Conditionally active anti-EPCAM antibodies, antibody fragments, and constructs incorporating them

Abstract

The present invention provides a conditionally active bispecific antibody comprising: an IgG antibody or antibody fragment binding to a 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; and at least one scFv antibody fragment binding to a T-lymphocyte protein attached to the C-terminus of at least one light chain of the IgG antibody or antibody fragment.

Description

Technical Field

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

[0002] Epithelial cell adhesion / activation molecules (EpCAM, also known as CD326, HEA125, MK-1, EGP-2, EGP34, GA733-2, KSA, TROP-1, KS1 / 4, and ESA) Figure 1 Due to its abundant and frequent expression in most cancers of different origins, it has become one of the initial and most important targets for immunotherapy in cancer treatment (Herlyn et al., Proceedings of the National Academy of Sciences). Proc Natl Acad Sci USA )》, 76:1438-1442, 1979; Went et al., Human Pathology ( Hum Pathol This molecule is a relatively small type I transmembrane glycoprotein, 314 amino acids (aa) in length, and is highly conserved throughout evolution. It has been reported to mediate calcium-independent homocellular adhesion (Litvinov et al., *Journal of Cell Biology*, 35:122-128, 2004). J Cell Biology (1994). The molecule consists of a short intracellular domain of 26 aa containing two binding sites for α-actin to interact with the actin cytoskeleton (Balzar et al., Molecular Cell Biology, 125:437-446). Mol Cell Biol. )》, 18(8): 4833-4843, 1998); 23-aa transmembrane domain; 242-aa extracellular domain (ECD); and 23-aa signal peptide cleaved from the mature form. The extracellular domain of EpCAM has three N-linked glycosylation sites. Glycosylation status differences between normal and malignant tissues have been reported in some types of cancer (Pauli et al., Cancer Communications, 18(8): 4833-4843, 1998). Cancer Lett (》, 193:25-32, 2003).

[0003] The extracellular domain contains three domains. The first two domains are believed to resemble epidermal growth factor (EGF)-like repeats, containing twelve cysteine ​​residues (Balza et al., *Molecular Cell Biology*, 21:2570-2580, 2001). However, some studies suggest that the second EGF-like repeat in EpCAM is actually the thyroglobulin (TY) domain (Linnenbach et al., *Proceedings of the National Academy of Sciences*, 86:27-31, 1989; Chong and Speicher, *Journal of Biochemistry*). J Biol Chem ( )》, 276:5804-5813,2001). The third domain is a unique cysteine-poor region (CPR) that is not associated with any known molecule (Baeuerle and Gires, British Journal of Cancer ( ) , 276:5804-5813,2001). Br J Cancer )》, 96:417-423, 2007. EpCAM plays an important role in preventing cell-cell adhesion and in cell signaling, migration, proliferation, and differentiation. Figure 1 ).

[0004] EpCAM expression in humans is epithelial cell specific. Except for squamous epithelium and some specific epithelial cell types, such as epidermal keratinocytes, hepatocytes, gastric parietal cells, and myoepithelial cells, most human epithelial cells express EpCAM (Balza et al., *Journal of Molecular Medicine*). J Mol Med )》, 77: 699-712, 1999: Momburg et al., Cancer Research ( Cancer Res ( )》, 47:2883-2891, 1987). Higher expression levels are usually observed in tumors originating from the epithelium (Balza et al., Journal of Molecular Medicine, 77: 699-712, 1999; Winter et al., American Journal of Pathology ( ) , 47:2883-2891, 1987). Am J Pathol( ), 163:2139-2148, 2003; Vent et al., Human Pathology, 35:122-128, 2004; Vent et al., British Journal of Cancer, 94:128-135, 2006). For example, EpCAM protein has been found to be expressed in a large number of human adenocarcinomas and squamous cell carcinomas (Vent et al., Human Pathology, 35:122-128, 2004). Current studies using immunohistochemical (IHC) staining and microarray techniques have found EpCAM expression in a large number of samples from patients with breast cancer, ovarian cancer, kidney cancer, esophageal cancer, colon cancer, gastric cancer, prostate cancer, and lung cancer (Spizzo et al., Breast Cancer Research and Treatment ( ), 163:2139-2148, 2003; Vent et al., Human Pathology, 35:122-128, 2004; Vent et al., British Journal of Cancer, 94:128-135, 2006). Breast Cancer Res Treat )》, 86:207-213, 2004; Spizo et al., Gynecologic Oncology ( Gynecol Oncol )》, 103:483-488, 2006; Stoecklein et al., BMC Cancer ( BMC Cancer )》, 6:165, 2006; Kimura et al., International Journal of Oncology ( Int J Oncol )》, 30:171-179, 2007; Vent et al., American Journal of Surgical Pathology ( Am J Surg Pathol ( ), 29:83-88, 2005; Vent et al., British Journal of Cancer, 94:128-135, 2006). The data highlight the potential utility of EpCAM as an immunotherapeutic target for the treatment of human cancers.

[0005] Since epithelial cells are known to be the most important cell type in the development of human malignant diseases, and more than 90% of all malignant tumors are of epithelial origin (Birchmeiera et al., Acta Anatomica Sinica),... Acta Anatomica )》, 156(3):217-226, 1996), therefore EpCAM is now considered one of the most frequently and extensively expressed tumor-associated antigens. It has been independently identified multiple times as an immunogenic tumor-associated antigen for monoclonal antibody development (Gottlinger et al., International Journal of Cancer ( ), 156(3):217-226, 1996). Int J Cancer ), 38:47-53, 1986; Edwards et al., Cancer Research ( Cancer Res )》, 46:1306-1317, 1986; Spurr et al., International Journal of Cancer, 38:631-636, 1986; Momburg et al., Cancer Research, 47:2883-2891, 1987; Schön et al., Journal of Research Dermatology (J Investig Dermatol )》, 102: 987-991, 1994; Bumol et al., Fusion Tumor ( Hybridoma ( )》, 7:407-415, 1988; Quak et al., Fusion Tumor, 9:377-387, 1990).

[0006] In fact, the first monoclonal antibody used in human cancer therapy was actually a murine IgG2a antibody, called mAb 17-1A (later named edrecolomab and Panorexs), which targets EpCAM (Sears et al., The Lancet). Lancet )》, 1(8275):762-765, 1982; Sears et al., Journal of Biological Response Modifiers ( J Biol Response Mod )》, 3(2):138-150, 1984). Subsequently, epcam-specific murine, chimeric and humanized monoclonal antibodies were tested preclinically and clinically in the form of native (naked) antibodies, hybrid bispecific (trifunctional) antibodies or conjugates with toxins, radioisotopes or cytokines (IL-2 or GM-CSF) used for cancer treatment (Velders et al., Cancer Research, 54(7):1753-1759, 1994; Raum et al., Cancer Immunology and Immunotherapy ( Cancer Immunol Immunother )》, 50(3):141-150, 2001; Elias et al., American Journal of Respiratory and Critical Care Medicine ( Am J Respir Crit Care Med )》, 150:1114-1122, 1994; Di Paolo et al., Clinical Cancer Research, 9:2837-2848, 2003; Andratschke et al., Anticancer Research ( Anticancer Res )》, 27(1A):431-436, 2007; Xiang et al., Cancer Research, 57(21):4948-4955, 1997; Schanzer et al., Journal of Immunotherapy ( J Immunother )》, 29(5):477-488, 2006; Wimberger et al., International Journal of Cancer ( Int J Cancer)》, 105(2):241-248, 2003; Amann et al., Cancer Research, 68(1):143-151, 2008). To date, many different immunotherapies targeting EpCAM are still in clinical trials (Boyle and Gill, British Journal of Cancer, 96:417-423, 2007). Data from clinical trials suggest that naked anti-EpCAM antibodies, such as ezolocumab (17-1A; Panorexs) and adecatumumab (MT201), have only limited antitumor activity (Punt et al., The Lancet, 360: 671-677, 2002), which may be exerted through activation of the complement system (CDC) and antibody-dependent cytotoxicity (ADCC) (Schwartzberg, Oncology Hematology Review ( Crit Rev Oncol Hematol )》, 40(1):17-24, 2001; Naundorf et al., International Journal of Cancer, 100(1):101-110, 2002; Prang et al., British Journal of Cancer, 92(2):342-349, 2005; Oberneder et al., European Journal of Cancer ( Eur J Cancer ( )》, 42(15):2530-2538, 2006). Antibodies conjugated to highly potent effector mechanisms (such as IL-2, PE toxin, or anti-CD3) appear to have better antitumor activity. However, some side effects limit the systemic use of these anti-EpCAM antibodies (Boyle and Gill, British Journal of Cancer, 96:417-423, 2007).

[0007] ING-1 is an engineered high-affinity human monoclonal antibody that targets EpCAM-positive cells. It has been used in a phase I clinical trial in patients with advanced adenocarcinoma that is difficult to treat with standard therapy, and data from this study suggest that antibodies with high affinity for EpCAM, despite their high cytotoxicity against tumor cells, may also induce rapid pancreatic toxicity, thus limiting their therapeutic window for systemic administration (De Bono et al., Clinical Cancer Research, 10(22):7555-65, 2004). Potential systemic toxicities associated with the therapeutic use of high-affinity anti-EpCAM antibodies can be reduced through pre-targeting strategies, including a chasing step to clear circulating antibodies at a predetermined time. Alternatively, the use of high-affinity anti-EpCAM antibodies can be limited to localized regional therapy.

[0008] Known side effects of anti-EpCAM antibodies are associated with the presence of EpCAM on normal epithelial cells (although at a lower density compared to tumor cells) (Kim et al., Clinical Cancer Research, 10:5464-5471, 2004; Osta et al., Cancer Research, 64:5818-5824, 2004). Therefore, increasing the affinity or specificity of anti-EpCAM antibodies does not reduce their effect in normal tissues expressing EpCAM, which would otherwise cause side effects.

[0009] This disclosure aims to provide anti-EpCAM antibodies or antibody fragments with reduced or minimal side effects, suitable for therapeutic and diagnostic uses, particularly for the diagnosis and treatment of cancer. Some of these anti-EpCAM antibodies or antibody fragments exhibit a higher binding affinity for EpCAM in tumors compared to EpCAM present in normal tissues. These anti-EpCAM antibodies or antibody fragments generally possess efficacy at least comparable to known anti-EpCAM antibodies. Furthermore, due to the relatively low binding affinity for EpCAM in normal tissues, the anti-EpCAM antibodies or antibody fragments of this disclosure exhibit reduced side effects compared to monoclonal anti-EpCAM antibodies known in the art. These advantages enable more selective targeting of EpCAM in tumors, and the selectivity of the antibody for EpCAM present in tumors allows for the use of higher doses of these anti-EpCAM antibodies or antibody fragments, thereby enabling more effective therapeutic treatment without a corresponding increase in undesirable side effects.

[0010] Antibodies have become a major therapeutic protein. Traditional antibodies typically bind to a single epitope on an antigen. Novel antibody constructs, called multispecific antibodies, have been developed to bind to more than one antigen or to more than one epitope 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. The EU and the US have approved two bispecific antibody drugs for the treatment of oncology (Catumaxomab™ and Blinatumab™). Due to their unique properties, multispecific antibodies have become attractive next-generation antibody therapeutics.

[0011] US 2013 / 0017200 discloses a method for synthesizing multispecific antibodies. A first antibody fragment having a free thiol group is obtained from a first parent antibody having a first monospecificity. This free thiol group is capable of reacting with a sulfur-reactive cross-linking agent to produce an antibody fragment-cross-linking agent moiety. The antibody fragment-cross-linking agent moiety reacts in pairs with each of two or more other antibody fragments obtained from other parent antibodies to produce multispecific antibodies, wherein the other parent antibodies have a different monospecificity than the first antibody fragment and each of the other antibody fragments has a free thiol group. Multispecific antibodies are suitable as novel therapeutic and diagnostic agents.

[0012] Brinkmann and Kontermann (“The making of bispecific antibodies”, [reference needed]) MABS The journal *[Journal Name]* (Vol. 9, 2017, pp. 182-212) investigated the formats of bispecific antibodies, including small molecules consisting only of the antigen-binding sites of two antibodies, molecules with IgG structures, and large, complex molecules composed of different antigen-binding parts typically combined with dimer modules. Depending on the application, bispecific antibodies can vary in the size, configuration, valence, flexibility, and geometry of their binding modules, as well as their distribution and pharmacokinetic properties. Together, these bispecific formats increase the diversity of antibodies that can be used to develop therapeutics for various indications. Examples of specific bispecific formats can be found in [Journal Name]. Figure 2 and 3 middle.

[0013] It is also desirable to develop suitable antibodies with conditional activity. Examples include antibodies that are almost inactive under normal physiological conditions but exhibit significantly higher activity under conditions other than normal physiological conditions (e.g., abnormal conditions), antibodies that are activated or inactivated in certain microenvironments (e.g., in the tumor microenvironment), or antibodies that are activated or inactivated over time. Other triggering conditions, besides temperature, that can induce antibody evolution or optimization include pH, osmolarity, molar osmolar concentration, oxidative stress, and electrolyte concentration. Other desired antibody properties that can be optimized during evolution, besides activity, include stability, half-life, chemical resistance, and proteolytic resistance.

[0014] Numerous strategies have been disclosed for evolving or engineering parental antibodies into mutant antibodies with desired properties. However, engineering or evolving parental antibodies to be inactive or nearly inactive (less than 10% activity, especially less than 5%) under normal physiological conditions while maintaining substantial activity under abnormal conditions requires the coexistence of one or more destabilizing mutations with activity-increasing mutations that do not counteract the destabilizing effect. The amount by which destabilizing mutations reduce antibody activity is expected to be greater than predicted by standard rules, such as the Q10 rule. Therefore, the ability to evolve proteins to function effectively (with higher activity) under specific abnormal conditions, such as lower temperatures or pH values, while remaining substantially inactive under their normal operating conditions, has yielded unexpectedly novel antibodies known as conditionally active antibodies.

[0015] The embodiments disclosed herein include a novel multispecific antibody having conditional activity for binding to the EpCAM antigen. This novel multispecific antibody leverages the flexibility and versatility of conventional multispecific antibodies while directing the activity, affinity, and / or cohesion of the multispecific antibody to the desired site, tissue, or organ in the subject. Consequently, these multispecific antibodies avoid or significantly reduce side effects associated with binding to normal cells, including (but not limited to) avoiding excessive cytokine production. Summary of the Invention

[0016] This document provides conditionally active antibodies or antibody fragments that bind to human EpCAM protein or epitopes of EpCAM protein, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region includes three complementarity-determining regions L1, L2, and L3, and the heavy chain variable region includes 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 Complementary decision regions of embodiments of this disclosure

[0018]

[0019] In several other embodiments, the conditionally active anti-EpCAM antibody and antibody fragment may be selected from combinations of the following: the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:53; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:54; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:55; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:56; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:57; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:58; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:59; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:60; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:61; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:53; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:61; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:54; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:55; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:56; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:57; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:58; the weight-variable region of SEQ ID NO:52 and the light chain variable region of Light chain variable region of SEQ ID NO:62; light chain variable region of SEQ ID NO:52 and SEQ ID NO:63; light chain variable region of SEQ ID NO:52 and SEQ ID NO:64; light chain variable region of SEQ ID NO:52 and SEQ ID NO:65; light chain variable region of SEQ ID NO:52 and SEQ ID NO:66; light chain variable region of SEQ ID NO:52 and SEQ ID NO:67; light chain variable region of SEQ ID NO:52 and SEQ ID NO:68; and light chain variable region of SEQ ID NO:52 and SEQ ID NO:69.

[0020] In other embodiments, the conditionally active anti-EpCAM antibody and antibody fragment may include a combination selected from: the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:70; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:71; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:72; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:73; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:74; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:75; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:76; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:77; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:78; SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:78; SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:79; SEQ ID NO:50 and the heavy chain variable region of SEQ ID NO:70; SEQ ID NO:51 ...0; SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:70; SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:70; SEQ ID NO:51 and the heavy chain variable region of SEQ The light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:79; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:80; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:81; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:82; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:83; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:84; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:85; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:86; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:87; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:88; SEQ ID The light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:89; 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.

[0021] The conditionally active antibody or antibody fragment of this disclosure exhibits increased binding affinity for EpCAM protein or EpCAM protein epitopes under abnormal conditions different from normal physiological conditions, and decreased binding affinity for EpCAM protein or EpCAM protein epitopes under normal physiological conditions. In one embodiment, the abnormal conditions are conditions in the tumor microenvironment, and the normal physiological conditions are conditions in the non-tumor microenvironment. In another embodiment, the conditions are pH values. In one specific embodiment, the abnormal conditions are pH 5.0 to 6.9 in the tumor microenvironment, and the normal physiological conditions are pH 7.0 to 7.6 in the non-tumor microenvironment. In another specific embodiment, the binding affinity of the conditionally active anti-EpCAM antibody or antibody fragment of this disclosure to EpCAM protein or EpCAM protein epitopes at pH 6.0 is increased compared to the binding affinity of the same conditionally active anti-EpCAM antibody or antibody fragment at pH 7.4.

[0022] In one embodiment, the conditionally active anti-EpCAM antibody or antibody fragment disclosed herein is obtained from an unconditionally active anti-EpCAM parental antibody. In a specific embodiment, the unconditionally active anti-EpCAM parental antibody has the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:52. In this embodiment, the binding affinity of the conditionally active anti-EpCAM antibody or antibody fragment disclosed herein to EpCAM protein or EpCAM protein epitopes at pH 5.0 to 6.9 in the tumor microenvironment is increased compared to the binding affinity of the same conditionally active anti-EpCAM antibody or antibody fragment at pH 7.0 to 7.6 in a non-tumor microenvironment, and the binding affinity to EpCAM protein or EpCAM protein epitopes at pH 7.0 to 7.6 is decreased compared to the binding affinity of the parental unconditionally active anti-EpCAM antibody or antibody fragment at pH 7.0 to 7.6. In specific embodiments, the binding affinity of the conditionally active anti-EpCAM antibody disclosed herein to EpCAM protein or EpCAM protein epitopes at pH 6.0 is increased compared to the binding affinity of the same conditionally active anti-EpCAM antibody at pH 7.4, and the binding affinity to EpCAM protein or EpCAM protein epitopes at pH 7.4 is decreased compared to the binding affinity of the parental unconditionally active anti-EpCAM antibody to EpCAM protein or EpCAM protein epitopes at pH 7.4.

[0023] In one embodiment, the ratio of the binding affinity of the conditionally active anti-EpCAM antibody or antibody fragment disclosed herein to the EpCAM protein or the epitope of the EpCAM protein at pH 6.0 to the binding affinity to the EpCAM protein or the epitope of the EpCAM protein at pH 7.4 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.

[0024] This document also provides multispecific antibodies comprising a conditionally active anti-EpCAM antibody or antibody fragment of the present disclosure and at least one scFv antibody fragment bound to a T-lymphocyte antigen, the scFv antibody fragment being 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.

[0025] In one embodiment, the anti-EpCAM antibody or antibody fragment of the multispecific antibody comprises a light chain variable region containing three complementarity-determining regions L1, L2, and L3, and a heavy chain variable region containing three complementarity-determining regions H1, H2, and H3. The light and heavy chain complementarity-determining regions of the conditionally active anti-EpCAM antibody or antibody fragment in the multispecific antibody can be obtained from the various embodiments disclosed in Table 1 above.

[0026] In several other embodiments, the conditionally active anti-EpCAM antibody or antibody fragment of the multispecific antibody may include combinations of the following: the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:53; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:54; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:55; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:56; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:57; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:58; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:59; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:60; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:61; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:53; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:61; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:54; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:55; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:56; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:57; the weight-variable region of SEQ ID NO:52 and the light chain variable region of SEQ ID NO:58; the weight-variable region of SEQ ID NO:52 and the light chain Light chain variable region of SEQ ID NO:62; light chain variable region of SEQ ID NO:52 and light chain variable region of SEQ ID NO:63; light chain variable region of SEQ ID NO:52 and light chain variable region of SEQ ID NO:64; light chain variable region of SEQ ID NO:52 and light chain variable region of SEQ ID NO:65; light chain variable region of SEQ ID NO:52 and light chain variable region of SEQ ID NO:66; light chain variable region of SEQ ID NO:52 and light chain variable region of SEQ ID NO:67; light chain variable region of SEQ ID NO:52 and light chain variable region of SEQ ID NO:68; and light chain variable region of SEQ ID NO:52 and light chain variable region of SEQ ID NO:69.

[0027] In other embodiments, the conditionally active anti-EpCAM antibody or antibody fragment of the multispecific antibody contains a combination of the following: the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:70; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:71; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:72; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:73; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:74; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:75; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:76; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:77; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:78; SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:78; SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:79; SEQ ID NO:50 and the heavy chain variable region of SEQ ID NO:70; SEQ ID NO:51 ... The light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:79; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:80; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:81; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:82; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:83; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:84; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:85; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:86; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:87; the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:88; SEQ ID The light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:89; 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.

[0028] In one embodiment, the conditionally active anti-EpCAM antibody or antibody fragment of the multispecific antibody disclosed herein is obtained from an unconditionally active anti-EpCAM parent antibody. In a specific embodiment, the unconditionally active anti-EpCAM parent antibody has the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:52. In this embodiment, the binding affinity of the conditionally active anti-EpCAM antibody or antibody fragment of the multispecific antibody disclosed herein to EpCAM protein or EpCAM protein epitopes at pH 5.0 to 6.9 in the tumor microenvironment is increased compared to the binding affinity of the same conditionally active anti-EpCAM antibody or antibody fragment at pH 7.0 to 7.6 in the non-tumor microenvironment, and the binding affinity to EpCAM protein or EpCAM protein epitopes at pH 7.0 to 7.6 is decreased compared to the binding affinity of the parental unconditionally active anti-EpCAM antibody or antibody fragment at pH 7.0 to 7.6. In specific embodiments, the binding affinity of the conditionally active anti-EpCAM antibody among the multispecific antibodies disclosed herein to the EpCAM protein or its epitope at pH 6.0 is increased compared to the binding affinity of the same conditionally active anti-EpCAM antibody at pH 7.4, and the binding affinity to the EpCAM protein or its epitope at pH 7.4 is decreased compared to the binding affinity of the parental unconditionally active anti-EpCAM antibody at pH 7.4.

