 2 μL of mouse anti-hEpCAM antibody (PE-conjugated mouse IgG) or PE-isotype mouse IgG to 100 μL of PBS containing 1% BSA. Add 100 μL of diluted PE antibody per tube and shake at 100 RPM on ice for 1 hour, protected from light. Wash cells three times with 150 μL of PBS. Fix cells with 4% PFA for 10 minutes at room temperature, then wash once with PBS. Suspend cells in 100 μL of PBS and analyze using a NovoCyte flow cytometer. BD Quantibrite™ PE bead standards were used to estimate the number of EpCAM molecules on the cell surface.

[0264] Binding analysis of BA3182 to EpCAM expressed in CHO-hEpCAM, CHO-cynoEpCAM, and HCT116 cells Harvest the cells as described in the "Cell Staining" section above and wash the cells once with PBS. The cell concentration is adjusted to 3 x 10 in pH 6.0 or pH 7.4 wash buffer. 6 Adjust to 3 x 10 cells / mL in 100 μL of pH 6.0 or pH 7.4 wash buffer. 5Aliquot the cells and transfer to a 96-well U-bottom plate. Spin down the cells and discard the buffer. Prepare 3-fold serial dilutions of the antibody in pH 6.0 or pH 7.4 FACS buffer, starting at 1500 nM for CHO-cynoEpCAM or 500 nM for CHO-hEpCAM and HCT116. Add 100 μL of the diluted test substance to the cells at 100 μL / well, mix gently, and then incubate on ice for 1 hour with shaking (100 rpm). Centrifuge the cells at 2000 rpm for 3 minutes at 4°C. Wash the cells twice with 150 μL of pH 6.0 or pH 7.4 wash buffer. Dilute goat anti-human IgG AF488 antibody 1:300 in pH 6.0 or pH 7.4 FACS buffer. Add 100 μL of the diluted antibody from the above step to the cells and incubate on ice for 45 minutes, protected from light. The cells are pelleted and washed three times with 150 μL of pH 6.0 or pH 7.4 wash buffer. The cells are fixed with 4% paraformaldehyde (PFA) diluted in 1× PBS for 10 minutes at room temperature, and then washed with 1× PBS. The cells are resuspended in 100 μL of 1× PBS. The cells are analyzed using a NovoCyte flow cytometer and software. 20,000 events are acquired for each sample.

[0265] Binding analysis of BA3182 to CD3 expressed on Jurkat T cells, human, cyno, rat, mouse, and dog PBMCs. Prepare a cell suspension as described in the "Cell Culture" section above. Wash the cells once with PBS. Adjust the cell concentration to 3 x 10 in pH 6.0 or pH 7.4 wash buffer. 6 Adjust to 3 x 10 cells / mL in 100 μL of pH 6.0 or pH 7.4 wash buffer. 5Aliquot the cells and transfer to a 96-well U-bottom plate. Spin down the cells and discard the buffer. Prepare 3-fold serial dilutions of the antibody in pH 6.0 or pH 7.4 FACS buffer, starting at 2500 nM. Add 100 μL / well of the diluted antibody to the cells, mix gently, and then incubate on ice with shaking (100 rpm) for 1 hour. Centrifuge the cells for 3 minutes at 2000 rpm at 4°C. Wash the cells twice with 150 μL of pH 6.0 or pH 7.4 wash buffer. Dilute goat anti-human IgG AF488 antibody 1:300 in pH 6.0 or pH 7.4 FACS buffer. Add 100 μL of diluted secondary antibody to the cells and incubate on ice for 45 minutes, protected from light. Pellet the cells and wash three times with 150 μL of pH 6.0 or pH 7.4 wash buffer. Fix cells with 4% PFA diluted in 1x PBS for 10 minutes at room temperature. Wash with 1x PBS and centrifuge at 2000 rpm for 3 minutes at 4°C. Resuspend cells in 100 μL of 1x PBS. Analyze cells using a NovoCyte flow cytometer and software. Acquire 20,000 events for each sample.

[0266] Data analysis: Median fluorescence intensity (MFI) and antibody concentration were used to generate a four-parameter nonlinear regression curve with variable slope using GraphPad Prism software version 9.2.0.

[0267] Results: At least three independent FACS experiments were performed for each cell line or PBMC tested. The gating strategy for determining positive cells is shown in Figures 12A-12C. The binding activity of BA3182 to CHO-hEpCAM cells, CHO-cynoEpCAM cells, and HCT116 cells is shown in Figures 13A-13C. The binding activity of BA3182 to Jurkat cells, human, and cynoPBMCs is shown in Figures 14A-14C. The expression level of EpCAM molecules on the cell surface is shown in Figures 15A-15B.

[0268] BA3182 bound well to EpCAM-expressing cells and produced good dose-response curves at pH 6.0 and pH 7.4 (Figures 13A-13C and Figures 14A-14C). The mean EC50 values ​​for BA3182 binding to EpCAM at the tumor microenvironment pH (pH 6.0) were 3.4 nM, 11.0 nM, and 5.9 nM for human and cyno EpCAM expressed in CHO cells and hEpCAM expressed in HCT116 cells, respectively (Table 6). The mean EC50 values ​​for BA3182 binding to EpCAM at normal physiological pH (pH 7.4) were 5.6 nM, 28.6 nM, and 8.3 nM for human and cyno EpCAM expressed in CHO cells and hEpCAM expressed in HCT116 cells, respectively (Table 6). BA3182 bound with lower affinity to the EpCAM antigen expressed on the cell surface at pH 7.4. No binding to native CHO cells was observed (data not shown).

[0269] The dose-response curves for BA3182 binding to CD3-expressing cells are shown in Figures 14A-14C. BA3182 bound to the CD3 antigen with high affinity at pH 6.0. At pH 7.4, BA3182 bound to CD3-expressing cells with significantly lower affinity. The mean EC50 values ​​for BA3182 binding to cell surface-expressed CD3 were 808.6 nM, 460.3 nM, and 161.6 nM for human PBMCs, cyno PBMCs, and Jurkat cells, respectively (Table 7). The mean EC50 values ​​for BA3182 binding activity at physiological pH (pH 7.4) were 2337.3 nM, 1717.3 nM, and 518.2 nM for human PBMCs, cyno PBMCs, and Jurkat cells, respectively (Table 7). No binding of BA3182 to rat, mouse, or dog PBMCs was observed (data not shown).

[0270] [Table 7]

[0271] [Table 8]

[0272] Example 21. Functional T cell activation bioassay In this example, the functional activity of the BA3182 bispecific antibody is demonstrated using a Promega T cell activation bioassay. EpCAM-expressing cell lines, HCT116, hEpCAM-expressing CHO cells, and cynoEpCAM-expressing CHO cells, were cultured in the presence of titrated concentrations of BA3182 and an isotype control bispecific antibody, followed by the addition of TCR / CD3 effector cells according to the supplier's protocol. When TCR / CD3 effector cells are bound via the BA3182 bispecific antibody in the presence of EpCAM-expressing cells, they activate the NFAT pathway, resulting in an NFAT-RE-mediated luminescent signal that is detected by the addition of Bio-Glo™ reagent and quantified with a luminometer. To determine the EC50 value of BA3182, relative luminescence unit (RLU) values ​​were analyzed using a nonlinear fit model (variable slope, 4 parameters) implemented in GraphPad Prism software. The average EC50 values ​​of BA3182 added to CHO cells expressing hEpCAM were 0.049 nM at pH 6.0 and 0.383 nM at pH 7.4. The average EC50 values ​​of BA3182 added to CHO cells expressing cynoEpCAM were 0.576 nM at pH 6.0 and 9.647 nM at pH 7.4. The average EC50 values ​​of BA3182 added to HCT116 cells were 0.273 nM at pH 6.0 and 1.988 nM at pH 7.4. These results demonstrated that BA3182 is highly potent in mediating T cell TCR / CD3 engagement at pH 6.0 (tumor microenvironment), but its efficacy is significantly reduced at physiological pH (pH 7.4).

[0273] material BA3182 RPMI-1640: ThermoFisher Gibco Cat# 11875-085, Lot# 2463433 DMEM: ThermoFisher Gibco Cat#11965-084, Lot#2346179 FBS: ThermoFisher Gibco Cat#16140-011, Lot#2372673RP 100 x NEAA: Gibco, Cat# 11140-050, Lot# 2337217 100mM Sodium Pyruvate: Gibco, Cat# 11360-070, Lot# 2323639 Bio-Glo™ Buffer: Promega, Cat# G719A, Lot# 0000473817 Bio-Glo™ Assay Substrate: Promega, Cat# G720A, Lot# 0000481126 Cell culture grade water: Cytiva Cat# SH30529.03, Lot# AH30009593 12N HCl:VWR, Cat#87003-251, Lot#4118020 50mL conical tube: BioPioneer Cat#CNT-50, Lot#10272021 96-well assay plate: Corning, Cat#3917, Lot#32021006 Syringe filter: Celltreat Cat# 229747, Lot# 211004-052-1A pH meter: ORION STAR A111, ThermoFisher, serial #J17162 Adhesive plate sealing film for microplates: E&K Scientific, Seal Plate, Cat# T396100, Lot# SG262G Shaker: MaxQ 2000 Sample Dilution Block: Corning, Cat# 3956, Lot# 07718000 Plate reader: Molecular Devices, SpectraMax i3X pH 6.0 Assay Buffer: DMEM + 1x NEAA (0.1 mM non-essential amino acid solution) + 1x sodium pyruvate (1 mM) + 10% FB, adjusted to pH 6.0 with 6 N HCl and sterilized with a 0.2 μm syringe filter. pH 7.4 Assay Buffer: DMEM + 1x NEAA (0.1 mM non-essential amino acid solution) + 1x sodium pyruvate (1 mM) + 10% FBS, adjusted to pH 7.4 with 6N HCl and sterilized with a 0.2 μm syringe filter. CHO-hEpCAM / CHO-cynoEpCAM medium: DMEM + 1x NEAA (0.1 mM non-essential amino acid solution) + 1x sodium pyruvate (1 mM) + 10% FBS + 1 mg / mL G418 HCT116 medium: McCoy's 5A+10%FBS CHO hEpCAM clone #9, BioAtla, passage #11. CHO cynoEpCAM clone #3, BioAtla, passage #13. HCT116, human colon cancer, ATCC, Cat#CCL-247(TM), passage #10. TCR / CD3 effector cells, Promega, T cell activation bioassay (NFAT), proliferation model, Cat# J1601, expanded / cryopreserved, passage #7.

[0274] method Prepare a 2x working solution of the test substance in pH 6.0 or pH 7.4 assay buffer at 500 nM for CHO hEpCAM cells and HCT116 cells, and 2500 nM for cynoEpCAM cells. Then, make 5-fold serial dilutions to obtain a total of 11 data points. Add 25 μL of the serially diluted 2x test substance solution to each well according to the plate layout. The final starting concentration is 250 nM or 1250 nM depending on the target cell. The day before the assay, seed EpCAM-expressing cells into a 96-well assay plate. 4x10 in culture medium 5 Add 100 µL of cells / mL to the plate, 4 x 10 4Seed cells / well. Incubate overnight at 37°C, 5% CO2. On the day of the assay, prepare 2x 5-fold serial dilutions of antibody in assay buffer for a total of 11 data points at pH 6.0 and pH 7.4, respectively. The initial antibody concentration for HCT116 and CHO hEpCAM cells was 250 nM. For CHO cynoEpCAM cells, the starting concentration was 1250 nM. Cover with sterile plate sealers and plate on ice. Thaw two vials of frozen TCR / CD3 effector cells in a 37°C water bath for 3 minutes. After gently mixing the cell suspension, transfer the contents to two 1.7 mL sterile Eppendorf tubes and spin down at 6000 rpm for 30 seconds in a tabletop microcentrifuge to pellet the cells. Aspirate the supernatant and resuspend one vial in 4 mL of pH 6.0 assay medium and the other in 4 mL of pH 7.4 assay medium. Carefully remove medium from the assay plate without disturbing the cells. Add 25 μL / well of 2x diluted antibody according to the layout. Add 25 μL / well of TCR / CD3 effector cell suspension according to the layout. Cover the plate and incubate for 6 hours at 37°C in 5% CO2. Prewarm Promega Bio-Glo™ buffer and substrate to room temperature. At the end of the 6-hour incubation, add Bio-Glo™ buffer to the substrate and mix well until the substrate is completely dissolved. Add 50 μL / well of Bio-Glo™ substrate to the assay plate according to the layout and incubate for 5 minutes on a 100 rpm shaker, protected from light, at room temperature. Record luminescence signals using a Molecular Devices SpectraMax i3X reader.

[0275] Data Analysis: Functional activity of test substances at pH 6.0 and pH 7.4 was determined by plotting bioluminescence units (RLU) against antibody concentration. EC50 values ​​were calculated using a nonlinear fit (variable slope, 4-parameter) model implemented in GraphPad Prism software version 9.2.0.

[0276] Results: Three independent experiments were performed for each cell type. The functional activity of BA3182 was measured using the Promega T cell activation bioassay. Results showed that BA3182 exhibited greater activity at an acidic pH (pH 6.0), which mimics the tumor microenvironment. In contrast, BA3182 was less active at normal physiological pH (neutral pH, pH 7.4) (Figures 16A, 16B, and 16C). The mean EC50 values ​​for BA3182 against CHO hEpCAM cells were 0.049 nM at pH 6.0 and 0.383 nM at pH 7.4 (Table 8). For CHO cynoEpCAM cells, the mean EC50 values ​​were 0.576 nM at pH 6.0 and 9.647 nM at pH 7.4 (Table 9). The mean EC50 of BA3182 against HCT116 cells was 0.273 nM at pH 6.0 and 1.988 nM at pH 7.4 (Table 10). No effect of the isotype control bispecific antibody was observed. Finally, no activity of BA3182 against native CHO cells was detected (data not shown).

[0277] [Table 9]

[0278] [Table 10]

[0279] [Table 11]

[0280] Example 22. Cytotoxicity test using human and cynomolgus monkey PBMCs In this example, the functional activity of the BA3182 bispecific antibody was demonstrated using an in vitro cytotoxicity assay in which human or cyno (cynomolgus monkey) peripheral blood mononuclear cells (PBMCs) were stimulated and activated in vitro with the BA3182 antibody to kill EpCAM-expressing cells. To this end, HCT116 or CHO cells expressing cynoEpCAM were treated with serial dilutions of the BA3182 antibody before the addition of human or cyno PBMCs. The co-cultures were incubated in medium at pH 6.5, which mimics the pH of the tumor microenvironment, or medium at pH 7.4, which mimics normal physiological pH. BA3182-mediated cytolysis was monitored in real time using Agilent xCELLigence real-time cell analysis technology (RTCA). The percentage of target cell lysis was calculated relative to untreated target cell proliferation at the indicated time points. The resulting percentage of cytolysis was plotted against BA3182 concentration. Fitting the data using a four-parameter nonlinear regression model with variable slope allowed for the determination of EC50 and EC20 values. The mean EC50 values ​​for BA3182-mediated lysis of HCT116 cells were 1.41 pM at pH 6.5 (using 10 different human PBMC donors) and 5.61 pM at pH 7.4 (using 6 different human PBMC donors). The mean EC20 values ​​for BA3182-mediated lysis of HCT116 cells were 0.54 pM at pH 6.5 (using 10 different human PBMC donors) and 4.09 pM at pH 7.4 (using 6 different human PBMC donors). The mean EC50 values ​​for BA3182-mediated lysis of CHO cells expressing cynoEpCAM by cyno PBMC were 17.39 pM at pH 6.5 and 184.73 pM at pH 7.4.

[0281] material BA3182 isotype control bispecific antibody was from Evitria (Zurich, Switzerland), lot number #10229, 3 mg / mL. RPMI-1640: ThermoFisher Gibco Cat# 11875-085, Lot# 2463433 DMEM: ThermoFisher Gibco Cat#11965-084, Lot#2346179 FBS: Sigma, Cat#12306C-500mL, Lot#16J367 100 x NEAA: Gibco, Cat# 11140-050 Lot# 2337217 100mM Sodium Pyruvate: Gibco, Cat# 11360-070, Lot# 2323639 Cell culture grade water: Corning Cat#25-055-CM, Lot#17216006 12N HCl:VWR, Cat#87003-251, Lot#4118020 50mL conical tube: BioPioneer Cat#CNT-50, Lot#10272021 E-Plate View 96: Agilent, Cat# 300601010, Lot# 20211132 Syringe filter: Celltreat Cat# 229747, Lot# 211004-052-1A pH meter: ORION STAR A111, ThermoFisher, serial #J17162 Trypan blue staining solution: Gibco Cat# 15250-061, Lot# 1861515 Adhesive plate sealing film for microplates: E&K Scientific, Seal Plate, Cat# T396100, Lot# SG262G Sample Dilution Block: Corning, Cat# 3956, Lot# 07718000 Agilent xCELLigence Real-Time Cell Analysis (RTCA) MP Analyzer Plate reader: SpectraMax i3X, Molecular Devices PBMC pH 6.5 assay medium: RPMI-1640 + 10% FBS, adjusted to pH 6.5 with 6N HCl, sterilized with a 0.2 μm syringe filter. PBMC pH 7.4 assay medium: RPMI-1640 + 10% FBS, adjusted to pH 7.4 with 6N HCl, sterilized with a 0.2 μm syringe filter. CHO cynoEpCAM medium: DMEM + 1x NEAA (0.1 mM non-essential amino acid solution) + 1x sodium pyruvate (1 mM) + 10% FBS + 1 mg / mL G418 HCT116 medium: McCoy's 5A+10%FBS CHO cynoEpCAM clone #3, BioAtla, passage #13 HCT116, Human Colon Cancer, ATCC, Cat#CCL-247(TM), Passage #11 Human PBMC: Precision for Medicine, Cat#93000-10M, Lot#13143; 2010113397; 2010113371; 2010113411; 2010113292; 2010113290; 201675377; 201877535; 201885639; 201897529 CynoPBMC: Worldwide Primates Inc, Cat#CA-10, Lot#207117-1

[0282] method Cell culture and plating. One day before the assay, detach the target cells and resuspend them in medium as a single cell solution. Count the cells using a hemocytometer using trypan blue staining. The target cell concentration was adjusted to 4 x 10 cells / mL. 4 Adjust the volume to 4 x 10 cells / mL and add 100 µL to a 96-well assay plate. 3 Seed cells / well. Incubate target cells overnight at 37°C, 5% CO2.