[0029] In one embodiment, the ratio of the binding affinity of the conditionally active anti-EpCAM antibody or antibody fragment of the multispecific antibody disclosed herein to the EpCAM protein or its epitope at pH 6.0 to that at pH 7.4 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. In one specific embodiment, the ratio of the binding affinity of the conditionally active anti-EpCAM antibody or antibody fragment of the multispecific antibody disclosed herein to the EpCAM protein or its epitope at pH 6.0 to that at pH 7.4 is at least about 6:1. In one embodiment, the binding affinity of the conditionally active anti-EpCAM antibody or antibody fragment in the multispecific antibodies disclosed herein to the EpCAM protein or the epitope of the EpCAM protein at pH 6.0 is at least about 8:1 to the binding affinity to the EpCAM protein or the epitope of the EpCAM protein at pH 7.4.

[0030] In one embodiment, the conditionally active anti-EpCAM antibody or antibody fragment in 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 still other embodiments, the anti-lymphocyte antigen scFv antibody or antibody fragment is linked to the C-terminus of the light chain of the conditionally active anti-EpCAM IgG antibody or antibody fragment.

[0031] In some embodiments, the scFv anti-lymphocyte antigen antibody or antibody fragment is an anti-CD3 scFv antibody. In some 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 an unconditionally active antibody or antibody fragment. In embodiments where the anti-CD3 scFv antibody or antibody fragment has conditional activity, the binding affinity of the anti-CD3 scFv antibody to the CD3 antigen is higher at pH 5.0 to 6.9 in the tumor microenvironment than the binding affinity of the same conditionally active anti-CD3 scFv antibody or antibody fragment at pH 7.0 to 7.6 in the non-tumor microenvironment. In a specific embodiment, the anti-CD3 scFv antibody or antibody fragment comprises the light chain variable region of SEQ ID NO: 101 and the heavy chain variable region of SEQ ID NO: 100. In another embodiment, the anti-CD3 scFv antibody comprises SEQ ID NO: 97. In one specific embodiment, the conditionally active anti-EpCAM / anti-CD3 multispecific antibody comprises the light chain of SEQ ID NO:98 and the heavy chain of SEQ ID NO:99.

[0032] In another embodiment, in addition to human EpCAM protein, the conditionally active anti-EpCAM antibody or antibody fragment in the multispecific antibody binds to cynomolgus macaque EpCAM protein, and the ratio of the binding affinity to cynomolgus macaque EpCAM protein at pH 6.0 to the binding affinity to cynomolgus macaque EpCAM protein at pH 7.4 is 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 one specific embodiment, the light chain variable regions CDR 1, 2, and 3 of the anti-EpCAM antibody or antibody fragment in the multispecific antibody have SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the heavy chain variable regions CDR have SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:45, respectively. In another embodiment, the anti-EpCAM antibody or antibody fragment in the multispecific antibody has the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of SEQ ID NO:91. In any of the foregoing embodiments, the multispecific antibody can be a bispecific antibody. In some embodiments, the bispecific antibody is an anti-EpCAM / anti-CD3 antibody. In these embodiments, the anti-CD3 antibody is an scFv antibody having the light chain variable region of SEQ ID NO:101 and the heavy chain variable region of SEQ ID NO:100. In a more specific embodiment, the anti-CD3 scFv antibody has the amino acid sequence of SEQ ID NO:97. In 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. Attached Figure Description

[0033] Figure 1 A schematic diagram illustrating the function of EpCAM in cancer metastasis and progression.

[0034] Figure 2 A schematic structure of a bivalent multispecific antibody is shown, which is a heterodimer having one arm for binding to EpCAM (Ag) and another arm for binding to CD3.

[0035] Figure 3 A schematic structure of a tetravalent multispecific antibody is shown, wherein the antibody is a homodimer, and each arm has a binding site for an antigen (Ag) and a binding site for CD3.

[0036] Figures 4A-4B Sequence alignment of an exemplary light chain variable region of the anti-EpCAM antibody disclosed herein.

[0037] Figure 5A ,5B Sequence alignment of the exemplary heavy chain variable region of the anti-EpCAM antibody disclosed herein with 5C.

[0038] Figure 6A This study demonstrates the binding affinity of BA3182 to human CD3 / human EpCAM at pH 6.0 and pH 7.4. The binding of BA3182 to human CD3 and human EpCAM was measured by ELISA at pH 6.0 and pH 7.4. The average OD values ​​from two replicas at pH 6.0 and pH 7.4 were plotted for different concentrations of BA3182. The plots show the dose-response binding curves of BA3182 to human CD3 / human EpCAM antigen at different pH values. Data represent three experiments. Y-axis: OD 450 nm. X-axis: BA3182 concentration.

[0039] Figure 6B This study demonstrates the binding affinity of BA3182 to human CD3 / cynomolgus monkey EpCAM at pH 6.0 and pH 7.4. The binding of BA3182 to human CD3 and cynomolgus monkey EpCAM was measured by ELISA at pH 6.0 and pH 7.4. The average OD values ​​from two replicates at pH 6.0 and pH 7.4 were plotted for different concentrations of BA3182. The plots show the dose-response binding curves of BA3182 to human CD3 / cynomolgus monkey EpCAM antigen at different pH values. Data represent three experiments. Y-axis: OD 450 nm. X-axis: BA3182 concentration.

[0040] Figure 7 This image shows the binding of the BA3182 bispecific antibody to human CD3 and human EpCAM at different pH values, as measured by a sandwich affinity ELISA. The average OD values ​​from two replicates at each tested pH value are also shown. Y-axis: OD 450 nm. X-axis: pH value.

[0041] Figure 8 The binding of BA3182 to human CD3 and the extracellular domain of EpCAM in humans, cynomolgus monkeys, rats, and mice is demonstrated at pH 6.0 and pH 7.4.

[0042] Figure 9A and 9BThe binding of BA3182 to the extracellular domains of human CD3 / human EpCAM and human CD3 / human Trop2 is shown at pH 6.0 and pH 7.4, respectively. The binding of BA3182 to recombinant human CD3 and (A) human EpCAM or (B) human Trop2 ECD was measured by sandwich ELISA at pH 6.0 and pH 7.4. Results of a representative experiment are presented. Black column: BA3182 bispecific antibody; white column: anti-Trop2 bispecific antibody.

[0043] Figure 10A and 10B The binding of BA3182 to the extracellular domains of human CD3 / human EpCAM and human CD3 / unrelated human antigens is shown 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 was measured by sandwich ELISA at pH 6.0 and pH 7.4. Results of a representative experiment are presented. Black column: BA3182 bispecific antibody; white column: positive control bispecific antibody targeting undisclosed unrelated antigens.

[0044] Figure 11A and 11B Demonstrates the binding of BA3182 to the extracellular domains of human EpCAM and human Trop2 at pH 6.0 and pH 7.4. The binding of BA3182 to the extracellular domains of human EpCAM at pH 6.0 and pH 7.4 was measured by affinity ELISA. Figure 11A ) and human Trop2 ECD ( Figure 11B The combination of ) is shown. The results of a representative experiment are presented. Black column: BA3182 bispecific antibody; patterned column: anti-Trop2 antibody.

[0045] Figure 12A , 12B The 12C diagram illustrates a representative selection strategy for FACS analysis of EpCAM-positive cells. Only FACS analysis of CHO-hEpCAM cells stained with the secondary antibody goat anti-hIgG1AF488 is shown. Figure 12A Density curves of live cell populations obtained by forward scattering (FSC) versus side scattering (SSC) are shown. Figure 12B A single cell population is shown under a live cell gate, determined by the ratio of forward scattering height (FSC-H) to forward scattering area (FSC-A). Figure 12C This is a histogram showing the cutoff gate used to identify AF488-positive single cells.

[0046] Figure 13A , 13BThe binding assays of BA3182 with EpCAM-expressing cells at pH 6.0 and pH 7.4 are shown. The binding assays of BA3182 with the EpCAM antigen expressed on the cell surface are also shown. Figure 13A The binding analysis of CHO hEpCAM cells and CHO cynoEpCAM cells was presented in [the study]. Figure 13B In, and the binding analysis with HCT116 cells was shown in Figure 13C Cells were stained with BA3182 at pH 6.0 and pH 7.4. The data shown represent three independent experiments. Y-axis: Median fluorescence intensity (MFI) value. X-axis: Antibody concentration. The initial concentration of BA3182 for staining CHO hEpCAM and HCT116 cells was 500 nM. For staining CHO cynoEpCAM cells, the initial concentration of BA3182 was 1500 nM.

[0047] Figure 14A , 14B The binding assays of BA3182 with CD3-expressing cells at pH 6.0 and pH 7.4 are shown. The binding assays of BA3182 with CD3 antigen expressed on the cell surface are also shown, with the analysis in human PBMCs shown. Figure 14A The analysis in cynomolgus monkey PBMCs is presented in [the following text is missing from the original] Figure 14B In, and the analysis in Jurkat cells is shown in Figure 14C 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 used for staining cells expressing CD3 was 2500 nM.

[0048] Figure 15A The standard curve of PE beads is shown.

[0049] Figure 15BThis study visualizes the expression levels of EpCAM antigen on the surface of CHO hEpCAM, CHO cynoEpCAM, and HCT116 cells. EpCAM expression levels on CHO-hEpCAM, CHO-cynoEpCAM, and HCT116 cells were assessed using the QantiBrite™ PE Quantification Kit from BD, which contains a mixture of beads loaded with known amounts of phycoerythrin (PE) molecules (high, intermediate, and low). A standard curve was generated using the log-geometric mean of PE fluorescence intensity from the beads and the number of PE molecules per bead, provided by the supplier. The number of PE molecules on EpCAM-expressing cells stained with the anti-hEpCAM PE conjugate antibody was calculated by extrapolating from the bead standard curve using the log-geometric mean of PE fluorescence on stained cells. The anti-hEpCAM antibody bound well to cells expressing human and cynomolgus macaque EpCAM.

[0050] Figure 16A , 16B BA3182 and 16C demonstrate in vitro functional activity against EpCAM-expressing target cells at pH 6.0 and pH 7.4. BA3182-mediated T cell activation is also observed at pH 6.0 and pH 7.4. Figure 16A CHO hEpCAM cells. Figure 16B CHO cynoEpCAM cells and ( Figure 16C HCT116 cells. The average RLU values ​​from two replicates are shown. Y-axis: Relative optical units (RLU). X-axis: Logarithmic antibody concentration (nM). Red circle: pH 6.0. Blue square: pH 7.4. Data represent three experiments.

[0051] Figure 17A , 17B BA3182 demonstrated in vitro cytotoxic activity against HCT116 / human PBMCs and cynoEpCAM-expressing CHO cells / cynomolgus monkey PBMCs at pH 6.5 and pH 7.4. At pH 6.5... and pH 7.4 Cell lysis of target cells expressing EpCAM mediated by PBMCs activated by BA3182 antibody. Figure 17A Displaying HCT116 cells / human PBMCs at pH 6.5. Figure 17B Displaying HCT116 cells / human PBMCs at pH 7.4, and Figure 17C Displaying CHOcynoEpCAM / cynomolgus monkey PBMCs. X-axis: Logarithmic concentration of BA3182, in pM; Y-axis: Percentage of cell lysis. At pH 6.5, N=10 and at pH 7.4, N=6. For the four batches of PBMCs, there were not enough cells available for testing at either pH value.

[0052] Figure 18A and 18B Demonstrating the effectiveness of BA3182 in the presence of HCT116 cancer cells. Figure 18A ) and isotype control antibody ( Figure 18B Human PBMCs were stimulated to induce IL-2. Cultures were maintained at 37°C and 5% CO2 for 48 hours, and the supernatant was collected. IL-2 cytokines were measured using the Human IL-2 Quantikine™ ELISA assay (R&D Systems, Andy Biotech). PBMCs from nine human subjects were tested. Y-axis: IL-2 concentration, in pg / mL. X-axis: Logarithmic concentration of BA3182 or isotype control antibody.

[0053] Figure 19A and 19B Demonstrating the effectiveness of BA3182 in the presence of HCT116 cancer cells. Figure 19A ) and isotype control antibody ( Figure 19B Human PBMCs were stimulated to induce INFγ. Cultures were maintained at 37°C and 5% CO2 for 48 hours, and the supernatant was collected. IL-2 cytokines were measured using the Human IFNγ Quantikine™ ELISA assay (Andy Biotech). PBMCs from 9 human subjects were tested. Y-axis: IFNγ concentration, in pg / mL. X-axis: Logarithmic concentration of BA3182 or isotype control antibody.

[0054] Figure 20A and 20B Demonstrating the effectiveness of BA3182 in the presence of HCT116 cancer cells. Figure 20A ) and isotype control antibody ( Figure 20B Human PBMCs were stimulated to induce IL-6. Cultures were maintained at 37°C and 5% CO2 for 48 hours, and the supernatant was collected. IL-2 cytokines were measured using the Human IL-6 Quantikine™ ELISA assay (Andy Biotech). PBMCs from 9 human subjects were tested. Y-axis: IL-6 concentration, in pg / mL. X-axis: Logarithmic concentration of BA3182 or isotype control antibody.

[0055] Figure 21A and 21B Demonstrating the effectiveness of BA3182 in the presence of HCT116 cancer cells. Figure 21A ) and isotype control antibody (Figure 21B Human PBMCs were stimulated to induce IL-10. Cultures were maintained at 37°C and 5% CO2 for 48 hours, and the supernatant was collected. IL-2 cytokines were measured using the Human IL-10 Quantikine™ ELISA assay (Andy Biotech). PBMCs from 9 human subjects were tested. Y-axis: IL-10 concentration, in pg / mL. X-axis: Logarithmic concentration of BA3182 or isotype control antibody.

[0056] Figure 22A and 22B This study demonstrates the induction of TNFα in human PBMCs in the presence of HCT116 cancer cells by stimulation with BA3182 and an isotype control antibody. Cultures were maintained at 37°C and 5% CO2 for 48 hours, and the supernatant was collected. IL-2 cytokines were measured using the HumanTNFα Quantikine™ ELISA assay (Andy Biotech). PBMCs from nine human subjects were analyzed. Y-axis: TNFα concentration, in pg / mL. X-axis: Logarithmic concentration of BA3182 or the isotype control antibody.

[0057] Figure 23 This paper demonstrates an ELISA assay that confirms the binding of human C1q protein to antibodies BA3182 and B12. B12 is used, a human IgG antibody targeting HIV envelope protein 120 (gp120). 1,k The antibody served as a positive control for this assay. The average OD values ​​from two replicates are shown. Data represent two experiments. Y-axis: OD at 450 nm. X-axis: C1q concentration, in nM.

[0058] Figure 24 This image shows SPR sensing plots of BA3182 binding to the extracellular domain of EpCAM in humans and cynomolgus monkeys at different pH values. The binding of BA3182 to EpCAM in humans and cynomolgus monkeys was measured by SPR at pH 6.0, pH 6.5, and pH 7.4. Binding curves from a representative experiment at each pH value are shown. Data were fitted using a 1:1 Langmuir binding model. Due to the engineered pH-dependent binding of BA3182, a maximum decrease in binding signal occurs from pH 6.0 to pH 7.4. Left column: Sensing plots of BA3182 binding to human EpCAM at pH 6.0 (top), pH 6.5 (middle), and pH 7.4 (bottom). Right column: Sensing plots of BA3182 binding to EpCAM in cynomolgus monkeys at pH 6.0 (top), pH 6.5 (middle), and pH 7.4 (bottom).

[0059] Figure 25 This image shows SPR sensing plots of BA3182 binding to the extracellular domain of CD3 in humans and cynomolgus monkeys at different pH values. The binding of BA3182 to CD3 in humans and cynomolgus monkeys was measured by SPR at pH 6.0, pH 6.5, and pH 7.4. Binding curves from a representative experiment at each pH value are shown. Data were fitted using a 1:1 Langmuir binding model. Due to the engineered pH-dependent binding of BA3182, a maximum decrease in binding signal occurs from pH 6.0 to pH 7.4. Left column: Sensing plots of BA3182 binding to human CD3 at pH 6.0 (top), pH 6.5 (middle), and pH 7.4 (bottom). Right column: Sensing plots of BA3182 binding to CD3 in cynomolgus monkeys at pH 6.0 (top), pH 6.5 (middle), and pH 7.4 (bottom).

[0060] Figure 26 The binding kinetics of BA3182 to human EpCAM under different pH conditions are demonstrated using SPR simulation software. Simulated sensor plots (top right) were generated using SPR simulation software as described in 3.6. The simulations show that the maximum signal reduction from pH 6.0 (top) to pH 6.5 (middle) and pH 7.4 (bottom) can be best simulated by the reduction of active ligands on the sensor surface.

[0061] Figure 27 The analysis of the binding kinetics of BA3182 to human CD3 at different pH values ​​is presented using SPR simulation software. Simulated sensor plots were generated using SPR simulation software as described in 3.6 (top right). The simulations show that the maximum signal reduction from pH 6.0 (top) to pH 6.5 (middle) and pH 7.4 (bottom) can be best simulated by the reduction of active ligands on the sensor surface.

[0062] Figure 28This section illustrates the binding of BA3182 to FcγRI (CD64). Sensing plots of BA3182 (top left) and IgG1-ctrl mAb (top right) are shown at the same scale. Bottom: Kinetic fit of BA3182 binding to FcγRI using a steady-state model. Sensing plots obtained by subtracting the reference point and buffer injection only from the test sensing plots. Experimental data for IgG1-ctrl mAb are fitted using a 1:1 binding model, and data for BA3182 are fitted using a steady-state model (blue: IgG1-ctrl mAb, red: BA3182). Molar concentrations are calculated using a molecular weight of 150 kDa for IgG1-ctrl mAb and 200 kDa for BA3182. The IgG1-ctrl antibody with the wild-type human IgG1 Fc domain binds to FcγRI with an affinity of 1.18 nM. The binding signal for BA3182 is significantly lower, and a signal is detected only at the three highest test concentrations. All binding curves reached saturation, so a steady-state model was used to analyze the data. However, the signal intensity and the differences between different concentrations were too low to calculate a meaningful K. D Data indicate that BA3182 does not bind to FcγRI.

[0063] Figure 29 This section illustrates the binding of BA3182 to FcγRIIa (CD32a). Sensing plots of BA3182 (top left) and IgG1-ctrl mAb (top right) are shown at the same scale. Bottom: Kinetic fit of experimental data for FcγRIIa using a steady-state model (blue: IgG1-ctrl mAb, red: BA3182). Sensing plots obtained by subtracting the reference point from the test sensing plot and by injecting buffer only. Molar concentrations were calculated using a molecular weight of 150 kDa for IgG1-ctrl mAb and 200 kDa for BA3182. The IgG1-ctrl antibody, possessing the wild-type human IgG1 Fc domain, binds to FcγRIIa with an affinity of 0.82 µM. No binding of BA3182 to FcγRIIa was detected, indicating that BA3182 does not interact with FcγRIIa.

[0064] Figure 30This section illustrates the binding of BA3182 to FcγRIIb / c (CD32b / c). Sensing plots of BA3182 (top left) and IgG1-ctrl mAb (top right) are shown at the same scale. Bottom: Kinetic fit of experimental data for FcγRIIb / c using a steady-state model (blue: IgG1-ctrl mAb, red: BA3182). Sensing plots obtained by subtracting the reference point from the test sensing plot and by injecting buffer only. Molar concentrations were calculated using a molecular weight of 150 kDa for IgG1-ctrl mAb and 200 kDa for BA3182. The IgG1-ctrl antibody, possessing the wild-type human IgG1 Fc domain, binds to FcγRIIb / c with an affinity of 4.2 µM. No binding of BA3182 to FcγRIIb / c was detected, indicating that BA3182 does not interact with FcγRIIb / c.

[0065] Figure 31 This image shows the binding of BA3182 to FcγIIIa CD16a (F158). Sensing plots of BA3182 (top left) and IgG1-ctrl mAb (top right) are shown at the same scale. Bottom: Kinetic fit of experimental data for FcγRIIIa using a steady-state model (blue: IgG1-ctrl mAb, red: BA3182). Sensing plots obtained by subtracting the reference point from the test sensing plot and by injecting buffer only. Molar concentrations were calculated using a molecular weight of 150 kDa for IgG1-ctrl mAb and 200 kDa for BA3182. The IgG1-ctrl antibody, containing the wild-type human IgG1 Fc domain, binds to FcγRIIIa with an affinity of 0.86 µM. No binding of BA3182 to FcγRIIIa was detected, indicating that BA3182 does not interact with FcγRIIIa.

[0066] Figure 32 This section illustrates the binding of BA3182 to FcγIIIb (CD16b). Sensing plots of BA3182 (top left) and IgG1-ctrl mAb (top right) are shown at the same scale. Bottom: Kinetic fit of experimental data for FcγRIIIb using a steady-state model (blue: IgG1-ctrl mAb, red: BA3182). Sensing plots obtained by subtracting the reference point from the test sensing plot and by injecting buffer only. Molar concentrations were calculated using a molecular weight of 150 kDa for IgG1-ctrl mAb and 200 kDa for BA3182. The IgG1-ctrl antibody, possessing the wild-type human IgG1 Fc domain, binds to FcγRIIIa with an affinity of 6.5 µM. No binding of BA3182 to FcγRIIIa was detected, indicating that BA3182 does not interact with FcγRIIIa.

[0067] Figure 33 Demonstrating the binding of BA3182 to FcRn. BA3182 binding to FcRn. Top: Sensing plots of BA3182 (left) and IgG1-ctrl mAb (right) at the same scale. Bottom: Kinetic fit of experimental data for FcRn using a steady-state model (blue: BA3182, red: IgG1-Ctrl mAb). Sensing plots obtained by subtracting the reference point from the test sensing plot and by injecting buffer only. Calculation of the molar concentration of FcRn using a molecular weight of 61.91 kDa. The IgG1-Ctrl antibody with the wild-type human IgG1 Fc domain binds to FcRn with an affinity of 366 nM. Similarly, BA3182 shows a binding affinity of 347 nM for FcRn.

[0068] Figures 34A-34H This study demonstrates cytokine release in human PBMC cultures stimulated with soluble BA3182 or CD3 / CD28 dynabeads at pH 6.5. Human PBMC cultures were stimulated with soluble BA3182 or CD3 / CD28 dynabeads at pH 6.5 for 48 hours in culture medium. Supernatants were collected, and cytokine concentrations were measured using a Luminex multiplex cytokine assay. Data show the levels (in pg / mL) of various cytokines in cultures of human PBMCs from nine different donors using CD3 / CD28 dynabeads: 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 However, these cytokines were not detected when treated with soluble BA3182.

[0069] Figures 35A-35H This study demonstrates cytokine release in human PBMC cultures stimulated with soluble BA3182 or CD3 / CD28 dinocilides at pH 7.4. Human PBMC cultures were stimulated with CD3 / CD28 dinocilides at pH 7.4 for 48 hours in culture medium. Supernatants were collected, and cytokine concentrations were measured using a Luminex multiplex cytokine assay. Data show the levels (in pg / mL) of various cytokines in cultures of human PBMCs from nine different donors using CD3 / CD28 dinocilides: 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 However, these cytokines were not detected when treated with soluble BA3182.

[0070] Figures 36A-36H This study demonstrates cytokine production in human PBMC cultures stimulated at pH 6.5 with immobilized BA3182 and anti-CD3 antibody (clonal line OKT3). Human PBMC cultures were stimulated at pH 6.5 with either immobilized BA3182 or OKT3 for 48 hours. Supernatants were collected, and cytokine concentrations were measured using a Luminex multiplex cytokine assay. Data show the levels of various cytokines (in pg / mL) in cultures of human PBMCs from nine donors: 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 ).

[0071] Figures 37A-37H This study demonstrates cytokine production in human PBMC cultures stimulated at pH 7.4 with immobilized BA3182 and anti-CD3 antibody (clonal line OKT3). Human PBMC cultures were stimulated with immobilized BA3182 or OKT3 at pH 7.4 for 48 hours in culture medium. Supernatants were collected, and cytokine concentrations were measured using a Luminex multiplex cytokine assay. Data show the levels of various cytokines (in pg / mL) in cultures of human PBMCs from nine donors: 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 ).

[0072] Figure 38The dose-response binding curves of BAP150.31-BF45 to human EpCAM antigen, measured by affinity ELISA, are displayed. The mean OD values ​​from two replicates are plotted at different concentrations of BAP150.31-BF45. X-axis: logarithmic concentration of BAP150.31-BF45 (ng / mL); Y-axis: OD value at 450 nm.

[0073] Figure 39 This study presents a serum concentration-time profile of BAP150.31-BF45 following a single IV administration of 1 mg / kg in mice. Mean serum concentrations of BAP150.31-BF45 following a single IV administration of 1 mg / kg were plotted at different serum sampling time points using PK Solver 2.0.

[0074] Figure 40 This study presents a serum concentration-time profile of BAP150.31-BF45 following a single intravenous administration of 10 mg / kg BAP150.31-BF45 in mice. Mean serum concentrations of BAP150.31-BF45 following a single intravenous administration of 10 mg / kg BAP150.31-BF45 were plotted at different sampling time points using PK Solver 2.0. Detailed Implementation

[0075] definition To aid in understanding the examples provided in this article, some frequently used terms are defined herein.

[0076] In conjunction with the quantity being measured, the term "about" as used herein refers to the normal variation in the measured quantity that would be expected by a person skilled in the art to match the measurement and operation to the purpose of the measurement and the accuracy of the measuring equipment used with respect to the quantity of interest. Unless otherwise specified, "about" means a variation of + / - 10% of the provided value.

[0077] As used herein, the term “abnormal condition” refers to a condition in a subject that exceeds the generally acceptable range of the condition. As used herein, the term “normal physiological condition” refers to a condition in a subject at a location, such as at the site of administration or the site of action, in a tissue or organ that is considered to be within the normal range.

[0078] As used herein, the term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its bound collateral (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its collateral Y is typically expressed by the dissociation constant (Kd). Affinity can be measured by commonly used methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described herein.

[0079] As used herein, the term "affinity-matured" antibody refers to an antibody that has such alterations in one or more variable regions of the heavy or light chain, compared to a parent antibody that does not have one or more alterations in one or more variable regions of the heavy or light chain, such alterations improving the antibody's affinity for the antigen.

[0080] As used herein, the term "amino acid" refers to any organic compound containing an amino group (--NH2) and a carboxyl group (--COOH); preferably in the form of a free group or as part of a peptide bond after condensation. "Twenty α-amino acids that form naturally encoded polypeptides" is understood in the art and refers to: 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).

[0081] As used herein, the term "antibody" refers to the complete immunoglobulin molecule capable of binding to epitopes of antigens, as well as fragments of immunoglobulin molecules, such as Fab, Fab', (Fab')2, Fv, and SCA fragments. These antibody fragments, which retain some of the selective binding ability of the antibodies from which they are derived to antigens (e.g., peptide antigens), can be prepared using methods well-known in the art (e.g., those described herein). Unless otherwise stated, the term antibody (antibody / antibodies) as used includes a functional fragment of an antibody. Antibodies can be used to separate prepared amounts of antigens by immunoaffinity chromatography. Various other uses of such antibodies are for the diagnosis and / or grading of diseases (e.g., tumorigenesis) and for therapeutic applications for treating diseases such as: tumorigenesis, autoimmune diseases, AIDS, cardiovascular diseases, infections, and similar diseases. Chimeric antibodies, human-like antibodies, humanized antibodies, or whole-human antibodies are particularly suitable for administration to human patients. The antibodies and antibody fragments of this disclosure can be obtained through the evolution or mutation of parental antibodies or antibody fragments having the same type of activity (e.g., binding activity or affinity for EpCAM proteins).

[0082] Fab fragments consist of monovalent antigen-binding fragments of antibody molecules and can be manufactured by digesting whole antibody molecules with papain to produce fragments consisting of complete light chains and a portion of heavy chains.

[0083] The Fab' fragment of an antibody molecule can be obtained by treating the whole antibody molecule with pepsin and then reducing it to produce a molecule consisting of the complete light chain and a portion of the heavy chain. Each antibody molecule treated in this way yields two Fab' fragments.

[0084] The (Fab')2 fragment of an antibody can be obtained by treating the entire antibody molecule with pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments linked together by two disulfide bonds.

[0085] Fv fragments are defined as genetically engineered fragments containing both light and heavy chain variable regions and expressed as two strands.

[0086] As used herein, the term "antibody fragment" refers to a molecule other than a complete antibody, including a portion of the antigen bound to the complete antibody. Examples of antibody fragments include (but are not limited to) Fv, Fab, Fab', Fab'-SH, F(ab')2; bifunctional antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0087] As used herein, the terms "anti-EpCAM antibody," "EpCAM antibody," and "EpCAM-bound antibody" refer to antibodies capable of binding to the EpCAM protein or an epitope of the EpCAM protein with sufficient affinity, thus making the antibody suitable as a diagnostic and / or therapeutic agent targeting EpCAM. In one embodiment, for example, as measured by radioimmunoassay (RIA), the binding degree of the anti-EpCAM antibody to unrelated, non-EpCAM proteins is less than about 10% of the binding of the antibody to EpCAM. In some embodiments, the dissociation constant (Kd) of the EpCAM-bound antibody is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM, 10 n ... -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 - 9 M to 10 -13 M). In some embodiments, in EpCAMs from different species, anti-EpCAM antibodies bind to epitopes of conserved EpCAMs, such as the extracellular domains of EpCAMs.

[0088] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune potential cells, or both. Those skilled in the art will understand that any macromolecule (including virtually all proteins or peptides, as well as polysaccharides, nucleic acids, or lipids) can act as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA comprising a nucleotide sequence or a portion thereof encoding a protein capable of eliciting an immune response also encodes the term "antigen" as 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 apparent that this disclosure includes (but is not limited to) the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are configured in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded entirely by a "gene." It is apparent that antigens can be produced, synthesized, or derived from biological samples. Such biological samples may include (but are not limited to) tissue samples, tumor samples, cells, or biological fluids.

[0089] As used herein, the term "binding" refers to the interaction between an antibody's variable region or Fv and an antigen, where such interaction depends on the presence of a specific structure on the antigen (e.g., an antigenic determinant or epitope). For example, an antibody's variable region or Fv recognizes and binds to a specific protein structure rather than a protein in general. As used herein, the term "specifically binding / binding specifically" means that an antibody's variable region or Fv binds or associates with a specific antigen with a more frequent, faster, longer duration, and / or greater affinity than with other proteins. For example, an antibody's variable region or Fv specifically binds to its antigen with a greater affinity, avidity, ease, and / or longer duration than it binds to other antigens. As another example, an antibody's variable region or Fv binds to cell surface proteins (antigens) with a generally greater affinity than it binds to related proteins or other cell surface proteins or antigens typically recognized by multireactive natural antibodies (i.e., naturally produced antibodies known to bind to a variety of antigens naturally found in humans). However, "specific binding" does not necessarily require exclusive binding or undetectable binding to another antigen, which is the meaning of the term "selective binding." In one instance, "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 said antigen at an equilibrium constant (KD) of 100 nM or less, such as 50 nM or less, such as 20 nM or less, such as 15 nM or less, or 10 nM or less, or 5 nM or less, 2 nM or less, or 1 nM or less.

[0090] As used herein, the terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals characterized by unregulated cell growth / proliferation. Examples of cancer include (but are not limited to) melanoma, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of these cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumors, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0091] As used herein, the term "cellular antigen" or "cell-associated antigen" refers to any protein, carbohydrate, or other component derived from or expressed by a cell capable of evoking an immune response. For example, the cell can be any cell in the subject, specifically cancer cells and senescent cells. Cellular antigens can be antigens on or inside the cell surface. The definition is intended to include (but is not limited to) proteins purified from the cell surface or cell membrane, or unique carbohydrate portions bound to the cell surface. The definition also includes antigens from the cell surface that require special treatment of the cell to be targeted by the antibodies of this disclosure.

[0092] As used herein, the terms "cellular proliferative disorder" and "proliferative disorder" refer to a disorder associated with an abnormal degree of cell proliferation. In one embodiment, the cellular proliferative disorder is cancer.

[0093] As used herein, the term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0094] As used herein, the term "conditionally active antibody" refers to an anti-EpCAM antibody or antibody fragment that is more active under conditions in the tumor microenvironment than under conditions in a non-tumor microenvironment. Conditions in the tumor microenvironment include a lower pH, higher lactate and pyruvate concentrations, lower oxygen levels, lower glucose concentrations, and slightly higher temperatures compared to the non-tumor microenvironment. For example, a conditionally active antibody is almost inactive at normal body temperature but active at the higher temperatures of the tumor microenvironment. On the other hand, a conditionally active antibody has lower activity in normal oxidized blood but is more active in the poorly oxidized environment present in a tumor. Furthermore, a conditionally active antibody has lower activity at normal physiological pH 7.0–7.6 or 7.2–7.6 but is more active at the acidic pH 5.0–6.9 or 6.0–6.8 present in the tumor microenvironment. Other conditions known to those skilled in the art in the presence of the tumor microenvironment may also be used as conditions for giving anti-EpCAM antibodies different binding affinities to EpCAM according to this disclosure.

[0095] As used herein, the term "bifunctional antibody" refers to a small antibody fragment having two antigen-binding sites, said fragment comprising a polypeptide chain (V) linked to the same polypeptide chain. H -V L The light chain variable domain (V) in ) L The heavy chain variable domain (V) H By using a connector that is too short to allow pairing between two domains on the same strand, the domain is forced to pair with a complementary domain of another strand, resulting in two antigen-binding sites.

[0096] As used herein, the term "detectable marker" refers to any substance that indicates the presence of an antigen in a sample by direct or indirect detection or measurement through physical or chemical means. Representative examples of applicable detectable markers include (but are not limited to) the following: molecules or ions that can be detected directly or indirectly based on absorbance, fluorescence, reflectance, light scattering, phosphorescence, or luminescence properties; molecules or ions that can be detected by their radioactive properties; and molecules or ions that can be detected by their nuclear magnetic resonance or paramagnetic properties. A group of molecules that can be detected indirectly based on absorbance or fluorescence includes, for example, various enzymes that cause a suitable substrate to be converted, for example, from a non-photoabsorbing molecule to a photoabsorbing molecule or from a non-fluorescent molecule to a fluorescent molecule.

[0097] As used herein, the term "diagnosis" refers to determining a subject's susceptibility to a disease or condition, determining whether a subject currently has a disease or condition, the prognosis of a subject with a disease or condition (e.g., identifying pre-metastatic or metastatic cancer status, cancer grade, or cancer response to therapy), and therapeutic dosages (e.g., monitoring the subject's condition to provide information about treatment effectiveness or efficacy). In some embodiments, the diagnostic methods of this disclosure are particularly suitable for detecting early-stage cancer.

[0098] As used herein, the term "effective function" refers to the biological activity attributable to the Fc region of an antibody, which varies from antibody isotype to antibody. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0099] As used herein, the term “effective amount” for a pharmaceutical preparation (e.g., a pharmaceutical formulation) refers to the amount that effectively achieves the desired therapeutic or preventative outcome at the required dose and time.

[0100] As used herein, the term "epitope" or "antigenic determinant" refers to a site on an antigen that binds to an antibody. Epitopes can be formed from consecutive amino acids (linear epitopes) or from non-continuous amino acids arranged in parallel within the ternary folds of a protein (conformational epitopes). Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from ternary folds are generally lost upon treatment with denaturing solvents. Epitopes can contain three or more amino acids. Epitopes typically consist of at least 5 to 7 amino acids (e.g., 5, 6, or 7 amino acids in an epitope), or at least 8 to 11 amino acids (e.g., 8, 9, 10, or 11 amino acids in an epitope), or more than 11 amino acids (e.g., 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in an epitope), or more than 20 amino acids (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in an epitope). Less frequently, epitopes may contain 31 to 40 amino acids. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, "Guidelines for Epitope Localization in Molecular Biology Methods" (…). Epitope Mapping Protocols in Methods in Molecular Biology ), Volume 66, edited by Glenn E. Morris (1996). Surface plasmon resonance is the preferred method for epitope localization on antigens.

[0101] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine residue (Lys447) of the Fc region may or may not be present. Unless otherwise stated herein, the amino acid residues of the Fc region or constant region are numbered 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 edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0102] As used herein, the term "framework" or "FR" refers to the variable domain residues excluding the complementarity-determining regions (CDR or H1-3 residues in the heavy chain and L1-3 residues in the light chain). A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, in V... H (or V)L In FR1, CDR and FR sequences are usually presented in the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0103] The terms "full-length antibody," "complete antibody," or "total antibody" refer to antibodies that include the antigen-binding variable region (V). H or V L Antibodies consist of a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains can be natural sequence constant domains (e.g., human natural sequence constant domains) or their amino acid sequence variants. Full-length antibodies can be classified into different “classes” depending on the amino acid sequence of their heavy chain constant domains. There are five main classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into “subclasses” (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different antibody classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known.

[0104] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to an amino acid sequence of an antibody produced by humans or human cells or derived from an antibody of a non-human origin using a human antibody lineage or other human antibody encoding sequence. This definition of human antibody specifically excludes humanized antibodies that include non-human antigen-binding residues.

[0105] As used herein, the term "humanized" antibody refers to a chimeric antibody comprising amino acid residues from a non-human CDR and amino acid residues from a human FR. In some embodiments, a humanized antibody will comprise at least one, and typically substantially all, of the two variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of the non-human antibody, and all or substantially all of the FRs correspond to the FRs of the human antibody. Optionally, a humanized antibody may comprise at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0106] "Individual," "patient," or "subject" refers to a human or animal. For example, a subject can be a mammal selected from domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as human and non-human primates, such as monkeys), rabbits, and rodents (such as mice and rats).

[0107] As used herein, the term "isolated" antibody is an antibody that has been isolated from its components in their natural environment. In some embodiments, antibodies are purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase high-performance liquid chromatography (HPLC)). A review of methods for assessing antibody purity can be found, for example, Flatman et al., Chromatography Journal B (…). J. Chromatogr (B), Volume 848, pp. 79-87, 2007.

[0108] As used herein, the term “metastasis” refers to all processes involving EpCAM that support the spread of cancer cells from the primary tumor, through lymphatic vessels and / or blood vessels, via blood circulation, and into distal foci in normal tissues elsewhere in the body (metastasis). Specifically, it refers to cellular events of tumor cells that constitute the basis of metastasis and are stimulated or mediated by EpCAM, such as proliferation, migration, fixation independence, apoptosis evasion, or angiogenesis factor secretion.

[0109] As used herein, the term “microenvironment” means any part or region of an tissue or body that has a persistent or temporary physical or chemical difference from other regions of the tissue or body. As used herein, for tumors, the term “tumor microenvironment” refers to the environment in which a tumor exists, encompassing the non-cellular regions within the tumor and the regions just outside the tumor tissue, but not including the intracellular compartments of the cancer cells themselves. Tumors and their microenvironments are closely related and constantly interact. Tumors can alter their microenvironment, and the microenvironment can influence the way tumors grow and spread. Typically, the tumor microenvironment has a low pH in the range of 5.0 to 7.0, or 5.0 to 6.9, or 5.8 to 6.8, or 6.2 to 6.8. On the other hand, standard physiological pH is in the range of 7.0 to 7.6 or 7.2–7.8. The tumor microenvironment is also known to have lower concentrations of glucose and other nutrients compared to plasma, but higher concentrations of lactate. Furthermore, the tumor microenvironment can have a temperature 0.3°C to 1°C higher than normal physiological temperature. The tumor microenvironment has been discussed in Gillies et al., “MRI of the Tumor Microenvironment,” *Journal of Magnetic Resonance Imaging*. Journal of Magnetic Resonance Imaging) Volume 16, pp. 430-450, 2002, which is incorporated herein by reference in its entirety. The term “non-tumor microenvironment” refers to the microenvironment of sites other than tumors.

[0110] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that, except for potentially variant antibodies (e.g., those containing naturally occurring mutations or variant antibodies that appear during the manufacture of a monoclonal antibody formulation, such variants are typically present in small amounts), the individual antibodies constituting said population are identical and / or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically contain different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to this disclosure can be prepared using a variety of techniques, including (but not limited to) fusion tumor methods, recombinant DNA methods, phage presentation methods, and methods using transgenic animals containing all or part of the human immunoglobulin loci.

[0111] As used herein, the term "multispecific antibody" refers to a full-length antibody, antibody fragment, or construct comprising one or more full-length antibodies and antibody fragments having at least two distinct binding sites, each capable of binding to the same or different epitopes. Constructs of engineered antibodies having two, three, or more (e.g., four, five, six, or seven) functional antigen-binding sites fall within the scope of multispecific antibodies (see, for example, US 2002 / 0004587A1 and Brinkman and Kontman, […]). MAbs (Volume 9, pp. 182-212, 2017).

[0112] As used herein, the term "instructions for use of a drug" refers to the instructions for use typically included in the commercial packaging of a therapeutic product, which contain information about indications, usage, dosage, administration, combination therapy, contraindications and / or warnings related to the use of such therapeutic product.

[0113] As used herein, the term "percentage of amino acid sequence similarity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after sequence alignment and the introduction of spacers (if necessary) to obtain the maximum percentage of sequence similarity, without considering any conserved substitutions as part of sequence similarity. Alignments for the purpose of determining the percentage of amino acid sequence similarity can be performed in various ways within the skill level of 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 the parameters applicable to the aligned sequences, including any algorithm required to achieve maximum alignment across the full length of the compared sequences.

[0114] As used herein, the term "drug formulation" means a preparation which is presented in a form that allows the bioactivity of the active ingredient contained therein to be effectively exerted and which does not contain any other components that would have unacceptable toxicity to the subject to whom the formulation will be administered.

[0115] As used herein, the term "pharmaceuticalally acceptable carrier" refers to a component of a drug formulation that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include (but are not limited to) buffers, excipients, stabilizers, or preservatives.

[0116] As used herein, the terms "purified" and "isolated" mean the presence of a molecule, based on an antibody or nucleotide sequence of this disclosure, in the absence of other biomolecules of the same type. As used herein, the term "purified" preferably means the presence of 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 of the same type of biomolecule. An "isolated" nucleic acid molecule encoding a specific polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode polypeptides; however, said molecule may contain some additional bases or portions that do not harmfully affect the essential characteristics of the composition.

[0117] As used herein, the term “recombinant antibody” refers to an antibody (e.g., chimeric, humanized, or human antibody or its antigen-binding fragment) expressed by a recombinant host cell comprising a nucleic acid encoding the antibody. Examples of “host cells” that produce recombinant antibodies include: (1) mammalian cells, such as Chinese hamster ovary (CHO), COS, myeloma cells (including Y0 and NSO cells), young hamster kidney (BHK) cells, HeLa cells, and Vero cells; (2) insect cells, such as sf9, sf21, and Tn5; and (3) plant cells, such as plants belonging to the genus *Nicotiana* (e.g., tobacco). Nicotiana tabacum (4) Yeast cells, such as those belonging to the genus *Saccharomyces* (e.g., *Saccharomyces cerevisiae*). Saccharomyces cerevisiae )) or Aspergillus species (e.g., Aspergillus niger) Aspergillus niger (5) Bacterial cells, such as Escherichia coli cells. Escherichia.coli ) or Bacillus subtilis cells ( Bacillus subtilis )wait.

[0118] As used in this article, the term "single-chain Fv" ("scFv") refers to a covalently linked V. H ::V L Heterodimers, typically expressed by gene fusion molecules, which contain V molecules linked by peptide-coding linkers. H and V L The encoding gene, “dsFv”, is a V gene stable by disulfide bonds. H ::V LHeterodimers. Divalent and multivalent antibody fragments can be spontaneously formed by binding to monovalent scFvs, or generated by coupling monovalent scFvs with a peptide linker (e.g., divalent sc(Fv)2).