[0283] PBMC resting Thaw PBMCs in a 37°C water bath for 3 minutes and add dropwise to 10 mL of warmed PBMC assay medium (pH 7.4), constantly swirling the conical tube to allow the cells to acclimate to the medium temperature. Pipette up and down with a 10 mL pipette until no clumps are visible. Harvest the cells by centrifugation at 1400 rpm for 10 minutes and discard the medium supernatant. Resuspend the cell pellet in 10 mL of fresh warm medium and dissociate the cells by pipetting. Incubate overnight (18-20 hours) at 37°C in a 5% CO2 incubator. Count the cell number and determine cell viability before plating. Harvest the cells by centrifugation at 1400 rpm for 10 minutes and discard the medium supernatant. Discard 4 x 10 cells in the specified volume of pH medium. 5 Resuspend cells to a density of 100 cells / mL.

[0284] Test antibody preparation and assay setup Test substances were serially diluted to 2x the starting concentration in assay medium at pH 6.5 or pH 7.4 to create stock solutions starting at 10 nM for HCT116 / human PBMC cells and 200 nM for CHOcyno EpCAM / cyno PBMC cells. Five-fold dilutions were performed to obtain a total of 11 data points. Serially diluted test substances were dispensed at 100 μL / well, and 100 μL / well of PBMC suspension was added. The final volume was 200 μL / well. To ensure complete lysis, the PBMC and target cell co-cultures were treated with 1% SDS. The cells were incubated at 37°C and 5% CO2 for 100 hours, with data collection every 15 minutes, yielding a total of 400 readings.

[0285] Results: The functional activity of BA3182 was measured using CD3-mediated killing of target cells by PBMCs. Human PBMCs from six healthy subjects were co-cultured with HCT116 cells and various concentrations of BA3182 for 120 hours. Cell proliferation was measured using XCELLigence real-time cell analysis technology. Cytotoxicity was calculated using the following formula:

number

[0286] [Table 12]

[0287] Example 23. PBMC cytokine release assay In this example, we investigated the release of IL-2, IL-6, IL-10, INFγ, and TNFα cytokines upon stimulation of human peripheral mononuclear cells (PBMCs) with various concentrations of BA3182 in the presence of EpCAM-expressing cells. To this end, HCT116 cancer cells were seeded onto tissue culture plates and cultured overnight at 37°C and 5% CO2. The next day, growth medium was removed, and human PBMCs from nine donors were added. Cocultures were established in medium at pH 6.5, representative of the tumor microenvironment pH. Serial dilutions of BA3182 or an isotype control antibody were added to the plates, and the cultures were maintained at 37°C and 5% CO2. After 48 hours, the plates were centrifuged to pellet the cells, and the supernatants were collected, transferred to new plates, and stored at -80°C until analysis of cytokine concentrations. Cytokine concentrations were measured using an R&D Systems Quantikine ELISA kit, and the EC50 values ​​of BA3182 were calculated using GraphPad Prism software. BA3182 was able to induce the release of IL-2 and INFγ cytokines in nine donors at pH 6.5. Baseline IL-6, IL-10, and TNFα cytokines were detected, and their levels varied between donors. No effect of BA3182 or isotype on the induction of these cytokines was observed.

[0288] material

[0289] [Table 13] *Isotype: Non-CAB human IgG anti-Hen egg white lysozyme (HEL)-containing anti-hCD3 scFv.

[0290] [Table 14] JPEG2026507181000019.jpg17170

[0291] [Table 15]

[0292] method Cell culture: HCT116 cells were maintained in McCoy's 5A medium supplemented with 10% FBS. Cells were routinely passaged twice weekly. Cells were harvested during exponential growth phase and counted for plating and measurement of EpCAM surface expression.

[0293] Cell staining: Before plating the target cells for the assay, EpCAM surface expression was confirmed by flow cytometry. To do this, the medium was removed from T75 HCT116 culture flasks, and the cell layer was briefly washed with PBS (pH 7.4). 1 mL of 0.25% trypsin-EDTA was added to each flask, and the cells were returned to 37°C until the cell layer was dispersed. The trypsin reaction was stopped with 9 mL of medium, and the cells were resuspended by gentle pipetting. For FACS staining, 2 × 10 cells were cultured. 5HCT116 cells were placed in a 1.5 mL Eppendorf tube and washed with PBS, pH 7.4. The cells were spun, the supernatant removed, and resuspended in 100 μL of PBS + 1% BSA containing 2 μL each of PE-anti-human EpCAM antibody clone 9C4 or PE-mouse IgG2bk isotype clone MPC-11. The cells were incubated on ice in the dark with shaking at 100 rpm for 1 hour. After incubation, the cells were washed three times with 1 mL of PBS (pH 7.4). The cells were then resuspended in 120 μL of PBS + 1% BSA and analyzed using a NovoCyte flow cytometer.

[0294] Incubation with antibodies: HCT116 cells were seeded into 96-well tissue culture plates at a density of 6,000 cells / well in McCoy's 5A medium supplemented with 10% FBS and incubated overnight at 37°C, 5% CO2. After overnight culture, growth medium was removed and human PBMCs were added at 30,000 cells / well in RPMI 1640 + 10% FBS medium at pH 6.5 (reflecting the tumor microenvironment). PBMCs were added at a 5:1 ratio to target cells. BA3182 or isotype control antibodies were serially diluted from 150 nM to 0.008 nM (3-fold dilutions) in RPMI 1640 + 10% FBS at pH 6.5 and added to the plates. Cultures were then maintained at 37°C, 5% CO2. After 48 hours of incubation with the test antibodies, the plates were centrifuged at 2000 rpm for 3 minutes at room temperature to pellet the cells, and the supernatants were transferred to new 96-well plates and stored at -80°C until analysis of cytokine concentrations.

[0295] Cytokine Measurement: Cytokine concentrations were measured using the Quantikine™ ELISA assay according to the manufacturer's protocol. Supernatants were diluted with assay diluent as needed to obtain values ​​within the linear range of the assay's standard curve. EC50 values ​​were calculated from the nonlinear four-parameter regression curve of the cytokine standards using Graph Pad Prism software version 9.0.

[0296] Results: BA3182 was able to induce the release of IL-2 and INFγ cytokines by human PBMCs cocultured with the human colon cancer cell line HCT116 at pH 6.5 (tumor microenvironment pH). Stimulation of human PBMCs with BA3182 in the absence of HCT116 cells did not induce a cytokine response (data not shown). An isotype control antibody (bispecific antibody anti-Hen egg white lysozyme / CD3) did not induce the release of IL-2 or INFγ (Figures 18A-18B and 19A-19B). Neither BA3182 nor the isotype antibodies had any measurable effect above background levels on the release of IL-6, IL-10, or TNFα (Figures 20-20B, 21A-21B, and 22A-22B). The EC50 values ​​measured for BA3182 for induction of IL-2 and IFNγ by PBMCS from nine human donors tested at pH 6.5 were low nanomolar. PBMCs from a tenth donor showed no response to any stimuli, including positive control CD3 / CD28-immobilized microbeads, when cultured with HCT116 cancer cells (data not shown), and this donor was therefore excluded from the study. The EC50 values ​​obtained for IL-2 and IFNγ induction by BA3182 at pH 6.5 varied depending on the donor and are shown in Table 15. This data demonstrates that the BA3182 antibody is a potent inducer of IL-2 and IFNγ production by human PBMCs in the presence of the EpCAM antigen in vitro. BA3182 does not induce the production of pro-inflammatory cytokines, such as IL-6 and TNF-α, or the anti-inflammatory cytokine IL-10 by human PBMCs.

[0297] [Table 16]

[0298] Example 24. C1q affinity ELISA In this example, a C1q affinity ELISA assay was used to measure the binding of human complement protein C1q to BA3182. ELISA assay plates were coated with BA3182 or a positive control antibody (B12). Serially diluted purified human C1q protein was added to the plate. C1q protein and antibody binding were detected by the addition of a sheep anti-human C1q-HRP-conjugated antibody. A colored product was generated by the addition of TMB colorimetric substrate. The optical density (OD) of each well was measured using a microplate reader. Data were analyzed, and a nonlinear four-parameter regression curve was generated using GraphPad Prism software. The results indicated weak binding of C1q to BA3182. This result was expected due to the mutation of the glycosylation site at position 297 from asparagine (N) to glutamine (Q). This mutation has been shown to be effective in completely inhibiting effector functions such as complement-dependent cytotoxicity (CDC).

[0299] material

[0300] [Table 17]

[0301] [Table 18]

[0302] Methods: ELISA plates were coated with 100 μL / well of BA3182 or B12 antibody at 3 μg / mL in carbonate-bicarbonate buffer and incubated overnight at 4°C. The next day, the plates were blocked with 300 μL / well of blocker casein buffer and incubated at room temperature for 1 hour. The plates were then washed three times with 300 μL / well of PBS containing 0.05% Tween 20. C1q was diluted half-logarithmically in blocker casein buffer (600, 189.75, 60.01, 18.98, 6.00, 1.90, 0.60, 0.19, 0.06, and 0.02 μg / mL), and 100 μL of each dilution was added to the plate and incubated at room temperature for 2 hours. The plates were then washed and incubated with 100 μL / well of a 1:200 dilution of the secondary antibody sheep anti-human C1q Ab-HRP for 1 hour at room temperature. After washing, 100 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added. The plates were incubated in the dark at room temperature for 5–20 minutes, and the enzymatic reaction was stopped with 50 μL of 2 M HCl. Absorbance was measured at 450 nm using a SpectraMax i3x Molecular Devices microplate reader. Data were analyzed, and a nonlinear four-parameter regression curve was generated using GraphPad Prism software version 9.0.