[0119] The term "therapeutic effective amount" for the antibodies disclosed herein means an amount of antibody sufficient to treat the cancer at a reasonable benefit / risk ratio applicable to any medical treatment. However, it should be understood that the total daily dosage of the antibodies and compositions of this disclosure will be determined by the attending physician within the bounds of proper medical judgment. The specific therapeutic effective dose level for any particular patient will depend on a number of factors, including the condition being treated and its severity; the activity of the specific antibody used; the specific composition used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and rate of excretion of the specific antibody; the duration of treatment; drugs used in combination with or concurrently with the specific antibody used; and similar factors well known in the medical field. For example, it is well known in the art to start the composition at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0120] As used herein, the term "treatment" refers to a clinical intervention that attempts to alter the natural course of a subject's disease, and may be performed to achieve prevention or during the clinicopathological course of the disease. The desired therapeutic effect includes, but is not limited to, preventing the onset or recurrence of disease, relieving symptoms, mitigating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease condition, and alleviating or improving prognosis. In some embodiments, the antibodies of this disclosure are used to delay disease development or slow disease progression.

[0121] As used herein, the term “tumor” refers to all tumorous cell growth and proliferation (whether malignant or benign), and all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative lesion,” and “tumor” are not mutually exclusive, as mentioned herein.

[0122] As used herein, the terms "variable region" or "variable domain" refer to the antibody heavy chain or light chain domain involved in antibody-antigen binding. The heavy chain and light chain variable domains of natural antibodies (V1 and V2, respectively) H and V L Generally, they have similar structures, and each domain includes four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). Single VH or V L The domain can be sufficient to confer antigen binding specificity. Furthermore, antibodies binding to specific antigens can use the V domain of antibodies binding to said antigens. H or V L Each domain is selected to screen complementary V L Alternatively, use libraries from the VH domain for separation. See, for example, Portolano et al., *Journal of Immunology*. J. Immunol.) Volume 150, pp. 880-887, 1993; Clarkson et al., Nature ( Nature) Volume 352, pp. 624-628, 1991.

[0123] As used herein, the term “unit dosage form” refers to a physically discrete unit suitable as a unit dose for a subject, each unit containing a predetermined amount of the conditionally active multispecific antibody of the present disclosure, sufficient to produce the desired therapeutic effect, and a pharmaceutically acceptable diluent, carrier, or mediator.

[0124] It should be noted that, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” as used herein and in the appended claims include multiple indicators. Furthermore, the terms “a or an,” “one or more,” and “at least one” are used interchangeably herein. The terms “comprising,” “including,” “having,” and “constructed from” are also used interchangeably.

[0125] Unless otherwise specified, all figures used in the specification and claims to indicate the quantity of components, such as molecular weight, percentage, ratio, reaction conditions, etc., should be understood to be modified in all cases by the term "about," regardless of whether the term "about" is present. Therefore, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximate values, which may vary depending on the desired characteristics sought to be obtained by the invention. At least, and without attempting to limit the application of equivalence to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques. Although the numerical ranges and parameters describing the broad scope of the invention are approximate values, the values ​​set forth in specific examples are reported as accurately as possible. However, any numerical value inherently contains some errors that are necessarily caused by the standard deviation found in the corresponding test measurements.

[0126] It should be understood that each component, compound, substituent or parameter disclosed herein should be interpreted as being disclosed alone or in combination with one or more of each other component, compound, substituent or parameter disclosed herein.

[0127] It should also be understood that the amounts or ranges of amounts / values ​​of each component, compound, substituent or parameter disclosed herein should be interpreted as being disclosed in combination with the ranges of amounts / values ​​or ranges of amounts / values ​​disclosed for any other component, compound, substituent or parameter disclosed herein, and therefore, any combination of two or more amounts or ranges of amounts / values ​​of components, compounds, substituents or parameters disclosed herein is also disclosed in combination with each other for the purposes of this specification.

[0128] It should also be understood that each range disclosed herein should be interpreted as a disclosure of each specific value having the same number of significant figures within the disclosed range. Therefore, ranges 1-4 should be interpreted as explicitly disclosing values ​​1, 2, 3, and 4. It should also be understood that each lower limit of each range disclosed herein should be interpreted as a combination of each upper limit of each range disclosed herein for the same component, compound, substituent, or parameter, and each specific value within each range. Therefore, this disclosure should be interpreted as a disclosure of all ranges obtained by combining each lower limit of each range with each upper limit of each range or with each specific value within each range, and by combining each upper limit of each range with each specific value within each range.

[0129] Furthermore, the specific amounts / values ​​of components, compounds, substituents, or parameters disclosed in this specification or examples should be interpreted as disclosures of a lower or upper limit of a range, and therefore can be combined with any other lower or upper limit of a range or specific amounts / values ​​of the same components, compounds, substituents, or parameters disclosed elsewhere in this application to form a range of said components, compounds, substituents, or parameters.

[0130] In one aspect, this disclosure provides isolated antibodies that specifically bind to EpCAM proteins, particularly human EpCAM proteins, comprising at least one heavy chain variable region containing three complementarity-determining regions H1, H2, and H3, and at least one light chain variable region containing three complementarity-determining regions L1, L2, and L3. Specific examples of suitable combinations of heavy and light chain complementarity-determining regions are provided in Table 1.

[0131] Exemplary combinations of light chain and heavy chain variable regions are also provided. These combinations include antibodies comprising: any one of the heavy chain variable region of SEQ ID NO:52 and the light chain variable regions of SEQ ID NO:53-69; or any one of the light chain variable region of SEQ ID NO:51 and the light chain variable regions of SEQ ID NO:70-96.

[0132] A comparison of various embodiments of the light chain variable region can be found in Figures 4A-4B In the comparison of various embodiments of the heavy chain variable region, one can see in Figures 5A-5B middle.

[0133] The heavy chain variable region and light chain variable region disclosed herein are obtained from parental (wild-type) antibodies using the methods disclosed in U.S. Patent No. 8,709,755. The methods for generating heavy chain variable regions and light chain variable regions, as well as the methods for generating antibodies and antibody fragments disclosed in U.S. Patent No. 8,709,755, are incorporated herein by reference.

[0134] Antibodies and antibody fragments containing these heavy chain and light chain variable regions can specifically bind to EpCAM, such as human EpCAM. Antibodies or antibody fragments containing a combination of one of these heavy chain variable regions and one of these light chain variable regions have been found to have a higher binding affinity for EpCAM in the tumor microenvironment (e.g., pH 6.0–6.8) compared to the pH in the non-tumor microenvironment (e.g., pH 7.0–7.6). Therefore, anti-EpCAM antibodies or antibody fragments exhibit a higher binding affinity for EpCAM in the tumor microenvironment than they do in the typical normal tissue microenvironment.

[0135] In another embodiment, this disclosure provides a multispecific antibody comprising at least one binding site for EpCAM and at least one binding site for tumor-reactive lymphocyte antigen. The multispecific antibody binds to at least one EpCAM epitope and the tumor-reactive lymphocyte antigen with greater affinity under a first physiological condition than under a second physiological condition. In one specific embodiment, 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 may be a condition within the tumor microenvironment. The multispecific antibody of this disclosure may be referred to as a conditionally active multispecific antibody.

[0136] In some embodiments, the conditionally active multispecific antibody is almost inactivated under normal physiological conditions but active under abnormal conditions, with an activity level optionally higher than that of the conditionally active multispecific antibody under normal physiological conditions or the parent antibody used to obtain the conditionally active multispecific antibody under normal physiological conditions. In another embodiment, the conditionally active multispecific antibody is almost inactivated at pH 7.0-7.6 but active at a lower pH of 5.0-6.8. In some cases, the conditionally active multispecific antibody is reversibly inactivated under normal physiological conditions. In another instance, the conditionally active multispecific antibody may have higher or lower activity in highly oxidized blood (e.g., after passing through the lungs) or in lower pH environments found in the tumor microenvironment. The conditionally active multispecific antibody can be used as a drug, therapeutic agent, or diagnostic agent.

[0137] Not wishing to be bound by theory, the conditionally active multispecific antibody of this disclosure binds to both target cells and tumor-reactive lymphocytes, thereby bringing the target cells into close proximity to the tumor-reactive lymphocytes. It is believed that this will promote the attack of tumor-reactive lymphocytes on the target cells, thereby inhibiting, damaging, or destroying the target cells. The therapeutic effect of inhibiting or removing tumor cells can be achieved by using the conditionally active multispecific antibody of this disclosure to guide reactive lymphocytes towards tumor cells, thereby inhibiting, destroying, and removing tumor cells in the subject.

[0138] The structure / format of multispecific antibodies can be Brinkman and Kontmann, as described in "Preparation of Bispecific Antibodies", MABs Any of the structures / formats described in [the book / article], Volume 9, pp. 182-212, 2017. Specifically, Brinkman and Kontman's [works / technologies]. Figure 2 Nineteen different structures / formats of bispecific antibodies are described. 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) attached and Fc-modified IgG; (9) modified Fc and CH3 fusion proteins; (10) attached IgG-HC fusions; (11) attached IgG-LC fusions; (12) attached 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.

[0139] In a specific embodiment, a multispecific antibody can be as follows: Figure 2 The divalent scFv-Fc heterodimer shown, or as Figure 3 The “butterfly-shaped” tetravalent homodimer is shown. In both structures, the reactive lymphocyte antigen is not limited to CD3, but is depicted only as a representative of tumor reactive lymphocyte antigens. Figure 2The multispecific antibody 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 to form only heterodimers, for example, by using a knock-in-hole technique. The first and second binding sites are scFv antibodies that bind to EpCAM and reactive lymphocyte antigens, respectively. One or both of the first and second binding sites have conditionally active binding activity against the corresponding antigen.

[0140] Figure 3 Conditionally active multispecific antibodies can have a full-length IgG antibody bound to EpCAM and a scFv antibody bound 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 can be a short alanine linker (Ala). n Serine linkers (Ser) n Hydrophilic linkers or glycine-serine-rich linkers are used. The heavy chain of the IgG antibody pairs with the light chain of the IgG antibody already linked to the scFv antibody, thereby forming half of a homodimer. This multispecific antibody has a "butterfly" structure.

[0141] In some embodiments, the multispecific antibody comprises an IgG antibody or fragment thereof bound to a tumor-reactive lymphocyte antigen and a single-chain antibody bound to EpCAM, also forming a... Figure 3 The “butterfly” configuration is shown. The single-chain antibody can be an scFv antibody. The scFv antibody can be linked to the C-terminus of an IgG antibody via a linker as described herein.

[0142] The binding sites of the multispecific antibodies disclosed herein each comprise a light chain variable region and a heavy chain variable region. The light chain variable region and the heavy chain variable region can be in a single-chain antibody format or in a double-chain format, for example, formed by the pairing of the light chain and the heavy chain. Figure 2 and 3 In a conditionally active binding site, one of the light chain and heavy chain variable regions is conditionally active, or both may be conditionally active. An illustrative 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. Other sequences of conditionally and unconditionally active anti-CD3 antibodies suitable for use in the multispecific antibodies of this disclosure can be found in WO 2019 / 241216, the disclosure of which is incorporated herein by reference.

[0143] The illustrative conditionally active anti-EpCAM antibody variable regions that can be used to construct the multispecific antibodies of this disclosure include combinations of any one of the heavy chain variable regions of SEQ ID NO:52 and the light chain variable regions of SEQ ID NO:53-69, and combinations of any one of the light chain variable regions of SEQ ID NO:51 and the heavy chain variable regions of SEQ ID NO:70-96.

[0144] In some other embodiments, it can be as follows Figure 2 The multispecific antibody shown herein has two variable regions forming a binding site for EpCAM and two other variable regions forming a binding site for a reactive lymphocyte antigen (e.g., CD3). These variable regions can be selected from light and heavy chain variable regions having the amino acid sequences provided herein. One or both binding sites must have conditional activity against their corresponding antigens. At each conditionally active binding site, at least one of the light and heavy chain variable regions exhibits increased affinity against its antigen under a first physiological condition (e.g., abnormal condition) compared to an affinity under a second physiological condition (e.g., normal physiological condition). Therefore, those skilled in the art can select appropriate light and heavy chain variable regions to construct a multispecific antibody as shown herein. Figure 2 The multispecific antibody shown. Figure 2 The heavy chain fragments in the antibody are selected from the constant region of IgG antibodies, including any subclass of IgG: IgG1, IgG2, IgG3, and IgG4.

[0145] In some other embodiments, it may be as follows Figure 3 The diagram illustrates the construction of multispecific antibodies. Similarly, the light chain variable regions and heavy chain variable regions in scFv antibodies and full-length IgG antibodies can also be selected from light chain and heavy chain variable regions having the amino acid sequences provided herein. At each conditionally active binding site, at least one of the light chain variable regions and heavy chain variable regions exhibits increased affinity for its antigen under a first physiological condition (e.g., abnormal condition) compared to an affinity under a second physiological condition (e.g., normal physiological condition). Therefore, those skilled in the art can select appropriate light chain variable regions and heavy chain variable regions provided herein to construct multispecific antibodies as shown. Figure 3 The multispecific antibody shown. Figure 3 The constant region is selected from the constant region of IgG antibodies, including any subclass of IgG: IgG1, IgG2, IgG3, and IgG4.

[0146] It is anticipated that the conditionally active anti-EpCAM antibodies or antibody fragments of this disclosure will exhibit fewer side effects compared to unconditionally active anti-EpCAM antibodies because their binding affinity to EpCAM is reduced in the normal tissue microenvironment. It is also anticipated that the anti-EpCAM antibodies or antibody fragments of this disclosure will have similar efficacy to monoclonal anti-EpCAM antibodies known in the art. This combination of properties, due to reduced side effects, allows for the use of higher doses of these anti-EpCAM antibodies or antibody fragments, thereby providing more effective therapeutic options.

[0147] In some embodiments, the abnormal condition is an acidic pH value in the range of about 5.0 to 7.0, or about 5.2 to about 6.8, or about 5.4 to about 6.8, or about 5.6 to about 6.8, or about 5.8 to about 6.8, or about 6.0 to about 6.8, or about 6.2 to about 6.8, or about 6.4 to about 6.8, or about 6.6 to about 6.8. In some embodiments, the acidic pH value may be in the range of about 6.4 to 7.0, or about 6.6 to 7.0, or about 6.8 to 7.0. Normal physiological conditions may be the normal physiological pH value of blood recognized in the art. In some embodiments, the normal physiological pH value of blood may be in the range of about 7.0 to about 7.8, or about 7.1 to about 7.7, or about 7.2 to about 7.6, or about 7.2 to about 7.5, or about 7.2 to about 7.4.

[0148] In some embodiments, the ratio of the affinity or affinity of the multispecific antibodies of this disclosure for EpCAM and / or tumor-reactive lymphocyte antigens (e.g., CD3) under abnormal conditions to the same affinity or affinity under normal physiological conditions is 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 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.

[0149] 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, hydrazide, acylhydrazide, aminourea, azide, or alkynyl groups. For suitable non-naturally occurring amino acids, see, for example, U.S. Patent No. 7,632,924. The term “non-naturally occurring amino acid” also includes amino acids produced by modification (e.g., post-translational modification) of naturally occurring amino acids, but not naturally incorporated into the growing polypeptide chain by the translation complex of a living organism. Examples of said non-naturally occurring amino acids include (but are not limited to) N-acetylglucosamine-L-serine, N-acetylglucosamine-L-threonine, and O-phosphotyrosine.

[0150] In some embodiments, the antibody is in the form of a "mimic" or "peptide mimic," which is composed entirely of synthetic amino acids, non-natural analogs, or chimeric molecules of partially naturally occurring amino acids and partially non-natural amino acid analogs. The mimic may also incorporate any amount of conserved substitutions of naturally occurring amino acids, as long as these substitutions do not substantially alter the antibody's structure and / or activity.

[0151] The mimicry form can contain any combination of non-natural structural components. In one aspect, the mimicry of this disclosure includes one or all of the following three structural groups: a) residue linking groups other than natural amide bonds (“peptide bonds”); b) non-natural residues replacing naturally occurring amino acid residues; and c) residues that induce secondary structure mimicry, i.e., induce or stabilize secondary structures, such as β-turns, γ-turns, β-sheets, α-helical conformations, etc. For example, a multispecific antibody can be characterized as a mimicry when all or some of its residues are chemically linked by means other than natural peptide bonds. Individual peptide mimicry residues can be linked by peptide bonds, other chemical bonds, or coupling, such as glutaraldehyde, N-hydroxysuccinimide ester, bifunctional maleic anhydride, N,N'-dicyclohexylcarbodiimide (DCC), or N,N'-diisopropylcarbodiimide (DIC). Linking groups that can replace traditional amide bonds (“peptide bonds”) 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), tetrazolium, thiazole, reverse amide, thioamide, and ester (see, for example, Spatola (1983), *Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins*). Peptides and Proteins"Peptide Backbone Modifications", 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, edited by B. Weinstein, New York: Marcell Dekker, pp. 257-267.

[0152] 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-pyrenealanine; D- or L-3-thienylalanine; D- or L-(2-pyridyl)-alanine; D- or L-(3-pyridyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D- (Trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-fluorophenylalanine; D- or L-p-biphenylphenylalanine; D- or L-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanine; and D- or L-alkylalanine, wherein the alkyl group may be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, tert-isobutyl, isopentyl, or a non-acidic amino acid. Aromatic rings of non-natural amino acids include, for example, thiazolyl, phenylthio, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrroleyl, and pyridyl aromatic rings.

[0153] Acidic non-natural amino acids can be generated by substitution, for example, with non-carboxylic acid ester amino acids while maintaining a negative charge, such as (phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g., aspartic or glutamic) can also be selectively modified by reacting with carbodiimides (R'~NC--N--R'), such as 1-cyclohexyl-3-(2-morpholino-(4-ethyl)carbodiimide or l-ethyl-3-(4-aza-onium-4,4-dimethylpentyl)carbodiimide. Aspartic or glutamic groups can also be converted to asparagine acyl and glutamic acyl residues by reacting with ammonium ions.

[0154] Basic non-natural amino acids can be generated by substitution with, for example, ornithine (other than lysine and arginine), citrulline, or (guanidino)-acetic acid or (guanidino)alkyl-acetic acid, wherein the alkyl group is as defined above. Nitrile derivatives (e.g., containing a CN-part replacing COOH) can be substituted with asparagine or glutamine. Asparagine acyl and glutamine acyl residues can be deaminated to yield the corresponding asparagine or glutamine residues. Arginine residue mimics can be generated by reacting arginyl with, for example, one or more conventional reagents under basic conditions, including, for example, phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or indantrione. Tyrosine residue mimics can be generated by reacting tyrosyl with, for example, an aromatic diazo compound or tetranitromethane. N-acetylimidazolium and tetranitromethane can be used to form O-acetyltyrosyl substances and 3-nitro derivatives, respectively. Cysteine ​​residue mimics can be generated by reacting a cysteyl residue with, for example, an α-haloacetic acid ester, such as 2-chloroacetic acid, or chloroacetamide and its corresponding amine, to yield a carboxymethyl or carboxyamidomethyl derivative. Cysteine ​​residue mimics can also be generated by reacting a cysteyl residue with, for example, the following: bromo-trifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid; chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide; methyl-2-pyridyl disulfide; p-chloromercuryl benzoate; 2-chloromercuryl-4-nitrophenol; or chloro-7-nitrobenzo[-oxa-1,3-diazole. Lysine mimics can be generated by reacting a lysyl group with, for example, succinic acid or other carboxylic anhydrides (and the amino-terminal residue can be modified). Lysine and other α-amino residue mimics can also be produced by reacting with imino esters, such as methyl pyridinium imide, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminases-catalyzed reactions with glyoxylates. Methionine mimics can be produced by reacting with, for example, methionine sulfoxide. Proline mimics include, for example, piperidinic acid, thiazolyl carboxylic acid, 3- or 4-hydroxyproline, dehydroproline, 3- or 4-methylproline, or 3,3-dimethylproline. Histidine residue mimics can be produced by reacting histidine acyl groups with, for example, diethyl pyrocarbonate or p-bromophenylacetyl bromide. Other mimics include, for example, those derived from: hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of serine or threonine residues; methylation of the α-amino group of lysine, arginine, and histidine; acetylation of the N-terminal amine; methylation of the main chain amide residues or substitution with an N-methyl amino acid; or amidation of the C-terminal carboxyl group.

[0155] The mimic form of an antibody can also contain one or more amino acids with opposite chirality. Thus, any amino acid that is naturally present in an L-configuration (which may also be referred to as R or S depending on the structure of the chemical entity) can be replaced by an amino acid of the same chemical structure type or peptide mimic but with opposite chirality (called D-amino acids), and these forms may also be referred to as R- or S-forms.

[0156] The mimicry form of the antibody can be synthesized using any protein chemical synthesis technique. In typical in vitro protein synthesis, a peptide is extended by one amino acid by forming a peptide bond between the peptide and an amino acid. The peptide bond formation is carried out using a conjugation reaction, which can use either native or non-native amino acids. Therefore, in this manner, non-native amino acids can be introduced into the antibodies of this disclosure to prepare mimicry.

[0157] In some embodiments, non-naturally occurring amino acids in the antibody can provide a link to macromolecules such as polymers, proteins, or fatty acids. In some embodiments, multispecific antibodies are linked (e.g., covalently linked) to polymers (e.g., polymers other than peptides). 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, polyolefin, or polyoxyalkylene polymers, as well as branched or unbranched polysaccharides, such as homopolysaccharides or heteropolysaccharides. Further examples of suitable polymers include ethylene-vinyl alcohol copolymers (often known by the generic name EVOH or the trade name EVAL); polybutyl methacrylate; poly(hydroxyvalerate); poly(L-lactic acid); polycaprolactone; poly(lactide-co-glycolic acid); poly(hydroxybutyrate); poly(hydroxybutyrate-co-valerate); poly(p-dioxanone); polyorthoesters; polyanhydrides; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolic acid-co-trimethylene carbonate); polyphosphates; polyphosphate urethanes; poly(amino acids); cyanoacrylates; poly(trimethylene carbonate); poly(imino carbonate); copoly(ether-ester). (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; polysiloxanes; polyesters; polyolefins; polyisobutylene and ethylene-α-olefin copolymers; acrylic polymers and copolymers; vinyl halide polymers and copolymers such as polyvinyl chloride; polyethylene ethers such as polyethylene methyl ether; polyvinylidene halides such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketone; polyethylene aromatic compounds such as polystyrene; polyethylene esters such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as ethylene-methyl methacrylate copolymers, acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers; polyamides such as Nylon 66 and polycaprolactam; alkyd resins; polycarbonate; polyoxymethylene; polyimide; polyether; epoxy resin; polyurethane; rayon; rayon-triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ether; amorphous Teflon™; poly(ethylene glycol); and carboxymethyl cellulose.

[0158] Examples of synthetic polymers include unsubstituted and substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), and their derivatives, such as substituted poly(ethylene glycol), such as methoxylated poly(ethylene glycol), and their derivatives. Suitable naturally occurring polymers include, for example, albumin, amylose, polydextrose, glycogen, and their derivatives.

[0159] The average molecular weight of the linking polymer can range from 500 Da to 50,000 Da, for example, from 5,000 Da to 40,000 Da, or from 25,000 Da to 40,000 Da. For example, in some embodiments where the multispecific antibody comprises a poly(ethylene glycol) (PEG) or methoxy poly(ethylene glycol) polymer, the molecular weight of the PEG or methoxy poly(ethylene glycol) polymer can range from 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.

[0160] 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 having a non-naturally occurring amino acid (containing an aminooxy, hydrazine, acylhydrazine, or aminourea group). As another example, an antibody can be linked to a water-soluble polymer by reacting an antibody containing an alkyne-containing amino acid with a water-soluble polymer containing an azide group. In some cases, the azide or alkyne group is linked to the PEG molecule via an amide bond.

[0161] In some embodiments, the macromolecule linked to the antibody is albumin. Albumin can be, for example, the albumin of the subject receiving the antibody. For instance, if the antibody is intended for use in humans, then human albumin can be linked to the antibody. If the antibody is intended for use in dogs, then canine albumin can be linked to a multispecific antibody. Generally, if the antibody is intended for use in a species, then albumin from said species is linked to the antibody.

[0162] Examples of linkers used to conjugate macromolecules to antibodies include glutaraldehyde, homobifunctional crosslinkers, or heterobifunctional crosslinkers. Glutaraldehyde crosslinks peptides via their amino moieties. Homobifunctional crosslinkers (e.g., homobifunctional imide esters, homobifunctional N-hydroxysuccinimide (NHS) esters, or homobifunctional thiol reactive crosslinkers) contain two or more identical reactive moieties and can be used in a single-step reaction process by adding the crosslinker to a solution containing a mixture of the macromolecule to be linked and the antibody. At a slightly alkaline pH, the imide ester reacts only with the primary amine to form an imide amide, and the overall charge of the crosslinked macromolecule and antibody is unaffected. Homofunctional thiol reactive crosslinking agents include bis(cis-butenedimide)hexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-bis-(3′,2′-pyridyldithio)propamidobutane (DPDPB).

[0163] Heterofunctional bifunctional crosslinking agents have two or more distinct reactive moieties (e.g., amine-reactive moieties and thiol-reactive moieties) and crosslink with one of the macromolecules and antibodies via the amine or thiol-reactive moieties, followed by a reaction with the other of the macromolecules and antibodies via the unreacted moieties. A variety of heterofunctional bifunctional haloacetyl crosslinking agents are available, such as pyridyl disulfide crosslinking agents. Carbodiimide is a classic example of a heterofunctional bifunctional crosslinking agent used to couple a carboxyl group to an amine to generate an amide bond.

[0164] Antibodies can be glycosylated, for example, by covalently linking to carbohydrate or polysaccharide moieties. Glycosylation of multispecific antibodies is typically performed via N-linking or O-linking.

[0165] N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue in an antibody. The tripeptide sequence “asparagine-X-serine” or “asparagine-X-threonine” (where X is any amino acid other than proline) is a recognition sequence used for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, if either of these tripeptide sequences is present in the antibody, a potential glycosylation site will be created. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine can also be used.

[0166] Adding glycosylation sites to antibodies can be achieved by altering their amino acid sequence to include one or more of the aforementioned tripeptide sequences (regarding N-linked glycosylation sites). The changes can also be made by adding or substituting one or more serine or threonine residues into the original antibody sequence (regarding O-linked glycosylation sites). Conversely, glycosylation sites can be removed by altering the amino acid composition within the native glycosylation sites of multispecific antibodies.

[0167] Antibodies can be covalently linked to another macromolecule (e.g., lipids, peptides, synthetic polymers, carbohydrates, etc.) using linkers selected from glutaraldehyde, homo-bifunctional cross-linking agents, or hetero-bifunctional cross-linking agents. Glutaraldehyde is cross-linked to the multispecific antibody via the amino group. Homo-bifunctional and hetero-bifunctional cross-linking agents are described in this application.

[0168] Linkers used to construct 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.

[0169] In some embodiments, in addition to binding to human EpCAM, the conditionally active anti-EpCAM antibodies of this disclosure also bind to non-human primates, such as cynomolgus monkeys (long-tailed macaques). Macaca fascicularis The ability to simultaneously bind to EpCAM proteins in both humans and non-human primates is beneficial for safety and efficacy testing because it allows for early testing in non-human primate subjects, not humans. In one specific embodiment, anti-EpCAM antibodies (including bispecific antibodies) bind to each of the human and cynomolgus monkey EpCAM proteins with an affinity at least 5, 4, or 3 times greater than that of the same antibody binding to rat or mouse EpCAM proteins. In another embodiment, the conditionally active bispecific antibodies of this disclosure bind to each of the human and cynomolgus monkey EpCAM proteins with an affinity at least 5 times greater than that of the same antibody binding to mouse or rat EpCAM proteins. In yet another embodiment, these antibodies bind to the cynomolgus monkey EpCAM protein with at least 45% or at least 50% of the binding affinity of the same antibody to the human EpCAM protein.

[0170] In the embodiments disclosed herein, the antigen-binding activity of an antibody or antibody fragment against the EpCAM protein may be higher at a value of one condition in a tumor microenvironment than at different values ​​of the same condition in a non-tumor microenvironment. In one embodiment, the condition is pH.

[0171] The conditionally active anti-EpCAM antibody or antibody fragment exhibits at least 70% of the same antigen-binding activity of the parent antibody or antibody fragment used to obtain it at pH 6.0, and its antigen-binding activity at pH 7.4 may be less than 50%, or less than 40%, or less than 30%, or less than 20%, or less than 10% compared to the same antigen-binding activity of the parent antibody or antibody fragment used to obtain it at pH 7.4. The antigen-binding activity may be, for example, binding to the EpCAM protein or binding to CD3.

[0172] In the previous embodiments, antigen binding activity could be measured by ELISA analysis.

[0173] These variants are obtained by means of methods described herein. 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, deletions, and / or insertions, and / or substitutions of residues in the amino acid sequences of the heavy and light chain variable regions. Any combination of deletions, insertions, and substitutions can be performed to obtain antibodies or antibody fragments of this disclosure, subject to the limitation that they possess the desired characteristics, such as binding to human EpCAM antigen and / or conditional activity.

[0174] In some embodiments, antibody or antibody fragment variants with one or more amino acid substitutions are provided. The relevant sites for inducing substitution mutations include the CDR and framework region (FR). Conserved substitutions are shown under the heading “Preferred Substitutions” in Table 2. More substantial variations are provided under the heading “Exemplary Substitutions” in Table 2 and are further described below regarding amino acid side chain categories. Amino acid substitutions can be introduced into the relevant antibody or antibody fragment and used to screen products for desired activities, such as preserved / improved antigen binding or reduced immunogenicity.

[0175] Table 2: Amino Acid Substitutions

[0176] Amino acids can be grouped according to their shared side chain characteristics: (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Alkaline: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatics: Trp, Tyr, Phe.

[0177] Non-conservative substitution will cause members of one of these categories to be replaced by members of another category.

[0178] One type of substitution variant involves replacing one or more complementary determinant region (CDR) residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant selected for further research will have modifications (e.g., improvements) in certain biological properties relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be conveniently generated, for example, using phage-based affinity maturation techniques (e.g., the techniques described herein). Simply put, one or more CDR residues are mutated, resulting in a variant antibody on a phage and screening for specific biological activities (e.g., binding affinity).

[0179] Alterations (e.g., substitutions) can be made in the CDR to, for example, improve antibody affinity. Such alterations can be made in CDR "hotspots" (i.e., residues encoded by codons that undergo frequent mutations during somatic cell maturation) (see, for example, Chowdhury, *Methods in Molecular Biology*). Methods Mol. Biol) Volume 207, pp. 179-196, 2008) and / or SDR (a-CDR), wherein the resulting variant V was tested H or V L Binding affinity. Affinity maturation achieved through the construction of secondary libraries and further selection from these libraries has been described, for example, in Hoogenboom et al., *Methods in Molecular Biology*. Methods in Molecular Biology) Volume 178, pp. 1-37, 2001. In some embodiments of affinity maturation, diversity is introduced into selected variable genes for maturation via any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-guided mutation induction). A secondary library is then generated. The library is subsequently screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-guided pathway, in which several CDR residues (e.g., 4 to 6 residues at a time) are randomly grouped. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutation induction or modeling. CDR-H3 and CDR-L3 are often targeted, especially.

[0180] In some embodiments, substitution, insertion, or deletion may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody or antibody fragment to bind to the antigen. For example, conserved changes that do not substantially reduce binding affinity (e.g., conserved substitutions as described herein) may be made within the CDR. Such changes may be outside the CDR “hotspot” or SDR. In the variant V described above... H and VL In some embodiments of the sequence, each CDR is unchanged or contains no more than one, two or three amino acid substitutions.

[0181] For example, Cunningham and Wells, Science ( Science) As described in Volume 244, pp. 1081-1085, 1989, a method for identifying antibody residues or regions that can target mutation induction is called "alanine-scan mutation induction." 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 replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction between the antibody or antibody fragment and the antigen is affected. Other substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody or antibody fragment and the antigen. These contact residues and adjacent residues can serve as targets for substitution candidates or be excluded from substitution candidate selection. Variants can be screened to determine if they contain the desired properties.

[0182] Amino acid sequence insertions include fusions of the N-terminus and / or C-terminus of a polypeptide ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other inserted variants of antibodies include fusions of the N-terminus or C-terminus of an antibody with an enzyme (e.g., targeting ADEPT) or a polypeptide that prolongs the antibody's serum half-life.

[0183] Consider one or more amino acid sequence modifications of the antibodies described herein. For example, it may be necessary to improve the binding affinity and / or other biological properties of the antibody. It is known that when humanized antibodies are produced by simply transferring the source from an antibody derived from a non-human animal, V... H and V L The CDR in the text is simply transplanted into the V of the human antibody. H and V L When produced from FR, the antigen-binding activity is reduced compared to the original antibody derived from non-human animals. It is believed that the V of non-human antibodies... H and V L Several amino acid residues are directly or indirectly related to antigen-binding activity not only in the CDR but also in the FR. Therefore, using V derived from human antibodies... H and V L Substituting different amino acid residues of FR into these amino acid residues will reduce binding activity. To address this issue, in antibodies transplanted with human CDRs, attempts are made to identify the V of the human antibody. H and V LThe amino acid residues in the FR (antigen-binding retrieval) sequence are directly related to the binding of the antibody, interact with the amino acid residues of the CDR (antigen-binding retrieval), or maintain the three-dimensional structure of the antibody and the amino acid residues directly related to binding to the antigen. Reduced antigen-binding activity can be increased by replacing identified amino acids with amino acid residues from the original antibody derived from non-human animals.

[0184] Modifications and alterations can be made in the structure of the antibodies disclosed herein and in the DNA sequence encoding them, and functional molecules encoding antibodies with the desired characteristics can still be obtained.

[0185] When making such changes in the amino acid sequence, the hydrophilicity index of the amino acid can be considered. The importance of the hydrophilic amino acid index in conferring the interacting biological functions of proteins is generally understood in the field. The relative hydrophilicity of amino acids is recognized to contribute to the secondary structure of the resulting protein, which in turn defines the protein's interactions with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, and their analogues. Each amino acid has been assigned a hydrophilic index based on its hydrophobicity and charge characteristics: 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).

[0186] Another objective of this disclosure also covers the functionally conserved variants of the antibodies disclosed herein.

[0187] Two amino acid sequences are considered "substantially homologous" or "substantially similar" when they are identical in more than 80%, preferably more than 85%, preferably more than 90% of the amino acids relative to the full length of the shorter sequence, or are similar (functionally identical) in more than about 90%, preferably more than 95%. Preferably, similar or homologous sequences are identified by alignment using, for example, a GCG (Genetics Computer Group, Program Manual for the GCG Package, 7th Edition, Madison, Wisconsin) stacking program or any of the sequence comparison algorithms such as BLAST, FASTA, etc.

[0188] For example, certain amino acids can be substituted by other amino acids in the protein structure without significant loss of activity. Because the interacting abilities and properties of proteins limit their biological function, certain amino acid substitutions can occur in the protein sequence, and of course in its DNA coding sequence, while still yielding a protein with similar properties. Therefore, it is anticipated that various changes can be made to the sequence of the antibody or antibody fragment disclosed herein, or to the corresponding DNA sequence encoding said antibody or antibody fragment, without significant loss of its biological activity.

[0189] In this field, it is known that certain amino acids can be substituted with other amino acids that have similar hydrophilicity indices or scores, and still produce proteins with similar biological activities, that is, proteins that are still biologically equivalent.

[0190] As outlined herein, amino acid substitutions are therefore generally based on the relative similarity of the substituents in the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, size, and similar forms. Exemplary substitutions considering a variety of the aforementioned characteristics are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0191] In some 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. Adding or removing glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0192] In the case of antibodies including the Fc region, the carbohydrates attached to it can be altered. Native antibodies produced by mammalian cells typically consist of branched-chain biantennary oligosaccharides, which are usually linked to Asn297 in the CH2 domain of the Fc region via N-bonds. See, for example, Wright et al. TIBTECH Volume 15, pp. 26-32, 1997. Oligosaccharides may comprise various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as trehalose of GlcNAc linked to the "backbone" of the bifacial oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of this disclosure may be modified to produce antibody variants with certain improved properties.

[0193] In one embodiment, an antibody variant is provided that lacks a fucosylated (direct or indirect) carbohydrate structure linked to the Fc region. For example, the amount of fucosy in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucosy is determined by calculating the average amount of fucosy at Asn297 within the sugar chain, relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) linked to Asn297, as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to the asparagine residue located approximately at position 297 (EU number of Fc region residues) in the Fc region; however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such trehalose-based variants may possess improved ADCC function. See, for example, U.S. Patent Publication No. 2003 / 0157108 (Presta, L.) and No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of disclosed antibody variants that are "defucosylated" or "fucose-deficient" include: US 2003 / 0157108; WO2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al., Journal of Molecular Biology ( J. Mol. Biol.) Volume 336, pp. 1239-1249, 2004; Yamane-Ohnuki et al., Biotechnology and Bioengineering ( Biotech. Bioeng.) Volume 87, pp. 614-622, 2004. Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., *Proceedings of the Biochemical and Biophysical Journal*). Arch. Biochem. Biophys )》 .Volume 249, pp. 533-545, 1986; U.S. Patent Application No. US 2003 / 0157108 A; and WO2004 / 056312 A1, particularly Example 11), and gene knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 gene knockout CHO cells (see, for example, Yamane-Ohnuki et al., Biotechnology and Bioengineering). 》， Volume 87, pp. 614-622, 2004; Kanda, Y. et al., Biotechnology and Bioengineering 》， Volume 94, pp. 680-688, 2006; and WO2003 / 085107).

[0194] Antibody variants further possess sharded oligosaccharides, for example, wherein the biantennary oligosaccharide linked to the Fc region of the antibody is sharded by GlcNAc. Such antibody variants may have reduced trehalose sizing and / or improved ADCC functionality. Examples of such antibody variants are described, for example, in WO 2003 / 011878; U.S. Patent No. 6,602,684; and US 2005 / 0123546. Antibody variants are also provided in which at least one galactose residue in the oligosaccharide is linked to the Fc region. Such antibody variants may have improved CDC functionality. These antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0195] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the anti-EpCAM antibody or antibody fragment provided herein, thereby creating Fc region variants. Fc region variants may include human Fc region sequences (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc regions) that include amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0196] Some embodiments cover antibody variants having some, but not all, effector functions, thereby making the antibody a desirable candidate for applications where the in vivo antibody half-life is critical, and certain effector functions (e.g., ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody does not have FcγR binding ability (and therefore may not have ADCC activity), but retains FcRn binding ability. Primary NK cells, used to regulate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annals of Immunology (…).Annu. Rev. Immunol Table 3, pp. 457-492, Vol. 9, 1991, p. 464. Non-limiting examples of in vitro analyses for assessing ADCC activity of relevant molecules are described in U.S. Patent No. 5,500,362 (see also, for example, Hellstrom et al., Proceedings of the National Academy of Sciences of the United States of America). Proc. Nat'l Acad. Sci See also Hellstrom, I. et al., *Proceedings of the National Academy of Sciences of the United States of America*, Vol. 83, pp. 7059-7063, 1986; and Hellstrom, I. et al., *Proceedings of the National Academy of Sciences of the United States of America*, Vol. 82, pp. 1499-1502, 1985; U.S. Patent No. 5,821,337 (see also Bruggemann et al., *Journal of Experimental Medicine*). J. Exp. Med (See, Vol. 166, pp. 1351-1361, 1987). Alternatively, non-radioactive analytical methods may be used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA); and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells suitable for this type of analysis include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of relevant molecules may be assessed in vivo in animal models, such as those disclosed in Clynes et al., Proceedings of the National Academy of Sciences, Vol. 95, pp. 652-656, 1998. C1q binding assays may also be performed to confirm that the antibody does not bind to C1q and therefore does not have CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC analysis can be performed (see, for example, Gazzano-Santoro et al., *Journal of Immunological Methods*). J. Immunol. Methods) Volume 202, pp. 163-171, 1996; Grugge, MS (Cragg, MS et al., Blood ( Blood) Volume 101, pp. 1045-1052, 2003; and Cragg, MS, and MJ Glennie, *Blood* ，Volume 103, pp. 2738-2743, 2004. Methods known in the field may also be used (see, for example, Petkova, SB, et al., *International Journal of Immunology*). Int'l. Immunol. (Volume 18, pp. 1759-1769, 2006) to determine FcRn binding and in vivo clearance / half-life.

[0197] Variants of antibodies or antibody fragments with reduced effector function include variants that substitute one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with residues 265 and 297 substituted with alanine (U.S. Patent No. 7,332,581).

[0198] This field describes certain antibody variants that exhibit increased or decreased binding to FcR. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al., *Journal of Biochemistry*). J. Biol. Chem. ( ), Volume 9, pp. 6591 to 6604, 2001).

[0199] In some embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbers of residues) of the Fc region.

[0200] In some embodiments, alterations are made in the Fc region that alter (i.e., improve or reduce) C1q binding and / or complement-dependent cytotoxicity (CDC), as exemplified by U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al. ， Journal of Immunology J. Immunol As described in Volume 164, pp. 4178-4184, 2000.

[0201] Prolonged half-life and increased interaction with neonatal Fc receptors (FcRn) responsible for transferring maternal IgG to the fetus (Guyer et al.) J. Immunol .), Volume 117, pp. 587-593, 1976, and Kim et al. Mol. Biol.)Antibodies binding to FcRn are described in US2005 / 0014934, Vol. 24, p. 249, 1994. Those antibodies include an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following 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, for example, substitution of Fc region residue 434 (US Patent No. 7,371,826). For other examples of Fc region variants, see Duncan and Winter, Nature, Vol. 322, pp. 738-740, 1988; U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0202] In some embodiments, it may be necessary to generate cysteine-engineered antibodies, such as “thioMAb”, wherein one or more residues of the anti-EpCAM antibody or antibody fragment are substituted with cysteine ​​residues. In specific embodiments, the substituted residues are located at accessible sites on the antibody. By substituting those residues with cysteine, the reactive thiol group is thereby positioned at an accessible site on the antibody and can be used to bind the antibody to other moieties (e.g., drug moieties or linker-drug moieties) to produce an immunoconjugate as further described herein. In some embodiments, any or more of the following residues may be substituted with cysteine: V205 (Kabat number) of the light chain; A118 (Eu number) of the heavy chain; and 5400 (Eu number) of the Fc region of the heavy chain. Cysteine-engineered antibodies can be generated as described, for example, in U.S. Patent No. 7,521,541.

[0203] In some embodiments, the anti-EpCAM antibodies or antibody fragments provided herein may be further modified to contain additional non-protein moieties known in the art and readily available. Moieties suitable for antibody or antibody fragment derivatization 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, carboxymethyl cellulose, polydextrose, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers) and polydextrose or poly(vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody or antibody fragment can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations such as the specific properties or functions of the antibody or antibody fragment to be improved (but not limited to), and whether the derivative will be used for therapy under specified conditions.

[0204] The anti-EpCAM antibody or antibody fragment of this disclosure, or a variant thereof, exhibits a higher binding affinity for EpCAM under tumor microenvironment conditions than under non-tumor microenvironment conditions. In one embodiment, both tumor and non-tumor microenvironment conditions are pH values. In one embodiment, the anti-EpCAM antibody or antibody fragment of this disclosure thus selectively binds to EpCAM at about pH 5.0–6.8, but exhibits a lower binding affinity for EpCAM at about pH 7.2–7.8, which is encountered in a normal non-tumor microenvironment. In another embodiment, the anti-EpCAM antibody or antibody fragment of this disclosure selectively binds to EpCAM at about pH 5.0–6.9, but exhibits a lower binding affinity for EpCAM at about pH 7.0–7.6, which is encountered in a normal non-tumor microenvironment. In yet another embodiment, the anti-EpCAM antibody or antibody fragment of this disclosure selectively binds to EpCAM at about pH 5.0–6.9, but exhibits a lower binding affinity for EpCAM at about pH 7.0–7.8, which is encountered in a normal non-tumor microenvironment. In another embodiment, the anti-EpCAM antibody or antibody fragment of this disclosure selectively binds to EpCAM at approximately pH 5.0–6.8, but will encounter lower binding affinity for EpCAM at approximately pH 7.2–7.6 in a normal non-tumor microenvironment. In one embodiment, in a screening assay, such as those described herein, the anti-EpCAM antibody or antibody fragment exhibits a higher binding affinity for EpCAM at pH 6.0 compared to pH 7.4.