[0303] Results: The molecular weight of 410 kDa was used to calculate the molar concentration of C1q, and the absorbance at 450 nm was used to generate a four-parameter regression curve for calculating the EC50 value. C1q protein bound strongly to the positive control antibody B12, generating a good dose-response curve (Figure 23). B12 binds to human IgG 1,k As this antibody is an anti-gp120 glycoprotein, C1q binding to this antibody was predicted. C1q bound weakly to BA3182 (Figure 23), suggesting that the affinity of C1q for the BA3182 antibody is low. The calculated EC50 value for the binding affinity of C1q to BA3182 was 12.89-fold higher than that of C1q to B12 (613.1 nM vs. 47.55 nM) (Table 16). The BA3182 antibody is a human IgG1 antibody containing the N297Q mutation.1,k It is a non-glycosylated variant, and this mutation has been shown to reduce the binding affinity of human IgG1 to the C1q protein and inhibit complement fixation and activation.

[0304] [Table 19]

[0305] Example 25. Analysis of binding kinetics by SPR This example aimed to determine the binding kinetics of BA3182 to human and cynomolgus EpCAM and CD3ε / δ heterodimers at pH 6.0, pH 6.5, and pH 7.4 using surface plasmon resonance (SPR). After immobilizing the ligand on the surface of a planar amine sensor chip, titrated concentrations of BA3182 were injected. The binding interaction between BA3182, the ligand, and the control surface was monitored in real time. Binding kinetics (association rate kd, dissociation rate ka, affinity K) were calculated. D ) was calculated using the 1:1 Langmuir model built into the analysis software.

[0306] BA3182 binds to human EpCAM with an affinity of approximately 1.2 nM under tumor microenvironment conditions (acidic pH). At normal physiological pH, the affinity is reduced 5.5-fold to 6.7 nM. Binding to cynomolgus monkey EpCAM shows a similar trend. The binding affinity is approximately 2-3 nM at acidic pH and is reduced 6-fold to approximately 12 nM at normal physiological pH. BA3812 binds to human CD3 with an affinity of 8-9 nM under tumor microenvironment conditions (acidic pH). At normal physiological pH, the affinity is approximately 35 nM. Binding to cynomolgus monkey CD3 shows a similar trend. The binding affinity is approximately 11 nM at acidic pH and is reduced approximately 3-fold to approximately 30 nM at normal physiological pH.

[0307] material antibody BA3182 was used at a stock concentration of 1.03 mg / mL.

[0308] [Table 20]

[0309] Buffer: SPR running buffers (pH 6.0, pH 6.5, and pH 7.4) containing sodium bicarbonate were prepared immediately before use using the following procedure, as the pH of the buffer changes during storage. PBST-SB-75 pH 6.0 Add 16.6 mL of 7.5% sodium bicarbonate solution to 1.483.4 mL of 1x PBS. 2. Add 250 μL of Tween-20. 3.2.19 g of NaCl is added. Adjust the pH to 5.9 with 4.6 N HCl. 5. Filter through a 0.22 μM PES bottle filter. 6. Degas using sonicator and vacuum for 5 minutes. 7. Prepare samples by taking 50 mL aliquots. PBST-SB-75 pH 6.5 Add 16.6 mL of 7.5% sodium bicarbonate solution to 1.483.4 mL of 1x PBS. 2. Add 250 μL of Tween-20. 3.2.19 g of NaCl is added. Adjust the pH to 6.4 with 4.6 N HCl. 5. Filter through a 0.22 μM PES bottle filter. 6. Degas using sonicator and vacuum for 5 minutes. 7. Prepare samples by taking 50 mL aliquots. PBST-SB-75 pH 7.4 Add 16.6 mL of 7.5% sodium bicarbonate solution to 1.483.4 mL of 1x PBS. 2. Add 250 μL of Tween-20. 3.2.19 g of NaCl is added. Adjust the pH to 7.35 with 4.6 N HCl. 5. Filter through a 0.22 μM PES bottle filter. 6. Degas using sonicator and vacuum for 5 minutes. 7. Prepare samples by taking 50 mL aliquots.

[0310] Sensor Regeneration Solution 10mM glycine pH 2.0 Methods: The SPR32 Pro instrument has eight channels (1–8) with four detection spots (A, B, C, and D) each. The 32 detection spots can be addressed individually or in groups. The surface of each new flat amine sensor chip was preconditioned using a built-in method prior to ligand immobilization, according to the manufacturer's recommendations. PBST (pH 7.4) was used as the running buffer for preconditioning and EpCAM or CD3 immobilization.

[0311] Detection spots A, B, and C were activated by injecting an EDC / NHS mixture (250 mM / 50 mM) at a flow rate of 25 μL / min for 240 seconds. No protein was immobilized on the control surface (detection spots 1A–8A). Human EpCAM-His was diluted to 0.5 μg / mL in 10 mM NaAc (pH 5.0) and injected at a flow rate of 25 μL / min for 240 seconds into detection spots 1B–8B. CynoEpCAM-His was diluted to 0.5 μg / mL in 10 mM NaAc (pH 5.5) and injected at a flow rate of 25 μL / min for 240 seconds into detection spots 1C–8C. Detection spots A, B, and C were blocked by injecting ethanolamine (1 M) at a flow rate of 25 μL / min for 240 seconds. Detection spots A, B, C, and D were activated by injecting an EDC / NHS mixture (250 mM / 50 mM) at a flow rate of 25 μL / min for 240 seconds. No protein was immobilized on the control surface (detection spots 1A–8A). Human CD3-hFc fusion protein was diluted to 1 μg / mL in 10 mM NaAc (pH 5.5) and injected at a flow rate of 25 μL / min for 240 seconds into detection spots 1D–8D. CynoCD3-His was diluted to 0.05 μg / mL in 10 mM NaAc (pH 5.5) and injected at a flow rate of 25 μL / min for 240 seconds into detection spots 1B–8B. Detection spots A, B, and C were blocked by injecting ethanolamine (1 M) at a flow rate of 25 μL / min for 240 seconds.

[0312] BA3182 was buffer-exchanged into 1x PBS using an Amicon Ultra-15 spin filter (150 kDa MWCO). 1 mL of BA3182 was added to the upper reservoir, followed by approximately 14 mL of 1x PBS. The solution was centrifuged at 4000 RPM for 12 minutes. The flow-through was discarded, and this procedure was repeated three more times. Protein concentration was measured by UV280. The buffer-exchanged BA3182 was diluted with running buffer to a starting concentration of 5 μg / mL. Two-fold serial dilutions were then performed over a total of seven dilution points (5 μg / mL to 0.078 μg / mL).

[0313] Serial dilutions of BA3182 (channel 8: highest concentration, channel 2: lowest concentration, channel 1: running buffer) were injected into detection spots A, B, and C (flow rate: 25 μL / min, contact time: 120 s; dissociation rate measurement: 120 s). The sensor surface was regenerated by injecting 10 mM glycine pH 2.0 (flow rate: 25 μL / min, contact time: 15 s). Analyte injections were repeated twice (a total of three analyte injections). Running buffer was injected as a blank analyte injection before and after the BA3182 injection.

[0314] Detection spot A, which contained no immobilized protein, was used as a control surface for background subtraction. Additionally, data using buffer alone (0 nM analyte) was subtracted from each run. The double-subtracted data were fitted with a 1:1 binding model using the provided analysis software, Sierra Analyzer R3 (Bruker). A molecular weight of 200 kDa was used to calculate the molar concentration of the analyte.

[0315] The kinetic binding data from a representative experiment at each test pH condition was entered into SPR simulation software (www.sprpages.nl\spr-simulation) to analyze the signal decline from pH 6.0 to pH 7.4. The input data into the software were the on / off rates and K calculated from the actual SPR experiments. D The parameters include the molecular weight of the analyte and ligand, the amount of analyte immobilized on the sensor surface, the binding and dissociation times, the maximum analyte concentration used, the analyte concentration, and the number of dilution steps. The program generates sensorgrams assuming that 100% of the immobilized ligand is capable of binding. In the case of random immobilization of the ligand, this is typically in the range of 30-40%. The actual percentage can be determined by lowering the active concentration until the maximum signal in the simulation matches that of the actual experiment. The active concentration of the ligand (EpCAM or CD3) at pH 6.0 was set to 100%.

[0316] Results: A total of three independent SPR experiments were performed at each pH. The same sensor chip was used for all pH conditions. The binding kinetics of BA3182 to recombinant human and cynomolgus EpCAM at pH 6.0, pH 6.5, and pH 7.4 are summarized in Table 1. Binding curves for representative experiments at each pH are shown in Figure 24. BA3182 binds to human EpCAM with an affinity of 1.30 nM at pH 6.0, 1.22 nM at pH 6.5, and 6.73 nM at pH 7.4 (Table 17), and to cynomolgus EpCAM with an affinity of 2.03 nM at pH 6.0, 2.93 nM at pH 6.5, and 11.5 nM at pH 7.4 (Table 17).

[0317] Simulation of the resulting sensorgrams indicates that the decrease in signal at higher pH is due to the loss of active ligand on the chip surface (Figure 26). This differential binding profile is expected since the antibody was designed to maintain full binding capacity in the tumor microenvironment while exhibiting reduced binding under physiological conditions. BA3182 bound to human and cynomolgus monkey EpCAM with comparable affinity (within 2-fold), indicating that cynomolgus monkeys are a suitable species for toxicity studies.

[0318] A total of three independent SPR experiments were performed at each pH. The same sensor chip was used for all pH conditions. The binding kinetics of BA3182 to recombinant human and cynomolgus CD3 at pH 6.0, pH 6.5, and pH 7.4 are summarized in Table 18. Binding curves for a representative experiment at each pH are shown in Figure 25.