[0205] In some embodiments, the dissociation constant (Kd) of the anti-EpCAM antibody or antibody fragment of this disclosure against EpCAM under tumor microenvironment conditions is approximately ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM). -8 M is lower, or 10 -8 M to 10 -13 M, or 10 -9 M to 10 -13M). In one embodiment, the ratio of the Kd of the antibody or antibody fragment to 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 the antibody or antibody fragment to EpCAM under tumor microenvironment conditions 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.

[0206] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA), which utilizes the Fab form of the relevant antibody and its antigen using the following analysis. The solution-binding affinity of the Fab to the antigen is measured by the lowest concentration of ( ) in the presence of a series of titrants of unlabeled antigen. 125 I) Labeling antigens to balance Fab, followed by capturing binding antigens with culture plates coated with anti-Fab antibodies (see, for example, Chen et al., *Journal of Molecular Biology*). J. J. Mol. 》, 293:865-881(1999)). To determine the analytical conditions, MICROTITER® multiwell plates (Thermo Scientific) were plated overnight with 50 mM sodium carbonate (pH 9.6) containing 5 µg / ml Cappel Labs anti-Fab antibody, and then blocked with PBS containing 2% (w / v) bovine serum albumin for two to five hours at room temperature (approximately 23°C). In absorbent-free plates (Nunc #269620), 100 pM or 26 pM [ 125I] The antigen was mixed with a serially diluted buffer of the associated Fab (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res., 57:4593-4599 (1997)). The associated Fab was then incubated overnight; however, incubation can continue for a longer period (e.g., about 65 hours) to ensure equilibration. The mixture was then transferred to a capture culture plate at room temperature for incubation (e.g., for one hour). The solution was then removed and the plate was washed eight times with PBS containing 0.1% polysorbate 20 (TWEEN-20®). When the plate was dry, 150 μL / well of scintillator (MICROSCINT-20™; Packard) was added and the plate was placed in a TOPCOUNT container. TM Use a gamma counter (Packard) to measure the gamma for several tens of minutes. Select the concentration of each Fab that provides a maximum binding of less than or equal to 20% for competitive binding analysis.

[0207] According to another embodiment, Kd is measured using surface plasmon resonance analysis with a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C using a fixed antigen CM5 chip of approximately 10 reaction units (RU). Briefly, the 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 obtain approximately 10 reaction units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. Regarding kinetic measurements, Fab was injected at a flow rate of approximately 25 µl / min into a solution containing 0.05% polysorbate 20 (TWEEN-20) at 25°C. TM The surfactant was used in two serial dilutions in PBS (PBST) (0.78 nM to 500 nM). The association rate (kJ / kM) was calculated by simultaneously fitting association and dissociation sensing spectra using a simple one-to-one Langmuir binding model (BIAcore® evaluation software version 3.2). on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) is proportional to k. off / kon To calculate. See, for example, Chen et al., *Journal of Molecular Biology*. Biol. Nat. Med )》 ， 293:865-881 (1999). If, based on the surface plasmon resonance analysis above, the on-rate of association exceeds 10... 6 M -1 s -1 The association rate can then be determined using a fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of PBS containing 20 nM anti-antigen antibody (Fab form) at 25°C in the presence of progressively increasing concentrations of antigen, such as in a stop-flow spectrophotometer with a stirred photocell (Aviv Instruments) or an 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic).

[0208] The anti-EpCAM antibody disclosed herein may be a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, an anti-EpCAM antibody fragment, such as Fv, Fab, Fab', Fab'-SH, scFv, a bifunctional antibody, a trifunctional antibody, a tetrafunctional antibody, or an F(ab')2 fragment, and a multispecific antibody formed from the antibody fragment, are employed. In another embodiment, the antibody is a full-length antibody, such as a complete IgG antibody or other antibody class or isotype as defined herein. For a review of certain antibody fragments, see Hudson et al., *Nature Medicine*. Front. Biosci .), Vol. 9, pp. 129-134, 2003. For a review of scFv fragments, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore (Springer-Verlag, New York), pp. 269-315 (1994); see also WO93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that include rescue receptor-binding epitope residues and have an extended in vivo half-life, see U.S. Patent No. 5,869,046.

[0209] The bifunctional antibodies disclosed herein may be bivalent or bispecific. For examples of bifunctional antibodies, see, for instance, EP 404,097; WO 1993 / 01161; Hudson et al., *Nature Medicine*. ， 9:129-134 (2003); and Hollinger et al., Proceedings of the National Academy of Sciences of the United States of America. ， Volume 90, pp. 6444-6448, 1993. Examples of trifunctional and tetrafunctional antibodies are also described in Hudson et al., *Nature Medicine*, Volume 9, pp. 129-134, 2003.

[0210] In some embodiments, this disclosure provides a single-domain antibody fragment comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In some embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1).

[0211] Antibody fragments can be prepared using various techniques, including (but not limited to) protein degradation and digestion of intact antibodies, as well as production via recombinant host cells (e.g., E. coli or bacteriophages). In some embodiments, the anti-EpCAM antibody disclosed herein may be a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and in the Proceedings of the National Academy of Sciences of Morrison et al. ， Volume 81, pp. 6851-6855, 1984. In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (e.g., monkey)) and a human constant region. In another example, a chimeric antibody is a "class-switching" antibody, wherein the class or subclass of the antibody has changed relative to the class or subclass of the parent antibody. A chimeric antibody contains its antigen-binding fragment.

[0212] In some embodiments, the chimeric antibodies of this disclosure are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein the CDR (or a portion thereof) is derived from a non-human antibody, and the FR (or a portion thereof) is derived from a human antibody sequence. Humanized antibodies may optionally also include at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0213] A review of humanized antibodies and their manufacturing methods can be found in publications such as Almagro and Fransson, *Forewords to the Biosciences*. Methods .)》, Volume 13, pp. 1619-1633, 2008, and further described in, for example, Riechmann et al., Nature, Volume 332, pp. 323-329, 1988; Queen et al., Proceedings of the National Academy of Sciences of the United States of America, Volume 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., Methods ( (Mol. Immunol.) (Description of SDR (a-CDR) transplantation), Vol. 36, pp. 25-34, 2005; Padlan ， Molecular Immunology Br. J. Cancer , Vol. 28, pp. 489–498, 1991 (description of “surface remodeling”); Dall'Acqua et al., Methods, Vol. 36, pp. 43–60, 2005 (description of “FR remodeling”); and Osbourn et al., Methods, Vol. 36, pp. 61–68, 2005; and Klimka et al., British Journal of Cancer ( Front. (Describing the “guided selection” approach to FR reorganization), Vol. 83, pp. 252-260, 2000.

[0214] Human scaffold regions that can be used for humanization include (but are not limited to): scaffold regions selected using the "best-fit" method (see, for example, Sims et al., *Journal of Immunology*, Vol. 151, p. 2296, 1993); scaffold regions derived from the common sequence of human antibodies with specific subgroups having light or heavy chain variable regions (see, for example, Carter et al., *Proceedings of the National Academy of Sciences*, Vol. 89, p. 4285, 1992; and Presta et al., *Journal of Immunology*, Vol. 151, p. 2623, 1993); human maturation (somatic mutation) scaffold regions or human germline scaffold regions (see, for example, Almagro and Fransson, *Forewords of Biological Sciences*). Biosci J. Biol. Chem .), Volume 13, pp. 1619-1633, 2008); and framework regions derived from screening FR libraries (see, for example, Baca et al., Journal of Biochemistry). J. Biol. Chem .), Volume 272, pp. 10678-10684, 1997 and Rosok et al., Journal of Biochemistry ( EMBO J (.), Volume 271, pp. 22611-22618, 1996).

[0215] In some embodiments, the anti-EpCAM antibody disclosed herein is multispecific, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody having binding specificity against at least two different sites. In some embodiments, one binding specificity is against EpCAM and the other against another antigen, such as CD3. A specific example of such an antibody is a bispecific antibody having binding specificity against both EpCAM and CD3+. A bispecific conditionally active antibody can be either monoconditionally active or biconditionally active. Thus, in the case of a monoconditionally active bispecific antibody, one binding site has conditional activity and the other does not, for example, wild-type (WT) EpCAM paired with conditionally active (CAB) CD3+ or CAB EpCAM paired with WT CD3+. In the case of a biconditionally active antibody, both binding sites have conditional activity, for example, in EpCAM×CAB CD3+.

[0216] Multispecific antibodies can exhibit single-conditional activity, dual-conditional activity, or triple-conditional activity. Therefore, any one or more binding regions of a multispecific antibody can possess conditional activity.

[0217] In some embodiments, bispecific antibodies can bind to two different epitopes of EpCAM. Bispecific antibodies can also be used to target cytotoxic agents to cells expressing EpCAM. Bispecific antibodies can be prepared in the form of full-length antibodies or antibody fragments.

[0218] Techniques for manufacturing multispecific antibodies include (but are not limited to) recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, *Nature*). ， Volume 305, pp. 537-540, 1983), WO 93 / 08829, and Traunecker et al. Science J.) 》 ， Volume 10, pp. 3655-3659, 1991), and engineered "knob-in-hole" structures (see, for example, U.S. Patent No. 5,731,168). Multispecific antibodies can also be prepared by: engineered electrostatic directing effects for preparing antibody Fc-heterodimers (WO 2009 / 089004A1); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980 and Brennan et al., *Science*). Figure 3 (See, for example, Kostelny et al., *Journal of Immunology*, Vol. 148, pp. 1547-1553, 1992); the use of "bifunctional antibody" techniques for preparing bispecific antibody fragments (see, for example, Hollinger et al., *Proceedings of the National Academy of Sciences*, Vol. 90, pp. 6444-6448, 1993); and the use of single-chain Fv (sFv) dimers (see, for example, Gruber et al., *Journal of Immunology*, Vol. 152, pp. 5368-5374, 1994); and the preparation of trispecific antibodies as described, for example, Tutt et al., *Journal of Immunology*, Vol. 147, pp. 60-69, 1991.

[0219] This article also includes engineered antibodies having three or more functional antigen-binding sites, including “octopus antibodies” (see, for example, US 2006 / 0025576A1).

[0220] The anti-EpCAM antibody or antibody fragment disclosed herein can be manufactured using recombinant methods and compositions, as detailed in US 2016 / 0017040.

[0221] The bispecific antibody disclosed herein comprises a combination of the anti-EpCAM antibody disclosed herein and an antibody that binds to a T-lymphocyte antigen. In one embodiment, the antibody may comprise an anti-EpCAM moiety containing a combination of complementarity-determining regions provided in Table 1. In other embodiments, the anti-EpCAM moiety of the bispecific antibody contains the heavy chain variable region of SEQ ID NO:52 and the light chain variable region of any one of SEQ ID NO:53-69. In another embodiment, the anti-EpCAM moiety of the bispecific antibody contains the light chain variable region of SEQ ID NO:51 and the heavy chain variable region of any one of SEQ ID NO:70-96. In some embodiments, the antibody that binds to the T-lymphocyte antigen is an scFv antibody. In some embodiments, the T-lymphocyte antigen antibody may be an anti-CD3 antibody. In a specific embodiment, the anti-CD3 antibody contains the light chain variable region of SEQ ID NO:101 and the heavy chain variable region of SEQ ID NO:100. In a more specific embodiment, the anti-CD3 scFv antibody comprises SEQ ID NO:99. Other sequences of anti-CD3 antibodies applicable to the multispecific antibodies of this disclosure can be found in WO 2019 / 241216.

[0222] In one embodiment, the bispecific antibody comprises an intact IgG molecule or fragment, such as a (Fab')2 fragment, and an scFv antibody attached to the C-terminus of the light chain of said IgG or IgG fragment, such as... Materials As shown in the figure. In one embodiment, the IgG portion of the bispecific antibody may comprise any anti-EpCAM antibody disclosed herein, and the scFv portion may comprise any anti-CD3 antibody disclosed herein. In another embodiment, the IgG portion of the bispecific antibody may comprise any anti-CD3 antibody disclosed herein, and the scFv antibody may comprise any anti-EpCAM antibody disclosed herein. In one specific embodiment, the IgG portion of the bispecific antibody comprises a light chain variable region comprising SEQ ID NO:1-3 and a heavy chain variable region comprising SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:45, while the scFv portion comprises a light chain variable region comprising SEQ ID NO:101 and a heavy chain variable region comprising SEQ ID NO:100. In another embodiment, the IgG portion of the bispecific antibody comprises a light chain variable region comprising SEQ ID NO:51 and a heavy chain variable region comprising SEQ ID NO:91, and an scFv fragment comprising SEQ ID NO:99.

[0223] In some embodiments, any anti-EpCAM antibody or antibody fragment provided herein can be used for quantitative or qualitative detection of the presence of EpCAM in a biological sample. In some embodiments, the biological sample comprises cells or tissues, such as breast, pancreas, esophagus, lung, and / or brain cells or tissues.

[0224] Another aspect of this disclosure relates to anti-EpCAM antibodies or antibody fragments of this disclosure, which are used for the diagnosis and / or monitoring of cancer or another disease in which EpCAM expression is increased or decreased relative to normal physiological levels at at least one site in the body.

[0225] In one embodiment, the antibody or antibody fragment of this disclosure may be labeled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule, or any other labeled molecule or substance known in the art. For example, the antibody or antibody fragment of this disclosure may be labeled with a radioactive molecule. For example, a suitable radioactive molecule may contain (but is not limited to) radioactive atoms for scintillation photography studies, such as... 123 I, 124 I, 111 In、 186 Re and 188 Re. The antibodies or antibody fragments disclosed herein can also be labeled with spin labels for nuclear magnetic resonance (NMR) imaging, such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. After antibody administration, the distribution of the radiolabeled antibody in 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 scanning.

[0226] The antibodies or antibody fragments disclosed herein may be used for the diagnosis and classification of cancers and diseases associated with EpCAM overexpression. Cancers associated with EpCAM overexpression may include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors (e.g., glioblastoma and neurofibroma), cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumors, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, sarcoma, hematologic malignancies (leukemia), astrocytomas, and various types of head and neck cancers or other EpCAM-expressing or overexpressing hyperproliferative diseases.

[0227] The antibodies or antibody fragments disclosed herein can be used to diagnose diseases other than cancer characterized by increased or decreased EpCAM expression. Both soluble and cellular forms of EpCAM can be used for such diagnoses. Typically, such diagnostic methods involve the use of biological samples obtained from patients. Biological samples encompass a variety of sample types obtained from subjects that can be used for diagnostic or monitoring analyses. Biological samples include (but are not limited to) blood and other liquid samples of biological origin, solid tissue samples (e.g., biopsy samples), or tissue culture media or cells derived therefrom and their progeny. For example, biological samples may contain cells obtained from tissue samples collected from subjects suspected of having cancer associated with EpCAM overexpression, and in preferred embodiments, from glioma, stomach, lung, pancreas, breast, prostate, kidney, liver, and endometrium. Biological samples encompass clinical samples, cells in cultures, cell supernatants, cell lysates, serum, plasma, biofluids, and tissue samples.

[0228] In a particular embodiment, a method for diagnosing cancer associated with EpCAM overexpression in a subject is provided, which is achieved by detecting EpCAM on cells from the subject using the antibody of the present invention. Specifically, the method may include the following steps: 1. To contact a subject's biological sample with an antibody or antibody fragment according to this disclosure under conditions suitable for the antibody or antibody fragment to form a complex with cells expressing EpCAM in the biological sample; and 2. Detect and / or quantify the complex, thereby indicating cancer associated with EpCAM overexpression.

[0229] To monitor cancer progression, the method can be repeated at different times to determine whether the antibodies bound to the sample increase or decrease, thereby determining whether the cancer has progressed, regressed, or stabilized.

[0230] In certain embodiments, this disclosure provides a method for diagnosing diseases associated with the expression or overexpression of EpCAM. Examples of such diseases may include human immune disorders, thrombotic diseases (thrombosis and atherosclerotic thrombosis), and cardiovascular diseases.

[0231] In one embodiment, an anti-EpCAM antibody or antibody fragment is provided for use in a diagnostic or detection method. In another embodiment, a method is provided for detecting the presence of EpCAM in a biological sample. In yet another embodiment, a method is provided for quantifying the amount of EpCAM in a biological sample. In some embodiments, the method includes contacting a biological sample with an anti-EpCAM antibody or antibody fragment as described herein 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 methods can be performed in vitro or in vivo. In one embodiment, an anti-EpCAM antibody or antibody fragment is used to select a subject eligible for treatment. In some embodiments, the therapy will comprise administering an anti-EpCAM antibody or antibody fragment to a subject.

[0232] In some embodiments, labeled anti-EpCAM antibodies or antibody fragments are provided. Labeling includes (but is not limited to) directly detectable labels or fractions (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and fractions indirectly detectable, such as via 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 and 131 I; fluorophores, such as rare earth chelates or luciferin and its derivatives; rhodamine and its derivatives; dansyl; umbelliferone; luciferases, such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456); luciferin; 2,3-dihydrophthalazinedione; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; glucosylamylase; lysozyme; sugar oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidases, such as uricase and xanthine oxidase, coupled to enzymes using hydrogen peroxide dye precursors (e.g., HRP, lactoperoxidase, or microperoxidase); biotin / antibiotic protein; spin labeling; phage labeling; stable free radicals, etc.

[0233] Anti-EpCAM antibodies or antibody fragments, as well as multispecific antibodies incorporating said antibodies or antibody fragments, possess cytotoxic activity. This cytotoxic activity extends to a wide variety of cell lines. Therefore, anti-EpCAM antibodies, their fragments, or multispecific antibodies are suitable for treating proliferative diseases associated with EpCAM expression. Antibodies, fragments, or multispecific antibodies can be used alone or in combination with any suitable agent or other conventional treatment.

[0234] The anti-EpCAM antibodies, antibody fragments, or multispecific antibodies disclosed herein can be used to treat diseases associated with EpCAM expression, overexpression, or activation. There are no specific limitations on the type of cancer or tissue that can be treated, except for the requirement of EpCAM expression. Examples include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors (e.g., glioblastoma and neurofibroma), cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumors, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, sarcoma, hematologic malignancies (leukemia), astrocytomas, and various types of head and neck cancers. Specific cancers include glioma, gastric cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, liver cancer, and endometrial cancer.

[0235] The anti-EpCAM antibodies, antibody fragments, or multispecific antibodies disclosed herein are potential activators of innate immune responses and therefore can be used to treat human immune disorders such as sepsis. The anti-EpCAM antibodies or antibody fragments disclosed herein can also be used as adjuvants for immunization, such as in vaccines and as anti-infective agents against, for example, bacteria, viruses, and parasites.

[0236] In various embodiments of the treatment methods described herein, anti-EpCAM antibodies, antibody fragments, or multispecific anti-EpCAM antibodies or antibody fragments may be delivered in a manner consistent with conventional methods associated with the management of the disease or condition for which treatment is sought. According to the disclosure herein, an effective amount of the antibody, antibody fragment, or multispecific antibody is administered to a subject in need of such treatment at a time and under conditions sufficient to prevent or treat the disease or condition. Therefore, one aspect of this disclosure relates to a method for treating a disease associated with EpCAM expression, comprising administering a therapeutically effective amount of the disclosed antibody, antibody fragment, or multispecific antibody to a subject in need.

[0237] For administration, the anti-EpCAM antibodies, antibody fragments, or multispecific antibodies of this disclosure can be formulated into pharmaceutical compositions. Pharmaceutical compositions comprising 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 containing a pharmaceutically acceptable carrier.

[0238] A pharmaceutically acceptable carrier is a substance that is tolerable to the receiving patient. Sterile phosphate-buffered saline is an 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.), *Remington's Pharmaceutical Sciences* (Mack Publishing Company, 19th ed., 1995)). The formulation may further contain one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss from the surface of the vial, etc.

[0239] The form, route of administration, dosage, and regimen of the pharmaceutical composition are, of course, determined by the condition to be treated, the severity of the disease, the patient's age, weight, and sex. Those skilled in the art can take these factors into account to formulate a suitable pharmaceutical composition. The pharmaceutical compositions disclosed herein can be formulated for topical, oral, non-enteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, and similar routes of administration.

[0240] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable mediator that allows for injection. These mediators may in particular be isotonic sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride and their analogs or mixtures of such salts), or dried, particularly freeze-dried compositions that, upon addition of, for example, sterile water or saline, allow for reconstitution into an injectable solution.

[0241] In some embodiments, a tonic agent, sometimes referred to as a "stabilizer," is present to regulate or maintain the surface tension of the liquid in the composition. When used with large charged biomolecules (e.g., proteins and antibodies), it is often referred to as a "stabilizer" because it can interact with charged groups on the side chains of amino acids, thereby reducing the likelihood of intermolecular and intramolecular interactions. The tonic agent may be present in any amount from 0.1% to 25% by weight of the pharmaceutical composition, for example, from 1% to 5% by weight. The tonic agent may comprise polyhydroxy sugar alcohols, such as triols or higher sugar alcohols, such as glycerol, erythritol, arabinitol, xylitol, sorbitol, or mannitol.

[0242] Additional excipients may include agents that can act as one or more of the following: (1) a build-up agent, (2) a solubility enhancer, (3) a stabilizer, and (4) an agent that prevents denaturation or adhesion to the container wall. Such excipients may include: polyhydroxy sugar alcohols (listed 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, sorbitol, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactose Alcohols, glycerols, cyclic alcohols (e.g., inositol), polyethylene glycols; sulfur-containing reducing agents, such as urea, glutathione, lipoic acid, sodium thioacetate, 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, and glucose); disaccharides (e.g., lactose, maltose, and sucrose); trisaccharides, such as raffinose; and polysaccharides, such as dextrin or dextran.