[0319] BA3182 binds to human CD3 with an affinity of 8.1 nM at pH 6.0, 9.1 nM at pH 6.5, and 35 nM at pH 7.4 (Table 18), and to cynomolgus CD3 with an affinity of 11.0 nM at pH 6.0, 11.8 nM at pH 6.5, and 28.6 nM at pH 7.4 (Table 18). At pH 7.4, the signal was within the noise level for most analyte concentrations, indicating that K D The binding data at pH 7.4 shown in Table 18 are based only on the highest concentration used.D The values ​​are within two-fold of the values ​​calculated using the entire dilution series. In addition to the decrease in binding affinity at higher pH, the maximum SPR signal decreases from pH 6.0 to pH 7.4 (Figure 25). Simulation of the resulting sensorgrams reveals that the decrease in signal at higher pH is due to the reduction in active ligands on the chip surface (Figure 27).

[0320] [Table 21]

[0321] [Table 22]

[0322] Example 26. Fcγ and FcRn receptor binding analysis by SPR In this example, the binding affinity (K ) of BA3182 to human FcγRI, FcγRIIa, FcγIIb / c, FcγRIIIa, FcγRIIIb, and FcRn receptors was measured using surface plasmon resonance (SPR) technology. D ) are shown. The extracellular domain of the Fc-γ receptor was captured on the chip using an anti-His tag antibody. Serial dilutions of BA3182 and a control IgG1 antibody were injected over eight detection spots containing the Fc-γ receptor and a control surface containing only the anti-His antibody. BA3182 does not bind to the Fc-γ receptor (as expected) because it has an aglycosylated Fc domain (N297Q mutation). Binding to FcRn at pH 6.0 was measured by immobilizing BA3182 or a control IgG1 on the chip surface and injecting serial dilutions of FcRn. BA3182 showed FcRn binding affinity comparable to that of the control antibody IgG1. Binding kinetics were calculated using the SICK Longmuir 1:1 model integrated into the analysis software.

[0323] material Antibody BA3182 IgG1-Ctrl (Lot #13578) manufactured by Evitria (Zurich, Switzerland)

[0324] [Table 23]

[0325] Methods: The SPR32 Pro instrument has eight channels (1-8), each with four detection spots (A, B, C, D). The 32 detection spots can be addressed individually or in groups. The surface of each new sensor chip was preconditioned using a built-in method prior to protein immobilization, according to the manufacturer's recommendations. PBST (pH 7.4) was used as the running buffer for preconditioning and all antibody immobilization.

[0326] All 32 spots on the preconditioned chip were activated by injecting a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) (250 mM / 50 mM) at a flow rate of 10 μL / min for 420 seconds. His-Tag antibody was diluted to 50 μg / mL in NaAc (pH 4.5) and injected at a flow rate of 10 μL / min for 360 seconds over all 32 detection spots. All 32 detection spots were blocked by injecting 1 M ethanolamine at a flow rate of 10 μL / min for 420 seconds.

[0327] A capture assay format was used for the binding study of BA3182 and Fcγ receptors. Fcγ receptors were diluted in running buffer and captured on spots B, C, or D of an anti-His sensor chip. BA3182 or an IgG1-control antibody (serial dilutions in running buffer) was then injected on spots AB, BC, or CD. A detection spot without captured protein served as a control surface. An injection of running buffer served as a blank. After each analyte injection, the His capture surface was regenerated. One injection of IgG1-control antibody and three injections of BA3182 were performed per assay.

[0328] FcγRI (CD64) capture assay Running buffer: Phosphate-buffered saline containing Tween-20 (PBST), pH 7.4 FcγRI (CD64): 50 nM in PBST (pH 7.4), injected onto the D spot (flow rate: 10 μL / min, contact time: 15 s). IgG1-ctrl: A two-fold dilution series in PBST (pH 7.4) was injected onto the CD spot starting at 6 μg / mL (flow rate: 20 μL / min, contact time 180 s; dissociation rate measured at 400 s). BA3182: 2-fold dilution series in PBST (pH 7.4) starting at 6 μg / mL injected onto CD spots (flow rate: 20 μL / min, contact time 180 s; dissociation rate measured at 400 s)

[0329] FcγRIIa H131 (CD32a) capture assay FcγRIIa H131 (CD32a): Running buffer was injected into the C-spot at 181.8 nM in HBS-EP+ (flow rate: 10 μL / min, contact time 60 s). IgG1-ctrl: A two-fold dilution series in HBS-EP+ was injected onto the BC spot starting from 1.5 mg / mL (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measurement 60 seconds). BA3182: A two-fold dilution series in HBS-EP+ was injected onto the BC spot starting at 1.5 mg / mL (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measurement 60 seconds). Regeneration: Inject 10 mM glycine, pH 2.0 into the BC spot (flow rate: 25 μL / min, contact time: 15 s).

[0330] FcγRIIb / c (CD32b / c) capture assay Running buffer: HEPES / NaCl buffer containing EDTA and surfactant P20 (HBS-EP+) FcγRIIb / c (CD32b / c): 192.3 nM in HBS-EP+ was injected into the C-spot (flow rate: 10 μL / min, contact time: 60 s). IgG1-ctrl: A two-fold dilution series in HBS-EP+ was injected onto the BC spot starting from 1.5 mg / mL (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measurement 60 seconds). BA3182: A two-fold dilution series in HBS-EP+ was injected onto the BC spot starting at 1.5 mg / mL (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measurement 60 seconds). Regeneration: Inject 10 mM glycine, pH 2.0 into the BC spot (flow rate: 25 μL / min, contact time: 15 s).

[0331] FcγRIIIa F158 (CD16a) capture assay FcγRIIIa (CD16a): 171.7 nM in HBS-EP+ was injected into the C-spot (flow rate: 10 μL / min, contact time: 60 s). IgG1-ctrl: A two-fold dilution series in HBS-EP+ was injected onto the BC spot starting from 1.5 mg / mL (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measurement 60 seconds). BA3182: A two-fold dilution series in HBS-EP+ was injected onto the BC spot starting at 1.5 mg / mL (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measurement 60 seconds). Regeneration: 10 mM glycine, pH 2.0, injected onto the BC spot (flow rate: 25 μL / min, contact time: 15 s).

[0332] FcγRIIIb (CD16b) capture assay FcγRIIIb (CD16b): 180.2 nM in HBS-EP+, injected into the C-spot (flow rate: 10 μL / min, contact time: 60 s) IgG1-ctrl: A two-fold dilution series in HBS-EP+ was injected onto the BC spot starting from 1.5 mg / mL (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measurement 60 seconds). BA3182: A two-fold dilution series in HBS-EP+ was injected onto the BC spot starting at 1.5 mg / mL (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measurement 60 seconds). Regeneration: 10 mM glycine, pH 2.0, injected onto the BC spot (flow rate: 25 μL / min, contact time: 15 s).

[0333] FcRn binding assay BA3182 and IgG1-control antibodies were immobilized on a flat amine sensor chip. All 32 spots on the preconditioned chip were activated by injecting the mixture EDC / NHS (250 mM / 50 mM) at a flow rate of 10 μL / min for 420 seconds. BA3182 and IgG1-control antibodies were both diluted to 0.1 μg / mL in NaAc (pH 5.5) and injected at a flow rate of 10 μL / min for 240 seconds into spot B (IgG1-control) or C (BA3182). All 32 detection spots were blocked by injecting ethanolamine (1 M) at a flow rate of 10 μL / min for 60 seconds. FcRn: A 3-fold dilution series in PBS+TWEEN pH 6.0 was injected onto the ABCD spot starting at 25 μg / mL (flow rate: 20 μL / min, contact time 60 seconds, dissociation rate measurement 60 seconds). Regeneration: 1x PBS, pH 7.4, injected into the ABCD spot (flow rate: 10 μL / min, contact time: 30 s)

[0334] Results: Figure 28 shows that an IgG1-control antibody with a wild-type human IgG1 Fc domain binds to FcγRI with an affinity of 1.18 nM. The binding signal of BA3182 was much lower, detected only at the three highest concentrations tested. Because all binding curves reached saturation, the data were analyzed using a steady-state model. However, the differences between signal intensities and concentrations were too small to yield a significant dissociation constant, K Dcannot be calculated. This data shows that BA3182 does not bind to FcγRI (CD64). Figure 29 shows that an IgG1-control antibody with a wild-type human IgG1 Fc domain binds to FcγRIIa with an affinity of 0.82 μM. No binding to BA3182 was detected, indicating that BA3182 does not interact with FcγRIIa (CD32a). Figure 30 shows that an IgG1-control antibody with a wild-type human IgG1 Fc domain binds to FcγRIIb / c with an affinity of 4.2 μM. No binding to BA3182 was detected, indicating that BA3182 does not interact with FcγRIIb / c (CD32b / c). Figure 31 shows that an IgG1-control antibody with a wild-type human IgG1 Fc domain binds to FcγRIIIa with an affinity of 0.86 μM. No binding to BA3182 was detected, indicating that BA3182 does not interact with FcγRIIIa (CD16a). Figure 32 shows that an IgG1-control antibody with a wild-type human IgG1 Fc domain binds to FcγRIIIa with an affinity of 0.86 μM. No binding to BA3182 was detected, indicating that BA3182 does not interact with FcγRIIIa (CD16b). Figure 33 shows that an IgG1-control antibody with a wild-type human IgG1 Fc domain binds to FcRn with an affinity of 366 nM. Similarly, BA3182 showed a binding affinity of 347 nM to FcRn.