[0243] Nonionic surfactants or detergents (also known as "wetting agents") can be used to help dissolve the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation, thereby exposing the formulation to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant may be present at concentrations ranging from about 0.05 mg / ml to about 1.0 mg / ml, preferably from about 0.07 mg / ml to about 0.2 mg / ml.

[0244] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitol monoethers (TWEEN®-20, TWEEN®-80, etc.), polylaurin 400, polyethylene glycol 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glyceryl monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Applicable anionic detergents include sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzyl chloride.

[0245] The dosage used for administration can be adjusted according to various parameters, and specifically according to the administration method used, the relevant pathology, or the required duration of treatment. To prepare the pharmaceutical composition, an effective amount of antibody, antibody fragment, or multispecific antibody can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0246] Suitable injectable drug forms may include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid in a manner readily injectable. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi.

[0247] Solutions of active compounds in the form of free bases or pharmacologically acceptable salts can be prepared by appropriate mixing with surfactants in water. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and in oils. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth.

[0248] The anti-EpCAM antibodies, antibody fragments, or multispecific antibodies disclosed herein can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed from the free amino groups of proteins) and are formed from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0249] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol and their analogues), suitable mixtures thereof, and vegetable oils. For example, this can be achieved by using a coating (e.g., lecithin), maintaining the desired particle size for the dispersion, and maintaining appropriate flowability by using surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and their analogues. In many cases, isotonic agents, such as sugars or sodium chloride, are preferred. The absorption of the injectable composition can be prolonged by using an absorption-retarding agent (e.g., aluminum monostearate and gelatin) in the composition.

[0250] The sterile injectable solution is prepared by incorporating the desired amount of the anti-EpCAM antibody, antibody fragment, or multispecific antibody described herein, as needed, together with one or more of the other components listed above, into a suitable solvent, followed by filtration and sterilization. Generally, a dispersion is prepared by incorporating various sterile active ingredients into a sterile medium containing an alkaline dispersion medium and any other desired components from those listed above. When sterile powder is used to prepare the sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying techniques, producing a powder of the active ingredient plus any additional desired components from its previously sterile filtered solution.

[0251] It also covers the preparation of larger or higher concentrations of solutions for direct injection, with the envisioned use of dimethyl sulfoxide (DMSO) as a solvent to generate extremely rapid penetration, delivering high concentrations of the active agent to small tumor regions.

[0252] When prepared, the solution containing anti-EpCAM antibodies, antibody fragments, or multispecific antibodies can be administered in a manner compatible with the dosage formulation and at a therapeutically effective amount. The formulation is readily available in various dosage forms, such as the injectable solutions described herein.

[0253] For administration non-enterically in aqueous solution form, the solution should, if necessary, be appropriately buffered and first diluted with sufficient physiological saline or glucose to make the liquid diluent isotonic. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, according to the invention, sterile aqueous media that can be used will be known to those skilled in the art. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous perfusion fluid, or injected at the recommended infusion site (see, for example, "Pharmaceutical Science of Remington," 15th edition, pp. 1035-1038 and 1570-1580). Some dose variation will inevitably occur depending on the condition, weight, and / or sex of the subject being treated. In any case, the person responsible for administration will determine the appropriate dose for the individual subject.

[0254] The antibodies, antibody fragments, or multispecific antibodies described herein may be formulated into therapeutic mixtures to deliver about 0.0001 to 10.0 mg, or about 0.001 to 5 mg, or about 0.001 to 1 mg, or about 0.001 to 0.1 mg, or about 0.1 to 1.0 mg, or even about 10 mg per dose. Multiple doses may also be administered at selected time intervals.

[0255] In some embodiments, the use of liposomes and / or nanoparticles is included in the introduction of antibodies or antibody fragments into host cells. The forms and uses of liposomes and / or nanoparticles are known to those skilled in the art.

[0256] Nanocapsules can generally encapsulate compounds in a stable and reproducible manner. To avoid side effects caused by intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) are generally designed using polymers that are degradable in vivo. This disclosure includes biodegradable polyalkylene cyanoacrylate nanoparticles that meet these requirements and are readily available.

[0257] Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilayered concentric bilayer vesicles (MLVs). MLVs typically have a diameter of 25 nm to 4 μm. Sonic processing of MLVs results in the formation of small monolayer vesicles (SUVs) with diameters ranging from 200 to 500 angstroms and containing an aqueous core. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0258] As described herein, pharmaceutical formulations containing anti-EpCAM antibodies, antibody fragments, or multispecific antibodies are prepared by mixing said antibody, antibody fragment, or multispecific antibody of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences, 16th ed., Osol, A. (1980)), in lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethylbenzyl ammonium chloride; hexahydroxyquaternary ammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butyl, or benzyl alcohol; alkyl p-hydroxybenzoate, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than about 10 residues). Polypeptides; 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, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming ions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0259] The exemplary pharmaceutically acceptable carriers described herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX). ® Baxter International, Inc. Certain exemplary sHASEGP and methods of use, comprising rHuPH20, are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more other glucosaminoglycanases (e.g., chondroitinase).

[0260] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations comprise those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter comprising histidine-acetate buffer.

[0261] Depending on the specific indications being treated, the formulations described herein may contain more than one active ingredient. Preferably, complementary ingredients that do not adversely affect each other may be combined into a single formulation. For example, in addition to the anti-EpCAM antibodies, antibody fragments, or multispecific antibodies disclosed herein, it may be necessary to provide EGFR antagonists (e.g., erlotinib), anti-angiogenic agents (e.g., VEGF antagonists, which may be anti-VEGF antibodies), or chemotherapeutic agents (e.g., paclitaxel or platinum preparations). These active ingredients are preferably present in a combination in amounts that effectively achieve the intended purpose.

[0262] Any anti-EpCAM antibody, antibody fragment, or multispecific antibody provided herein can be used in a treatment method. In one aspect, an anti-EpCAM antibody, antibody fragment, or multispecific antibody is provided for use as a pharmaceutical agent. In other aspects, an anti-EpCAM antibody or antibody fragment is provided for treating cancers such as breast cancer, non-small cell lung cancer, pancreatic cancer, brain cancer, pancreatic cancer, brain cancer, kidney cancer, ovarian cancer, gastric cancer, leukemia, endometrial cancer, colon cancer, prostate cancer, thyroid cancer, liver cancer, osteosarcoma, and / or melanoma. In some embodiments, an anti-EpCAM antibody, antibody fragment, or multispecific antibody is provided for use in a treatment method. In some embodiments, this disclosure provides an anti-EpCAM antibody, antibody fragment, or multispecific antibody for use in a method of treating an individual with cancer, comprising administering an effective amount of the anti-EpCAM antibody, antibody fragment, or multispecific antibody to said individual. In other embodiments, this disclosure provides an anti-EpCAM antibody, antibody fragment, or multispecific antibody for inhibiting angiogenesis, inhibiting cell proliferation, inhibiting tumor vascularity (e.g., intratumoral vascularity or tumor-associated vascularity), and / or inhibiting tumor matrix function.

[0263] In another aspect, the present invention provides the use of anti-EpCAM antibodies or antibody fragments for the manufacture or preparation of pharmaceutical agents. In one embodiment, the pharmaceutical agent is used to treat cancer (in some embodiments, breast cancer, non-small cell lung cancer, pancreatic cancer, brain cancer, pancreatic cancer, brain cancer, 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 pharmaceutical agent is used in a method of treating cancer, the method comprising administering an effective amount of the pharmaceutical agent to a subject suffering from cancer. In one such embodiment, the method further comprises administering an effective amount of at least one other therapeutic agent, such as the therapeutic agent described herein, to an individual. In another embodiment, the pharmaceutical agent is used to inhibit angiogenesis, inhibit cell proliferation, inhibit immune function, inhibit tumor vascularity (e.g., intratumoral vascularity or tumor-associated vascularity), and / or inhibit tumor matrix function. In another embodiment, a method of using an agent to inhibit angiogenesis, inhibit cell proliferation, inhibit tumor angiogenesis (e.g., intratumoral or tumor-associated blood vessels), and / or inhibit tumor matrix function in an individual, the method comprising administering to the individual an amount of the agent capable of effectively inhibiting angiogenesis, inhibiting cell proliferation, promoting immune function, inducing inflammatory cytokine components (e.g., from tumor-associated macrophages), inhibiting tumor angiogenesis development (e.g., intratumoral or tumor-associated blood vessels), and / or inhibiting tumor matrix function. According to any of the above embodiments, "individual" can be a human. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one other therapeutic agent, such as those described herein. In another embodiment, the agent is used to inhibit angiogenesis, inhibit cell proliferation, inhibit immune function, inhibit tumor angiogenesis (e.g., intratumoral or tumor-associated blood vessels), and / or inhibit tumor matrix function. In another embodiment, the method of using the agent in a subject to inhibit angiogenesis, inhibit cell proliferation, inhibit tumor vascularization (e.g., intratumoral vessels or tumor-associated vessels), and / or inhibit tumor matrix function includes administering an effective amount of the agent to the subject to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce the secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), inhibit tumor vascular development (e.g., intratumoral vessels or tumor-associated vessels), and / or inhibit tumor matrix function. The "subject" according to any of the above embodiments may be a human.

[0264] In another aspect, the present invention provides pharmaceutical formulations comprising any one of the anti-EpCAM antibodies, antibody fragments, or multispecific antibodies provided herein, for example, in any of more than one treatment method. In one embodiment, the pharmaceutical formulation comprises any one of the anti-EpCAM antibodies, antibody fragments, or multispecific antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any one of the anti-EpCAM antibodies, antibody fragments, or multispecific antibodies provided herein and at least one additional therapeutic agent, such as those described below.

[0265] In the treatments described herein, the antibodies, antibody fragments, or multispecific antibodies of this disclosure may be used alone, in the form of immune conjugates, or in combination with other agents. For example, the antibodies of this disclosure may be co-administered with at least one additional therapeutic agent. In some embodiments, the other therapeutic agent is an anti-angiogenic agent. In some embodiments, the other therapeutic agent is a VEGF antagonist (in some embodiments, an anti-VEGF antibody, such as bevacizumab). In some embodiments, the other therapeutic agent is an EGFR antagonist (in some embodiments, erlotinib). In some embodiments, the other therapeutic agent is a chemotherapeutic agent and / or a cell growth inhibitor. In some embodiments, the other therapeutic agent is paclitaxel (e.g., paclitaxel) and / or a platinum agent (e.g., carboplatin). In some embodiments, the other therapeutic agent is an agent that enhances the patient's immunity or immune system.

[0266] The aforementioned combination therapies encompass both combined administration (where two or more therapeutic agents are contained in the same or corresponding formulation) and monotherapy, in which case the antibody, antibody fragment, or multispecific antibody may be administered before, simultaneously with, and / or after other therapeutic agents and / or adjuvants. Antibodies or antibody fragments may also be used in combination with radiation therapy and / or surgical interventions.

[0267] Anti-EpCAM antibodies, antibody fragments, or multispecific antibodies may be formulated, administered, and given in accordance with good medical practice. In this case, factors to consider include the specific condition being treated, the specific mammal being treated, the individual patient's clinical presentation, the cause of the condition, the site of drug delivery, the method of administration, the timing of administration, and other factors known to the medical practitioner. Antibodies, antibody fragments, or multispecific antibodies may not be formulated with, but may optionally be formulated with, one or more currently used agents for the treatment of the condition in question. The effective amount of such other agents depends on the amount of antibody, antibody fragment, or multispecific antibody present in the formulation, the type of condition or treatment, and other factors as described above. These agents are typically used at the same dose and via the route of administration as described herein, or at any dose and via any route of administration determined empirically / clinically.

[0268] The appropriate dose of an antibody, antibody fragment, or multispecific antibody (when used alone or in combination with one or more other additional therapeutic agents) for the prevention or treatment of disease will depend on the type of disease to be treated, the type of antibody, antibody fragment, or multispecific antibody, the severity and course of the disease, whether the antibody or antibody fragment is given for prophylactic or therapeutic purposes, prior therapy, 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 may be administered to the patient appropriately in a single dose or over a series of treatments. Depending on the type and severity of the disease, an initial candidate dose of approximately 1 μg of antibody or antibody fragment per kilogram of patient body weight to 40 mg of antibody or antibody fragment per kilogram of patient body weight may be used, for example, by one or more separate administrations or by continuous infusion. When repeated administration over several days or longer, treatment generally continues until the necessary suppression of disease symptoms occurs, depending on the condition. Such doses may be given intermittently, for example, weekly or every three weeks (e.g., so that the patient receives approximately two to approximately twenty doses, or for example, approximately six doses of antibody or antibody fragment). A higher starting dose may be administered initially, followed by one or more lower doses. However, other dosing regimens may be applicable. The course of this therapy can be easily monitored using routine techniques and analysis.

[0269] Enhancing the host's immune function to combat tumors is a growing concern. Conventional methods include (i) APC enhancement, such as (a) injection into tumors encoding DNA encoding foreign MHC allogeneic antigens, or (b) transfection of biopsied tumor cells with genes that increase the probability of tumor recognition of immune antigens (e.g., immunostimulatory cytokines, GM-CSF, co-stimulatory molecules B7.1, B7.2), (iii) recipient-mediated cellular immunotherapy, or treatment with activated tumor-specific T cells. Recipient-mediated cellular immunotherapy involves isolating tumor-infiltrating host T-lymphocytes, for example, by in vitro expansion of the population via stimulation with IL-2 or the tumor, or both. Additionally, dysfunctional isolated T cells can be activated in vitro by administration of the anti-PD-L1 antibody of this disclosure. The co-activated T cells can then be re-administered to the host. One or more of these methods can be used in combination with administration of the antibody, antibody fragment, or multispecific antibody of this disclosure.

[0270] Traditional cancer treatments include: (i) radiation therapy (e.g., radiotherapy, X-ray therapy, irradiation) or the use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy can be administered externally via external beam radiotherapy (EBRT) or internally via proximal therapy; (ii) chemotherapy or the use of cytotoxic drugs that generally affect rapid cell differentiation; (iii) targeted therapy or agents that specifically affect dysregulated cancer cell proteins (e.g., tyrosine kinase inhibitors imatinib, gefitinib; monoclonal antibodies, photodynamic therapy); (iv) immunotherapy, or enhancing the host's immune response (e.g., vaccines); (v) hormone therapy, or blocking hormones (e.g., when the tumor is hormone-sensitive); (vi) angiogenesis inhibitors, or blocking angiogenesis and growth; and (vii) palliative care, or treatments aimed at improving the quality of care to reduce pain, nausea, vomiting, diarrhea, and bleeding. For example, analgesics such as morphine and oxycodone, and antiemetics such as ondansetron and aspirin, may allow for more aggressive treatment options.

[0271] In cancer treatment, any of the conventional treatments described herein for treating cancer may be administered before, after, or concurrently with the administration of the anti-EpCAM antibody, antibody fragment, or multispecific antibody disclosed herein. Additionally, the anti-EpCAM antibody, antibody fragment, or multispecific antibody may be administered before, after, or concurrently with conventional cancer treatments, such as the administration of tumor-binding antibodies (e.g., monoclonal antibodies, toxin-binding monoclonal antibodies) and / or chemotherapy agents.

[0272] In another aspect of this disclosure, an article is provided comprising an anti-EpCAM antibody, an antibody fragment, or a multispecific antibody, and other materials suitable for treating, preventing, and / or diagnosing the aforementioned conditions. The article includes a container and a label or instruction leaflet on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from various materials, such as glass or plastic. The container contains the composition alone or in combination with another composition effective in treating, preventing, and / or diagnosing the condition, and may have a sterile dispensing port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an antibody, antibody fragment, or multispecific antibody of this disclosure. The label or instruction leaflet indicates that the composition is used to treat the selected condition. Furthermore, the article may include (a) a first container containing a composition comprising an anti-EpCAM antibody, an antibody fragment, or a multispecific antibody; and (b) a second container containing a composition comprising another cytotoxic agent or other therapeutic agent. The article in this embodiment of the present disclosure may further include an instruction leaflet indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article may further include a second (or third) container comprising a pharmaceutically acceptable buffer, such as bactericidal water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0273] This disclosure also provides kits comprising at least one antibody, antibody fragment, or multispecific antibody of this disclosure. Kits containing antibodies, antibody fragments, or multispecific antibodies of this disclosure can be used to detect EpCAM expression (increased or decreased) or for therapeutic or diagnostic analysis. Kits of this disclosure may contain antibodies coupled to a solid support (e.g., tissue culture plates or beads (e.g., agarose beads)). Kits containing antibodies are available for in vitro detection and quantification of EpCAM, for example, in ELISA or Western blotting. Such antibodies suitable for detection may be labeled, for example, with fluorescent or radioactive labels.

[0274] The kit may further include instructions for use. In some embodiments, the instructions include those required by the U.S. Food and Drug Administration or other applicable agencies for in vitro diagnostic kits. 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 some embodiments, the kit further includes one or more chromatographic compounds. In other embodiments, the kit further includes one or more compounds for preparing samples for spectroscopic analysis. In other embodiments, the kit further includes a comparative reference substance to interpret the presence or absence of EpCAM based on indicator strength, chromatographic or other physical properties.

[0275] The following examples are illustrative rather than limiting of the anti-EpCAM antibody of the present invention. Other suitable modifications and adjustments to various conditions and parameters commonly encountered in the art and apparent to those skilled in the art are within the scope of this invention.

[0276] Example Examples 1-15 concerning the preparation of conditionally active antibodies are described in WO 2017 / 078839.

[0277] For Examples 16-28, exemplary antibodies (BA3182 or BAP150.31-BF45) containing SEQ ID NO: 98 and 99 were used.

[0278]

[0279] Example 16. Affinity ELISA This example demonstrates the binding activity of the BA3182 bispecific antibody to recombinant human CD3 and the extracellular domain (ECD) of human EpCAM or cynomolgus monkey EpCAM using enzyme-linked immunosorbent assay (ELISA). ELISA was performed using buffer at pH 6.0 (tumor microenvironment pH) or pH 7.4 (normal physiological pH). Serially diluted BA3182 was added to wells coated with the extracellular domains of recombinant CD3 ε and δ heterodimers. The bound BA3182 was quantified using a conjugate of the human or cynomolgus monkey EpCAM extracellular domain with mouse Fc and a conjugate of anti-mouse IgG antibody with horseradish peroxidase (HRP), the conjugate being reacted with a 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric substrate to produce a stained product. The absorbance (OD) at 450 nm in each well was recorded. 450The binding activity of BA3182 to human CD3 and human or cynomolgus monkey EpCAM was proportional to the amount of BA3182 bound to the human CD3 complex and human or cynomolgus monkey EpCAM, with a dynamic range of 0.0847 to 50000 pM. The EC50 values ​​of BA3182 binding to human CD3 and human or cynomolgus monkey EpCAM at different pH values ​​were calculated using a nonlinear fitting model (variable slope, four parameters) built into GraphPad Prism™ software. The EC50 values ​​of BA3182 binding activity to human CD3 and human EpCAM were 297.83 pM at pH 6.0 and 4155.67 pM at pH 7.4. The EC50 values ​​of BA3182 binding activity to human CD3 and cynomolgus monkey EpCAM were 543.47 pM at pH 6.0 and 29415.33 pM at pH 7.4. The results showed that the binding activity of BA3182 to CD3 and / or EpCAM at normal physiological pH (pH 7.4) was significantly weaker than that at the tumor microenvironment pH (pH 6.0).

[0280] Antigen: BA3182, 1.03 mg / mL Antibody: Recombinant human CD3 ε and δ heterodimers, BioVision, catalog number P11830-500, batch number 7C19P11830.

[0281] Recombinant human EpCAM fused with mouse Fc (human EpCAM-mFc), Evitria, lot number 12919-SEC.

[0282] Recombinant cynomolgus monkey EpCAM fused with mouse Fc (cynomolgus monkey EpCAM-mFc), BioAtla, lot number 20072.

[0283] Reagents: Goat anti-mouse HRP antibody, Promega Pharmaceuticals, catalog number W402B, batch number 0000465785.

[0284] Methods Carbonate-bicarbonate buffer capsules, Sigma, catalog number C3041-100CAP, lot number SLBZ3401.

[0285] 10X PBS, Gibco, catalog number 70011-044, lot number 2323767.

[0286] Bovine albumin (BSA), Sigma Aldrich, catalog number A9647-100G, lot number SLCH8436.

[0287] Sodium bicarbonate (7.5% solution), Gibbec Company, catalog number 25082-094, batch number 2336825.

[0288] Tween-20, Sigma-Aldrich, catalog number P9416-50ML, lot number SLCJ0231.

[0289] TMB Chromogen Solution, Life Technology, Catalog No. 002023, Batch No. 05123211-7.

[0290] 12N HCl: VWR, catalog number 87003-251, batch number 4118020.

[0291] ELISA analysis plate, Thermo Scientific Nunc, catalog number 269787, lot number 1223409.

[0292] Carbonate-bicarbonate coating buffer: Dissolve the contents of one carbonate-bicarbonate buffer capsule in 100 mL of sterile water.

[0293] PBS buffer: Dilute 10X PBS buffer to 1X with distilled water.

[0294] pH ELISA incubation buffer: Add sodium bicarbonate and BSA to PBS buffer to achieve a final sodium bicarbonate concentration of 2.5 g / L and a BSA concentration of 1%, and adjust the pH to 6.0 or 7.4 using 1N HCl.

[0295] pH Wash Buffer: Add sodium bicarbonate and Tween-20 to PBS buffer to achieve a final sodium bicarbonate concentration of 2.5 g / L and a Tween-20 concentration of 0.1%. Adjust the pH to 6.0 or 7.4 using 1N HCl.

[0296] Termination solution: 1N HCl ELISA Assay Perform 3-fold serial dilutions of BA3182 in culture buffer at pH 6.0 and pH 7.4. The starting concentration of BA3182 for the hCD3 / hEpCAM-mFc complex is 50 nM, and the starting concentration of BA3182 for the hCD3 / cynomolgus macaque EpCAM-mFc complex is 150 nM.

[0297] Results Spread ELISA plates at 100 μL / well using carbonate-bicarbonate spread buffer containing 1 µg / mL hCD3 antigen.