[0335] Example 27. Inducible Cytokine Release Assay The purpose of this example was to determine whether BA3182 can mediate CD3 T cell activation in human PBMCs in the absence of EpCAM-expressing cells. Human PBMCs were incubated with soluble or immobilized BA3182 for 48 hours. Soluble BA3182 was tested at 5 nM, 1.67 nM, and 0.56 nM. Assays were performed in medium at pH 6.5, which mimics the pH of the tumor microenvironment, or medium at pH 7.4, which mimics normal physiological pH. A panel of cytokines (IL-2, IL-4, IL-1β, IL-6, IL-10, IL-17, IFNγ, and TNFα) was measured using a Luminex multiplex assay. Human T cell activating factor and CD3 / CD28 Dynabeads for T cell proliferation and activation were included as positive controls for cytokine responses in human PBMCs. A total of nine human PBMC donors were tested. No detectable levels of cytokines were observed when human PBMCs were stimulated with soluble BA3182. IFNγ and TNFα were detected in cultures stimulated with immobilized BA3182, whereas low levels of IL-2, IL-4, IL-17, and IL-10 cytokines were detected in PBMC cultures from three to four human donors using immobilized BA3182. Immobilized OKT3 anti-CD3 antibody induced cytokine responses in all human PBMC donors tested. Cytokine production induced by immobilized BA3182 was significantly lower than that induced by immobilized OKT3 anti-CD3 antibody at pH 7.4. All cytokines except IL-6 were detectable in cultures stimulated with immobilized OKT3 anti-CD3 antibody (IL-6 was detected only in PBMC cultures from two human donors). All human PBMC donors tested showed elevated cytokine levels after activation with CD3 / CD28 Dynabeads, regardless of pH conditions. Overall, the cytokine levels induced by immobilized BA3182 were significantly lower than those induced by immobilized OKT3 anti-CD3 antibody. These results suggest that BA3182 induces a weak cytokine response.

[0336] material antibody BA3182 (1.03 mg / mL) Anti-human CD3 monoclonal antibody, clone OKT3: Tonbo, Cat#40-0037-U500, Lot#P0037072518404 PMBC cells. Precision for Medicine, Cat#93000-10M, 9 Donors, Lot#13143, 2010113371, 2010113397, 2010113292, 2010113411, 201675377, 201877535, 201885639, 201897529 Reagents and equipment RPMI-1640: ThermoFisher Gibco Cat# 11875-085, Lot# 2463433 FBS: Sigma, Cat#12306C-500mL, Lot#16J367 PBS: Gibco, Cat# 10010-023, Lot# 2430050 Cell culture grade purified water: Corning Cat#25-055-CM, Lot#17216006 12N HCl:VWR, Cat#87003-251, Lot#4118020 50mL conical tube: BioPioneer Cat#CNT-50, Lot#10272021 96-well clear flat-bottom tissue culture plate: Corning, Cat# 3596, Lot# 19620012 Syringe filter: Celltreat Cat# 229747, Lot# 211004-052-1A pH meter: ORION STAR A111, ThermoFisher, serial #J17162 Trypan blue staining solution: Gibco Cat# 15250-061, Lot# 1861515 Adhesive plate sealing film for microplates: E&K Scientific, Seal Plate, Cat# T396100, Lot# SG262G Sample Dilution Block: Corning, Cat# 3956, Lot# 07718000 Dynabeads CD3 / CD28: ThermoFisher, Cat# 11131D, Lot# 00637008 Luminex Multiplex Cytokine Kit: R&D Systems, Cat#FCSTM03-8, Lot#1644401 and 1667482 Luminex LX200: Luminex, SN#LX10021050422 PBMC pH 6.5 assay medium: RPMI-1640 + 10% FBS, adjusted to pH 6.5 with 6N HCl, sterilized with a 0.2 μm syringe filter. PBMC pH 7.4 assay medium: RPMI-1640 + 10% FBS, adjusted to pH 7.4 with 6N HCl, sterilized with a 0.2 μm syringe filter.

[0337] method Plate Coating One day before the assay, coat the wells with 100 μL / well of 20 μg / mL anti-hCD3 antibody, clone OKT3 or BA3182, in PBS buffer. Cover with a plate sealer and incubate overnight at 4°C. PMBC suspended Thaw PBMCs in a 37°C water bath for 3 minutes and add dropwise to 10 mL of warmed PBMC assay medium (pH 7.4). Continually swirl the conical tube to allow the cells to acclimate to the medium temperature. Pipette up and down with a 10 mL pipette until no clumps are visible. Harvest the cells by centrifugation at 1400 rpm for 10 minutes and discard the medium supernatant. Resuspend the cell pellet in 10 mL of fresh warm medium and separate the cells by pipetting. Incubate overnight (18-20 hours) at 37°C in a 5% CO2 incubator. Harvest the cells by centrifugation at 1400 rpm for 10 minutes and discard the medium supernatant. Dissolve 1 x 10 cells in the specified volume of pH medium. 6 Resuspend cells to a density of 100 cells / mL. Test substance preparation and assay set-up After removing the BA3182 and anti-hCD3 antibody solutions from the wells, wash twice with 100 μL / well of PBS to remove unbound antibody. Prepare CD3 / CD28 Dynabeads according to the manufacturer's instructions by washing once with at least 1 mL of PBS solution. Resuspend in PBS solution to the appropriate volume and add 1 x 10 5 Add 2.5 μL of beads per PBMC cell / well. Prepare soluble BA3182 at the desired concentrations of 5, 1.67, and 0.56 nM using assay medium at pH 6.5 or pH 7.4. Dispense 100 μL of pH assay medium per well into wells coated with BA3182 and anti-hCD3 antibody. Dispense 100 μL of BA3182 per well into uncoated wells. Add 100 μL of the PBMC suspension prepared above per well. Bring the final volume to 200 μL per well. Incubate at 37°C with 5% CO2 for 48 hours. Perform cytokine multiplex assays using the culture supernatants according to the kit manual, and collect data using a Luminex LX100 unit.

[0338] Data Analysis: Cytokine concentrations were determined using a Luminex Multiplex Cytokine Kit from R&D Systems and a Luminex LX100 instrument from Luminex. Standard curves for each analyte were generated by creating a 5-parameter logistic (5-PL) curve fit using xPonent software. Supernatants were diluted with assay diluent as needed to obtain values ​​in the linear range of the assay's standard curve.

[0339] Results: Soluble BA3182 did not induce detectable levels of any of the cytokines tested, including IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17, and TNF-α. Cytokine concentrations were below the minimum detectable concentration or similar to cytokine levels present in cultures of human PBMCs in the absence of activating antibodies (Figures 34A-34H and 35A-35H). All cytokines were detected at quantifiable levels in supernatants from PBMC cultures activated with CD3 / CD28 Dynabeads. Cytokine levels were donor-dependent. Immobilized BA3182 induced lower levels of cytokines compared with OKT3 anti-CD3 antibody in PBMC cultures at pH 6.5 and pH 7.4. Of the cytokines tested, only IFN-γ and TNF-α were detectable. Induction of IL-6 by immobilized BA3182 was observed in only one donor at pH 7.4, but it was significantly lower than the levels induced by immobilized OKT3 anti-CD3 antibody. IL-1β, IL-4, IL-17, and IL-10 were induced by immobilized BA3182 in some cultures of human PBMCs at pH 6.5 and pH 7.4, but the levels of these cytokines were generally lower than those induced by immobilized OKT3 in PBMC cultures from the same donors. All cytokines tested were consistently detected in cultures stimulated with immobilized OKT3 anti-CD3 antibody, except for IL-6, which was detectable only in PBMC cultures from two human donors (Figures 36A–36H and 37A–37H). These results suggest that BA3182 induces a weak cytokine response in the absence of the EpCAM antigen.

[0340] Example 28. Pharmacokinetic analysis of anti-EpCAM bispecific antibodies after intravenous administration to female BALB / c nude mice. This example demonstrates the pharmacokinetic analysis of BAP150.31-BF45. Female BALB / c nude mice received a single IV dose of either 1 mg / kg or 10 mg / kg of BAP150.31-BF45, followed by collection of serum samples at different time points. BAP150.31-BF45 concentrations in the collected serum samples were measured by affinity ELISA, which captures BAP150.31-BF45 using plates coated with recombinant human EpCAM. The ELISA results showed that serum BAP150.31-BF45 concentrations increased with increasing doses of BAP150.31-BF45. Furthermore, descriptive PK parameters were established by pharmacokinetic analysis using serum BAP150.31-BF45 concentration-time plots. Overall, the systemic exposure of BAP150.31-BF45 increased with increasing doses. The half-life of BAP150.31-BF45 in mouse serum was calculated to be 58.7 hours based on a 1 mg / kg dose and 47 hours based on a 10 mg / kg dose.

[0341] material antibody BAP150.31-BF45, 0.9 mg / mL reagent Target antigen, human EpCAM-his protein, 2.485 mg / mL, diluted to 1 μg / mL in coating buffer. Anti-human IgG-HRP, Promega, cat#W403B, lot#0000297692. Coating buffer, Sigma, cat#C3041-100CAP. Mouse serum. 3,3',5,5' Tetramethylbenzidine (TMB), Thermofisher, Cat# 002023, Lot# 03069141-7. Bovine serum albumin (BSA), VWR, cat#332-25G, lot#20D0656194. Tween-20, Sigma, cat# P1379-500mL, lot# SLBS7482. HCl, General-Reagent, cat#G81788B, lot#P1972715. Nunc™ MicroWell™ 96-well microplate, Thermofisher, cat#269787. Assay buffer, PBS pH 6.0 containing 1% BSA. Wash buffer, PBS pH 6.0 containing 0.05% Tween-20. Standard dilution buffer, PBS pH 6.0 containing 1% BSA and 1:200 diluted normal mouse serum.

[0342] method standard For the affinity ELISA assay, three-fold serial dilutions of BAP150.31-BF45 were performed in assay buffer at pH 6.0. BAP150.31-BF45 standard concentrations ranged from 2830 to 0.05 ng / mL. ELISA assay Coat a Nunc-Immuno MaxiSorp 96-well plate with 100 μL of 1 μg / mL human EpCAM-his antigen in coating buffer. Cover with a plate sealer and incubate overnight at 4°C. Empty the plate and tap the residual liquid on a paper towel. Add 200 μL of assay buffer per well and shake at 200 RPM for 5 minutes at room temperature. Empty the plate and tap the residual liquid on a paper towel. Repeat the wash step three times. Add 200 μL of assay buffer per well. Shake at 200 RPM for 1 hour at room temperature. Empty the plate and tap the residual liquid on a paper towel. Dilute each of the standard antibodies to 2.83 μg / mL in standard dilution buffer, then make 3-fold serial dilutions in standard dilution buffer. Dilute test serum samples in PBS / 1% BSA assay buffer according to the layout. Add 100 μL of standard antibody and test serum sample to each well according to the plate layout. Shake at 200 RPM for 1 hour at room temperature. Empty the plate and gently tap the residual liquid on a paper towel. Add 200 μL of wash buffer per well and shake at 200 RPM for 5 minutes at room temperature. Repeat the wash step three times. Add 100 μL of goat anti-human IgG-HRP diluted 1:2500 in assay buffer per well. Shake at 200 RPM for 1 hour at room temperature. Empty the plate and gently tap the residual liquid on a paper towel. Add 200 μL of wash buffer per well. Repeat the wash step four times. Add 80 μL of TMB substrate solution to each well. Incubate at room temperature, protected from light, for the specified development time (3 minutes). Add 80 μL of 1N HCl to stop the enzymatic reaction. The optical density (OD) is measured at 450 nm using a Multiskan™ Sky 51119770DP or equivalent.

[0343] Data Analysis: OD values ​​obtained with various concentrations of BAP150.31-BF45 were analyzed using GraphPad Prism software to generate a four-parameter nonlinear regression curve with a variable slope. The concentrations of BAP150.31-BF45 in mouse serum samples were determined by extrapolation from the BAP150.31-BF45 standard curve. Noncompartmental analysis of BAP150.31-BF45 plasma concentrations was performed using PK Solver 2.0.

[0344] In vivo stability study: BALB / c nude mice were randomly divided into two groups of 4 mice per group and intravenously administered BAP150.31-BF45 at 1 mg / kg and 10 mg / kg. The dose was 10 mL / kg. Detailed information on animal grouping and sampling can be found in Table 19. The body weight of all animals was monitored throughout the entire study period, and no weight l...

Claims

1. A conditionally active bispecific antibody comprising: an IgG antibody or antibody fragment that binds to human EpCAM protein, the IgG antibody or antibody fragment comprising a light chain variable region having three complementarity-determining regions L1, L2, and L3, and a heavy chain variable region having three complementarity-determining regions H1, H2, and H3; and at least one scFv antibody fragment that binds to a T lymphocyte protein linked to the C-terminus of at least one light chain of the IgG antibody or antibody fragment, The IgG light chain variable region has the following sequence: i) SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 3; ii) SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:3; iii) SEQ ID NO: 9, SEQ ID NO: 2 and SEQ ID NO: 3; iv) SEQ ID NO: 10, SEQ ID NO: 2 and SEQ ID NO: 3; v) SEQ ID NO: 1, SEQ ID NO: 11 and SEQ ID NO: 3; vi) SEQ ID NO: 1, SEQ ID NO: 12 and SEQ ID NO: 3; vii) SEQ ID NO: 1, SEQ ID NO: 13 and SEQ ID NO: 3; viii) SEQ ID NO: 1, SEQ ID NO: 14 and SEQ ID NO: 3; ix) SEQ ID NO: 1, SEQ ID NO: 15 and SEQ ID NO: 3; x) SEQ ID NO: 1, SEQ ID NO: 16 and SEQ ID NO: 3; xi) SEQ ID NO: 1, SEQ ID NO: 17 and SEQ ID NO: 3; xii) SEQ ID NO: 1, SEQ ID NO: 18 and SEQ ID NO: 3; xiii) SEQ ID NO: 1, SEQ ID NO: 19 and SEQ ID NO: 3; xiv) SEQ ID NO: 1, SEQ ID NO: 20 and SEQ ID NO: 3; xv) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 21; xvi) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 22, and xvii) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 23 a light chain variable region having L1, L2, and L3 complementarity determining regions, each having A conditionally active bispecific antibody, wherein the IgG heavy chain variable region complementarity determining regions H1, H2, and H3 have the following sequences: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

2. A conditionally active bispecific antibody comprising: an IgG antibody or antibody fragment that binds to human EpCAM protein, the IgG antibody or antibody fragment comprising a light chain variable region having three complementarity-determining regions L1, L2, and L3, and a heavy chain variable region having three complementarity-determining regions H1, H2, and H3; and at least one scFv antibody fragment that binds to a T lymphocyte protein linked to the C-terminus of at least one light chain of the IgG antibody or antibody fragment, The IgG heavy chain variable region has the following sequence: i) SEQ ID NO: 24, SEQ ID NO: 5 and SEQ ID NO: 6; ii) SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 6; iii) SEQ ID NO: 26, SEQ ID NO: 5 and SEQ ID NO: 6; iv) SEQ ID NO: 27, SEQ ID NO: 5 and SEQ ID NO: 6; v) SEQ ID NO: 28, SEQ ID NO: 5 and SEQ ID NO: 6; vi) SEQ ID NO: 29, SEQ ID NO: 5 and SEQ ID NO: 6; vii) SEQ ID NO: 30, SEQ ID NO: 5 and SEQ ID NO: 6; viii) SEQ ID NO: 31, SEQ ID NO: 5 and SEQ ID NO: 6; ix) SEQ ID NO: 4, SEQ ID NO: 32 and SEQ ID NO: 6; x) SEQ ID NO: 4, SEQ ID NO: 33 and SEQ ID NO: 6; xi SEQ ID NO: 4, SEQ ID NO: 34 and SEQ ID NO: 6; xii) SEQ ID NO: 4, SEQ ID NO: 35 and SEQ ID NO: 6; xiii) SEQ ID NO: 4, SEQ ID NO: 36 and SEQ ID NO: 6; xiv) SEQ ID NO: 4, SEQ ID NO: 37 and SEQ ID NO: 6; xv) SEQ ID NO: 4, SEQ ID NO: 38 and SEQ ID NO: 6; xvi) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 39; xvii) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 40; xviii) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 41; xix) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 42; xx) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 43, xxi) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 44; xxii) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 45; xxiii) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 46; xxiv) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 47; xxv) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 48; xxvi) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 49, and xxvii) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 50 a heavy chain variable region having H1, H2, and H3 complementarity determining regions, each having A conditionally active bispecific antibody, wherein the IgG light chain variable region complementarity determining regions L1, L2, and L3 have the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

3. 2. The conditionally active bispecific antibody of claim 1 , wherein the IgG heavy chain variable region has the sequence of SEQ ID NO: 52 and the IgG light chain variable region has a sequence selected from the group consisting of SEQ ID NOs: 53 to 69.

4. 3. The conditionally active bispecific antibody of claim 2, wherein the IgG light chain variable region has the sequence of SEQ ID NO: 51 and the IgG heavy chain variable region has a sequence selected from the group consisting of SEQ ID NOs: 70 to 96.

5. 5. The conditionally active bispecific antibody of claim 4, wherein the IgG light chain variable region complementarity determining regions L1, L2, and L3 have the sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the IgG heavy chain complementarity determining regions H1, H2, and H3 have the sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 45, respectively.

6. The conditionally active bispecific antibody of any one of claims 1 to 5, wherein the IgG antibody or antibody fragment is derived from a non-conditionally active parent anti-EpCAM antibody.

7. 7. The conditionally active bispecific antibody of claim 6, wherein the IgG antibody or antibody fragment has a higher binding affinity for the EpCAM protein at a tumor microenvironment pH of 5.0 to 6.9 compared to the binding affinity for the EpCAM protein at a non-tumor microenvironment pH of 7.0 to 7.6, and the conditionally active antibody or antibody fragment has a lower binding affinity for the EpCAM protein at a non-tumor microenvironment pH of 7.0 to 7.6 compared to the binding affinity of the parent antibody for the EpCAM protein at the non-tumor microenvironment pH of 7.0 to 7.

6.

8. 8. The conditionally active bispecific antibody of claim 1, wherein the IgG antibody or antibody fragment has a higher binding affinity to EpCAM protein at a tumor microenvironment pH of 5.0 to 6.9 compared to its binding affinity to EpCAM protein at a non-tumor microenvironment pH of 7.0 to 7.

6.

9. 8. The conditionally active bispecific antibody of claim 1 , wherein the IgG antibody or antibody fragment has a ratio of binding affinity for human EpCAM protein at a pH of 6.0 to binding affinity for the human EpCAM protein at a pH of 7.4 of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 15:1, or at least about 20:

1.

10. 10. The conditionally active bispecific antibody of claim 9, wherein the IgG antibody or antibody fragment has a ratio of binding affinity for human EpCAM protein at a pH of 6.0 to binding affinity for said human EpCAM protein at a pH of 7.4 of at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 15:1, or at least about 20:

1.

11. The conditionally active bispecific antibody according to any one of claims 1 to 10, wherein the scFv antibody fragment binds to CD3 protein.

12. 12. The conditionally active bispecific antibody of claim 11 , wherein the scFv antibody fragment has a greater binding affinity to the CD3 protein at a tumor microenvironment pH of 5.0 to 6.9 compared to its binding affinity to the CD3 protein at a non-tumor microenvironment pH of 7.0 to 7.

6.

13. 13. The conditionally active bispecific antibody of claim 11 or 12, wherein the scFv antibody fragment is derived from a non-conditionally active parent anti-CD3 antibody.

14. 14. The conditionally active bispecific antibody of claim 13, wherein the scFv antibody fragment has a higher binding affinity to the CD3 protein at a tumor microenvironment pH of 5.0 to 6.9 compared to the binding affinity to the CD3 protein at a non-tumor microenvironment pH of 7.0 to 7.6, and the conditionally active scFv fragment has a lower binding affinity to the CD3 protein at a non-tumor microenvironment pH of 7.0 to 7.6 compared to the binding affinity of the parent antibody to the CD3 protein at the non-tumor microenvironment pH of 7.0 to 7.

6.

15. The conditionally active bispecific antibody of claim 11 , wherein the scFv antibody fragment has the sequence of SEQ ID NO:

97.

16. 9. The conditionally active bispecific antibody of claim 8, comprising a light chain having the sequence of SEQ ID NO: 98 and a heavy chain having the sequence of SEQ ID NO:

99.

17. 14. The conditionally active bispecific antibody of any one of claims 11 to 13, wherein the scFv antibody fragment has a greater binding affinity to the CD3 protein at a tumor microenvironment pH of 6.0 compared to its binding affinity to the CD3 protein at a non-tumor microenvironment pH of 7.

4.

18. 18. The conditionally active bispecific antibody of any one of claims 1 to 17, wherein the IgG antibody or antibody fragment has a ratio of binding affinity for cynomolgus EpCAM protein at a pH of 6.0 to binding affinity for the cynomolgus EpCAM protein at a pH of 7.4 of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 15:1 or at least about 20:

1.

19. 18. The conditionally active bispecific antibody of any one of claims 11 to 17, wherein the IgG antibody or antibody fragment has a ratio of binding affinity for cynomolgus monkey EpCAM protein at a pH of 6.0 to binding affinity for the cynomolgus monkey EpCAM protein at a pH of 7.4 of at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 15:1 or at least about 20:

1.

20. A conditionally active antibody or antibody fragment that binds to human EpCAM protein, comprising a light chain variable region having three complementarity determining regions L1, L2, and L3, and a heavy chain variable region having three complementarity determining regions H1, H2, and H3; The light chain variable region has the following sequence: i) SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 3; ii) SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:3; iii) SEQ ID NO: 9, SEQ ID NO: 2 and SEQ ID NO: 3; iv) SEQ ID NO: 10, SEQ ID NO: 2 and SEQ ID NO: 3; v) SEQ ID NO: 1, SEQ ID NO: 11 and SEQ ID NO: 3; vi) SEQ ID NO: 1, SEQ ID NO: 12 and SEQ ID NO: 3; vii) SEQ ID NO: 1, SEQ ID NO: 13 and SEQ ID NO: 3; viii) SEQ ID NO: 1, SEQ ID NO: 14 and SEQ ID NO: 3; ix) SEQ ID NO: 1, SEQ ID NO: 15 and SEQ ID NO: 3; x) SEQ ID NO: 1, SEQ ID NO: 16 and SEQ ID NO: 3; xi) SEQ ID NO: 1, SEQ ID NO: 17 and SEQ ID NO: 3; xii) SEQ ID NO: 1, SEQ ID NO: 18 and SEQ ID NO: 3; xiii) SEQ ID NO: 1, SEQ ID NO: 19 and SEQ ID NO: 3; xiv) SEQ ID NO: 1, SEQ ID NO: 20 and SEQ ID NO: 3; xv) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 21; xvi) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 22, and xvii) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 23 a light chain variable region having L1, L2, and L3 complementarity determining regions, each having A conditionally active antibody or antibody fragment, wherein the heavy chain variable region comprises three complementarity determining regions H1, H2 and H3 having the sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

21. A conditionally active antibody or antibody fragment that binds to human EpCAM protein, comprising a light chain variable region having three complementarity determining regions L1, L2, and L3, and a heavy chain variable region having three complementarity determining regions H1, H2, and H3; The heavy chain variable region has the following sequence: i) SEQ ID NO: 24, SEQ ID NO: 5 and SEQ ID NO: 6; ii) SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 6; iii) SEQ ID NO: 26, SEQ ID NO: 5 and SEQ ID NO: 6; iv) SEQ ID NO: 27, SEQ ID NO: 5 and SEQ ID NO: 6; v) SEQ ID NO: 28, SEQ ID NO: 5 and SEQ ID NO: 6; vi) SEQ ID NO: 29, SEQ ID NO: 5 and SEQ ID NO: 6; vii) SEQ ID NO: 30, SEQ ID NO: 5 and SEQ ID NO: 6; viii) SEQ ID NO: 31, SEQ ID NO: 5 and SEQ ID NO: 6; ix) SEQ ID NO: 4, SEQ ID NO: 32 and SEQ ID NO: 6; x) SEQ ID NO: 4, SEQ ID NO: 33 and SEQ ID NO: 6; xi SEQ ID NO: 4, SEQ ID NO: 34 and SEQ ID NO: 6; xii) SEQ ID NO: 4, SEQ ID NO: 35 and SEQ ID NO: 6; xiii) SEQ ID NO: 4, SEQ ID NO: 36 and SEQ ID NO: 6; xiv) SEQ ID NO: 4, SEQ ID NO: 37 and SEQ ID NO: 6; xv) SEQ ID NO: 4, SEQ ID NO: 38 and SEQ ID NO: 6; xvi) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 39; xvii) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 40; xviii) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 41; xix) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 42; xx) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 43, xxi) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 44; xxii) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 45; xxiii) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 46; xxiv) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 47; xxv) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 48; xxvi) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 49, and xxvii) SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 50 a heavy chain variable region having H1, H2, and H3 complementarity determining regions, each having A conditionally active antibody or antibody fragment, wherein the light chain variable region complementarity determining regions L1, L2, and L3 are the sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

22. 21. The conditionally active antibody or antibody fragment of claim 20, wherein the heavy chain variable region has the sequence of SEQ ID NO: 52 and the light chain variable region has a sequence selected from the group consisting of SEQ ID NOs: 53-69.

23. 22. The conditionally active antibody or antibody fragment of claim 21, wherein the light chain variable region has the sequence of SEQ ID NO: 51 and the heavy chain variable region has a sequence selected from the group consisting of SEQ ID NOs: 70-96.

24. 24. The conditionally active antibody or antibody fragment of claim 23, wherein the light chain variable region complementarity determining regions L1, L2, and L3 have the sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the heavy chain complementarity determining regions H1, H2, and H3 have the sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 45, respectively.

25. 25. The conditionally active antibody or antibody fragment of any one of claims 20 to 24, having a higher binding affinity to human EpCAM protein at a tumor microenvironment pH of 5.0 to 6.9 compared to the binding affinity to human EpCAM protein at a non-tumor microenvironment pH of 7.0 to 7.

6.

26. 25. The conditionally active antibody or antibody fragment of any one of claims 20 to 24, obtained from a non-conditionally active parent anti-EpCAM antibody.

27. 27. The conditionally active antibody of claim 26, wherein the antibody or antibody fragment has a higher binding affinity for the EpCAM protein at a tumor microenvironment pH of 5.0 to 6.9 compared to the binding affinity for the EpCAM protein at a non-tumor microenvironment pH of 7.0 to 7.6, and the conditionally active antibody or antibody fragment has a lower binding affinity for the EpCAM protein at a non-tumor microenvironment pH of 7.0 to 7.6 compared to the binding affinity of the parent antibody for the EpCAM protein at the non-tumor microenvironment pH of 7.0 to 7.

6.

28. 25. The conditionally active antibody or antibody fragment of any one of claims 20 to 24, having a ratio of binding affinity for human EpCAM protein at a pH of 6.0 to binding affinity for said human EpCAM protein at a pH of 7.4 of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 15:1, or at least about 20:

1.

29. 29. The conditionally active antibody or antibody fragment of claim 28, having a ratio of binding affinity for human EpCAM protein at a pH of 6.0 to binding affinity for said human EpCAM protein at a pH of 7.4 of at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 15:1 or at least about 20:

1.

30. 7. The conditionally active bispecific antibody or antibody fragment of claim 1, wherein the binding affinity of said conditionally active bispecific antibody or antibody fragment to human and cynomolgus monkey EpCAM proteins is at least 5 times greater than the binding affinity of said same conditionally active bispecific antibody or antibody fragment to rat or mouse EpCAM proteins, respectively.

31. 31. The conditionally active bispecific antibody or antibody fragment of claim 30, wherein the binding affinity of the conditionally active bispecific antibody or antibody fragment to cynomolgus monkey EpCAM protein is at least 50% of the binding affinity of the same conditionally active antibody or antibody fragment to human EpCAM protein.

32. 32. The conditionally active bispecific antibody or antibody fragment of claim 31 , comprising a light chain comprising the sequence of SEQ ID NO: 98 and a heavy chain comprising the sequence of SEQ ID NO: 99.

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