[0298] Cover the plate with a sealing film and incubate overnight at 4°C.

[0299] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0300] Wash each well twice by dispensing 200 µL of culture buffer to each well at pH 6.0 or pH 7.4 and completely aspirating the contents.

[0301] Add 200 µL of incubation buffer to each well at pH 6.0 or pH 7.4. Cover the plate with a sealing film and place it at room temperature on a plate shaker set to 200 rpm for 60 minutes.

[0302] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0303] Prepare 3-fold serial dilutions of BA3182 in culture buffer starting at 50 nM or 150 nM at pH 6.0 or pH 7.4.

[0304] Add 100 μL / well of diluted BA3182 to the plate in duplicate.

[0305] Cover the board with a sealing film and place it on a board oscillator (set to 200 rpm) at room temperature for 60 minutes.

[0306] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0307] Each well was washed three times by dispensing 200 µL of washing buffer to each well at pH 6.0 or pH 7.4 and completely aspirating the contents.

[0308] Dilute human EpCAM-mFc to 1 µg / mL in culture buffer at pH 6.0 or pH 7.4.

[0309] Dilute EpCAM-mFc of cynomolgus monkeys to 2 µg / mL in culture buffer at pH 6.0 or pH 7.4.

[0310] Add the above diluted 1 µg / mL human EpCAM-mFc or 2 µg / mL cynomolgus macaque EpCAM-mFc to each well at a concentration of 100 µL / well.

[0311] Cover the board with a sealing film and place it at room temperature on a board oscillator set to 200 rpm for 60 minutes.

[0312] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0313] Each well was washed three times by dispensing 200 µL of washing buffer to each well at pH 6.0 or pH 7.4 and completely aspirating the contents.

[0314] Goat anti-mouse HRP secondary antibody was diluted 1:2500 in culture buffer at pH 6.0 or pH 7.4.

[0315] Add more than 100 µL of diluted goat anti-mouse HRP secondary antibody to each well.

[0316] Cover the board with a sealing film and place it on a board oscillator (set to 200 rpm) at room temperature for 60 minutes.

[0317] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0318] Each well was washed three times by dispensing 200 µL of washing buffer to each well at pH 6.0 or pH 7.4 and completely aspirating the contents.

[0319] Dispense the TMB substrate solution into all wells of the plate at a rate of 50 μL / well. Incubate wells containing human EpCAM-mFc for 5 minutes at room temperature. Incubate wells containing cynomolgus macaque EpCAM-mFc for 10 minutes.

[0320] Add 1N HCl to all wells of the plate at a rate of 50 μL / well. Read the plate at 450 nm using a Molecular Device SpectraMaxi3X microplate reader.

[0321] The mean EC50 values ​​of the binding activity of BA3182 to human CD3 / human EpCAM-mFc or cynomolgus monkey EpCAM-mFc were calculated using a nonlinear fitting model (variable slope, four parameters) built into GraphPad Prism software, version 9.2.0.

[0322] Figure 6A A total of three independent ELISA assays were performed. An overview of the EC50 values ​​of BA3182 against recombinant human CD3 / human EpCAM-mFc and human CD3 / cynomolgus macaque EpCAM-mFc at pH 6.0 and pH 7.4 is shown in Tables 3 and 4. Binding curves from representative assays are shown in… Materials and 6BThe EC50 values ​​of BA3182 at pH 6.0, measured by ELISA, were 297.8 pM (human CD3 / human EpCAM-mFc) and 543.37 pM (human CD3 / cynomolgus monkey EpCAM-mFc). At pH 7.4, the EC50 of BA3182 for human CD3 / human EpCAM-mFc was 4155.67 pM and for human CD3 / cynomolgus monkey EpCAM-mFc was 29415.33 pM. Based on these results, it was observed that BA3182 binds to CD3 and EpCAM antigens with higher affinity at the tumor microenvironment pH (pH 6.0) and much lower affinity at the physiological pH (pH 7.4).

[0323] Table 3: Binding affinity of BA3182 to human CD3 / human EpCAM-mFc at pH 6.0 and pH 7.4

[0324] Table 4: Binding affinity of BA3182 to human CD3 / cynomolgus macaque EpCAM-mFc at pH 6.0 and pH 7.4

[0325] Example 17. pH range ELISA This example demonstrates the binding activity of the BA3182 bispecific antibody to the extracellular domains of recombinant human CD3 and human EpCAM within a pH range including buffered solutions (pH 6.0 to pH 7.4), simulated tumor microenvironment pH (pH 6.0 to 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 bound to the extracellular domain of recombinant human CD3 ε / δ heterodimer immobilized in wells. The bound BA3182 was quantified using a conjugate of the human EpCAM extracellular domain with mouse Fc and a conjugate of anti-mouse IgG antibody with horseradish peroxidase (HRP), the conjugate being reacted with a 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric substrate to produce a stained product. The absorbance (OD) in each well at 450 nm was measured. 450 The amount of BA3182 bound to the human CD3 complex and human EpCAM was proportional to the amount in buffer solutions at different pH values. Data analysis showed that the pH inflection point for BA3182 (50% binding activity compared to pH 6.0) was pH 6.66, with 90% binding activity at pH 6.25 (tumor microenvironment pH). Significantly weaker BA3182 binding activity was detected at pH 7.4 (normal physiological pH).

[0326] Methods antigen: a) Recombinant human CD3 ε and δ heterodimers, BioVision, catalog number P1183-500, batch number 7C19P11830.

[0327] b) Recombinant human EpCAM (EpCAM-mFc) fused with mouse Fc, Evectel, lot number 12919-SEC.

[0328] Antigen coating buffer, distilled water, Millipore.

[0329] Goat anti-mouse HRP antibody, Promega Pharmaceuticals, catalog number W402B, batch number 0000465785.

[0330] Sodium bicarbonate, Sigma-Aldrich, catalog number S5761-500G, lot number BCCD6088.

[0331] PBS (1X): Cellgro, catalog number R21-040-CV, batch number 17321021.

[0332] Tween-20: Sigma Corporation, catalog number P1379-500ML, lot number SLBS7482.

[0333] Bovine albumin (BSA), VWR, catalog number 0332, batch number 20D0656194.

[0334] TMB Chromogen Solution, Life Sciences Company, Catalog No. 002023, Batch No. 08228211-7.

[0335] HCl, Titansci, catalog number G81788B, lot number P1972715.

[0336] ELISA analysis plate, Corning Incorporated, catalog number 42592, lot number 00821030.

[0337] PBS buffer: 0.144 g / L KH₂PO₄, 9 g / L NaCl, 0.795 g / L Na₂HPO₄, pH 7.4 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 using 1N HCl.

[0338] pH ELISA wash buffer, pH 6.0, 6.2, 6.5, 6.7, 7.0, and 7.4: Add 0.15 g sodium bicarbonate and 0.6 g BSA to 60 mL of 1X PBS buffer containing 0.1% Tween-20. Adjust the pH to 6.0, 6.2, 6.5, 6.7, 7.0, and 7.4 using 1N HCl.

[0339] Termination solution: 1N HCl, add 83.3 mL to 1L of distilled water.

[0340] Data Analysis. EA1 was first diluted to 100 nM in various pH incubation buffers, and then diluted to 1.5 nM.

[0341] Spread 100 µL of distilled water containing 1 µg / mL of recombinant CD3 ε and δ complex antigen onto an ELISA plate.

[0342] Cover the plate with a sealing film and incubate overnight at 4°C.

[0343] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0344] Each well was washed twice by dispensing 200 µL of culture buffer at various pH values ​​into each well and completely aspirating the contents.

[0345] Add 200 µL of incubation buffer at various pH values ​​to each well, cover the plate with a sealing film, and place it on a plate shaker (set to 200 rpm) at room temperature for 60 minutes.

[0346] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0347] The test substance was serially diluted to 1.5 nM in incubation buffers at various pH values.

[0348] Add BA3182 to the board at a rate of 100 μL / well.

[0349] Cover the board with a sealing film and place it on a board oscillator (set to 200 rpm) at room temperature for 60 minutes.

[0350] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0351] Each well was washed three times by dispensing 200 µL of washing buffer at various pH values ​​into each well and completely aspirating the contents.

[0352] Human EpCAM-mFc was diluted to 1 µg / mL in culture buffers at various pH values.

[0353] Human EpCAM-mFc diluted to 1 µg / mL in culture buffers of various pH values ​​was added to each well at a concentration of 100 µL / well.

[0354] Cover the board with a sealing film and place it on a board oscillator (set to 200 rpm) at room temperature for 60 minutes.

[0355] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0356] Each well was washed three times by dispensing 200 µL of washing buffer at various pH values ​​into each well and completely aspirating the contents.

[0357] The anti-mouse IgG HRP secondary antibody was diluted 1:2500 in various pH culture buffers.

[0358] Add 100 µL of anti-mouse IgG HRP secondary antibody diluted in incubation buffers of various pH values ​​to each well.

[0359] Cover the board with a sealing film and place it on a board oscillator (set to 200 rpm) at room temperature for 60 minutes.

[0360] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0361] Each well was washed three times by dispensing 200 µL of washing buffer at various pH values ​​into each well and completely aspirating the contents.

[0362] Dispense the TMB substrate solution into all wells of the plate at a rate of 5 μL / well. Incubate at room temperature for 5 minutes.

[0363] Add 1N HCl to all wells of the plate at a rate of 50 μL / well. Read the plate at 450 nm using a Molecular Device SpectraMax190 microplate reader.

[0364] Results. The average OD values ​​(from two replicates) for different pH points were plotted using GraphPad Prism software. EC50 and EC90 were calculated using a variable slope, 4-parameter curve fitting. The inflection point of the pH curve (where 50% binding activity was observed) equals the EC50 of the fitted equation. The binding activity at pH 6.0 was set to 100%. Using a nonlinear regression from findingECanything and a parameter set specific to EC90, interpolation was performed on the fitted curve to obtain the pH value at which 90% binding activity was achieved.

[0365] Figure 7Table 5 summarizes the binding activity of BA3182 to recombinant human CD3 and human EpCAM in various pH buffers. Binding curves from representative experiments are shown in [Table 5]. Figure 7 The pH inflection point for BA3182 binding activity was pH 6.66. This pH inflection point is the pH at which 50% pH-dependent binding of BA3182 was observed. 90% of the BA3182 binding activity was achieved at pH 6.25 (the pH of the tumor microenvironment). Furthermore, weaker BA3182 binding activity was detected at normal physiological pH (pH 7.4). Materials ).

[0366] Table 5: pH inflection points (EC50) and EC90 values ​​of pH-dependent binding activity of BA3182 to human CD3 / human EpCAM-mFc

[0367]

[0368] Example 18. Cross-species affinity ELISA This example demonstrates the binding activity of the BA3182 bispecific antibody against recombinant human CD3 and the extracellular domains of EpCAM in humans, cynomolgus monkeys, rats, and mice. The binding activity of BA3182 to human CD3 and EpCAM antigens from different species was assessed using a sandwich enzyme-linked immunosorbent assay (ELISA). ELISA was performed using analytical buffers at pH 6.0 (tumor microenvironment pH) and pH 7.4 (normal physiological pH). BA3182 was added at a concentration of 1.5 nM to wells containing immobilized recombinant human CD3 ε / δ heterodimer extracellular domains. The amount of BA3182 bound was quantified using a conjugate of the extracellular domains of EpCAM in humans, cynomolgus monkeys, rats, or mice with mouse Fc (EpCAM-mFc) and a conjugate of anti-mouse IgG antibody with horseradish peroxidase (HRP), which was then reacted with a TMB colorimetric substrate to produce a colored product. The absorbance (OD) at 450 nm in each well was recorded. 450 The binding activity of BA3182 was proportional to the amount bound to the human CD3 complex and the EpCAM antigen. BA3182 exhibited strong binding activity to the human CD3 complex and human or cynomolgus monkey EpCAM at pH 6.0, but weaker binding activity at pH 7.4. For rat and mouse EpCAM, very low or no binding activity of BA3182 was observed at both pH values.

[0369] Methods Antibody: BA3182 antigen: a) Recombinant human CD3 ε and δ heterodimers, BioVision, catalog number P1183-500, batch number 7C19P11830.

[0370] b) Recombinant human EpCAM fused with mouse Fc (human EpCAM-mFc), Evectel, lot number 12919-SEC.

[0371] c) Recombinant cynomolgus monkey EpCAM fused with mouse Fc (cynomolgus monkey EpCAM-mFc), Bioyara Corporation, lot number 22007.

[0372] d) Recombinant rat EpCAM fused with mouse Fc (rat EpCAM-mFc), Bioyara Corporation, lot number 22009.

[0373] e) Recombinant mouse EpCAM fused with mouse Fc (mouse EpCAM-mFc), Bioyara Corporation, lot number 22011.

[0374] Antigen coating buffer, distilled water, Millipore.

[0375] Goat anti-mouse HRP antibody, Promega Pharmaceuticals, catalog number W402B, batch number 0000465785.

[0376] PBS (1x), Cellgro, catalog number R21-040-CV, lot number 17321021.

[0377] Bovine albumin (BSA), VWR, catalog number 0332, batch number 20D0656194.

[0378] Sodium bicarbonate, Sigma-Aldrich, catalog number S5761-500G, lot number BCCD6088.

[0379] Tween-20, Sigma Corporation, catalog number P1379-500ML, lot number SLBS7482.

[0380] TMB Chromogen Solution, Life Sciences Company, Catalog No. 002023, Batch No. 08228211-7.

[0381] HCl, Titan Technologies, catalog number G81788B, batch number P1972715.

[0382] ELISA analysis plate, Corning Incorporated, catalog number 42592, batch number 01919010.

[0383] pH meter, Alalis pH400.

[0384] Oscillator, Kylin-Bell TS-2.

[0385] Board reader, Thermo Fisher Multiskan™ Sky 51119770DP, SN: 1530-800210C.

[0386] PBS buffer: 0.144 g / L KH2PO4, NaCl 9 g / L, 0.795 g / L Na2HPO4, pH 7.4.

[0387] pH ELISA incubation buffer: Add 0.5 g sodium bicarbonate and 2 g BSA to 200 mL 1X PBS buffer. Adjust the pH to 6.0 or 7.4 using 1N HCl.

[0388] pH ELISA Wash Buffer: Add 0.5 g sodium bicarbonate and 0.1% Tween-20 to 200 mL 1X PBS buffer. Adjust the pH to 6.0 or 7.4 using 1N HCl.

[0389] Termination solution: 1N HCl, add 83.3 ml to 1L of distilled water.

[0390] Results. BA3182 was first diluted to 100 nM in PBS, and then diluted to 1.5 nM in incubation buffers at pH 6.0 and pH 7.4.

[0391] Spread 100 µL of distilled water containing 1 µg / mL of recombinant CD3 ε and δ complex antigen onto an ELISA plate.

[0392] Cover the plate with a sealing film and incubate overnight at 4°C.

[0393] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0394] Wash each well twice by dispensing 200 µL of pH 6.0 or pH 7.4 culture buffer into each well and completely aspirating the contents.

[0395] Add 200 µL of pH 6.0 or pH 7.4 incubation buffer to each well. Cover the plate with a sealing film and place it on a plate shaker (set to 200 rpm) at room temperature for 60 minutes.

[0396] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0397] The test substance was serially diluted to 1.5 nM in culture buffer at pH 6.0 or pH 7.4.

[0398] Add the diluted test substance to the plate at a rate of 100 μL / well.

[0399] Cover the board with a sealing film and place it on a board oscillator (set to 200 rpm) at room temperature for 60 minutes.

[0400] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0401] Each well was washed three times by dispensing 200 µL of pH 6.0 or pH 7.4 washing buffer into each well and completely aspirating the contents.

[0402] Dilute human, cynomolgus monkey, rat, or mouse EpCAM-mFc to 1 µg / mL in culture buffer at pH 6.0 or pH 7.4.

[0403] Add human, cynomolgus monkey, rat, or mouse EpCAM-mFc diluted to 1 µg / mL in culture buffer at pH 6.0 or pH 7.4 to each well at a concentration of 100 µL / well.

[0404] Cover the board with a sealing film and place it on a board oscillator (set to 200 rpm) at room temperature for 60 minutes.

[0405] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0406] Each well was washed three times by dispensing 200 µL of pH 6.0 or pH 7.4 washing buffer into each well and completely aspirating the contents.

[0407] The anti-mouse IgG HRP secondary antibody was diluted 1:2500 in culture buffer at pH 6.0 or pH 7.4.

[0408] Add 10 µL of anti-mouse IgG HRP secondary antibody diluted in incubation buffers of various pH values ​​to each well.

[0409] Cover the board with a sealing film and place it on a board oscillator (set to 200 rpm) at room temperature for 60 minutes.

[0410] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0411] Each well was washed three times by dispensing 200 µL of pH 6.0 or pH 7.4 washing buffer into each well and completely aspirating the contents.

[0412] Dispense the TMB substrate solution into all wells of the plate at a rate of 50 μL / well. Incubate at room temperature for 5 minutes.

[0413] Add 1N HCl to all wells of the plate at a rate of 50 μL / well. Read the plate at 450 nm using a microplate reader.

[0414] Figure 8 Three independent ELISA assays were performed in total. Representative assays demonstrating the binding activity of BA3182 to recombinant human CD3 and EpCAM in humans, cynomolgus monkeys, rats, or mice at pH 6.0 and pH 7.4 are presented in [the assay description]. Materials BA3182 exhibited strong binding activity to human CD3 and human or cynomolgus monkey EpCAM at the tumor environment pH (pH 6.0), but the binding activity was significantly weaker at pH 7.4. Weak binding or no binding of BA3182 to rat and mouse EpCAM was observed at both pH values.

[0415] Example 19. Specific ELISA This example demonstrates the binding activity of the BA3182 bispecific antibody against recombinant human CD3, human EpCAM, human trophoblast cell surface antigen 2 (Trop2), and unrelated human antigens. Trop2 and EpCAM belong to the tumor-associated calcium signaling gene family. They share high sequence similarity in their extracellular domains and transmembrane regions. The undisclosed unrelated human antigens do not share sequence similarity with the human EpCAM molecule. The binding activity of BA3182 against these antigens was assessed using affinity and sandwich enzyme-linked immunosorbent assay (ELISA). Analyses were performed using buffer at pH 6.0 (tumor microenvironment pH) or pH 7.4 (normal physiological pH). BA3182 was captured with recombinant human EpCAM or Trop2 (both fused with his tag) immobilized in wells, followed by detection of conjugates of anti-human IgG antibodies with horseradish peroxidase (HRP) using affinity ELISA. In a sandwich ELISA assay, BA3182 was captured using the extracellular domain of a recombinant human CD3 ε / δ heterodimer immobilized in wells, followed by the addition of human EpCAM, human Trop2, or an unrelated antigen (all fused to mouse Fc). The bound complex was detected using a conjugate of anti-mouse IgG antibody and HRP, which reacted with a 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric substrate to produce a stained product. The absorbance (OD) in each well at 450 nm was measured. 450The binding affinity of BA3182 to recombinant human EpCAM, Trop2, or unrelated antigen proteins was proportional to the amount of EpCAM, Trop2, or unrelated antigen proteins. For human Trop2, very low or no binding was observed in pH 6.0 or pH 7.4 buffers. Anti-human Trop2 specific antibodies (affinity ELISA assay) or bispecific antibodies (sandwich ELISA assay) were included as positive controls to confirm binding affinity to the human Trop2 antigen independent of pH buffer. Similarly, no binding of BA3182 to unrelated antigens was observed. Therefore, the binding activity of the BA3182 bispecific antibody to recombinant human EpCAM is confirmed to be target-specific.

[0416] Methods Antibody: BA3182 antigen: a) Recombinant human CD3 ε and δ heterodimers, BioVision, catalog number P1183-500, batch number 7C19P11830.

[0417] b) Recombinant human EpCAM fused with mouse Fc, Evectel, lot number 12919-SEC.

[0418] c) Recombinant human Trop2 fused with mouse Fc, Bioyara Corporation, lot number 21042.

[0419] d) Recombinant unrelated human antigen fused with mouse Fc, Bioyatra, lot number 21044.

[0420] e) Recombinant human EpCAM conjugated with his tag, Bioyara Corporation, lot number 21058.

[0421] f) Recombinant human Trop2 conjugated with his tag, Bioyara Corporation, lot number 19044.

[0422] Positive control antibody for Trop2, Bioatra Biotechnology, batch number PB01.

[0423] Positive control bispecific antibody for Trop2, BioAttra, lot number 860650.

[0424] Positive control bispecific antibody against unrelated human antigens, BioAttra, lot number 21-10458.

[0425] Antigen coating buffer, distilled water, Millipore.

[0426] Carbonate-bicarbonate buffer capsules, Sigma-Aldrich, catalog number C3041-100CAP, batch number SLBZ3401.

[0427] Goat anti-human IgG HRP antibody, Promega Pharmaceuticals, catalog number W403B, lot number 0000423844 Goat anti-mouse IgG HRP antibody, Promega Pharmaceuticals, catalog number W402B, batch number 0000465785.

[0428] PBS (1x): Cellgro, catalog number R21-040-CV, lot number 17321021.

[0429] Bovine albumin (BSA), VWR, catalog number 0332, batch number 20D0656194.

[0430] Sodium bicarbonate, Sigma-Aldrich, catalog number S5761-500G, lot number BCCD6088.

[0431] Tween-20, Sigma Corporation, catalog number P1379-500ML, lot number SLBS7482.

[0432] TMB Chromogen Solution, Life Sciences Company, Catalog No. 002023, Batch No. 08228211-7.

[0433] HCl, Titan Technologies, catalog number G81788B, batch number P1972715.

[0434] ELISA analysis plate, Corning Incorporated, catalog number 42592, batch number 01919010.

[0435] pH meter, Alalis pH400.

[0436] Oscillator, Kylin-Bell TS-2.

[0437] Board reader, Thermo Fisher Multiskan™ Sky 51119770DP, SN: 1530-800210C.

[0438] PBS buffer: 0.144 g / L KH2PO4, NaCl 9 g / L, 0.795 g / L Na2HPO4, pH 7.4.

[0439] Carbonate-bicarbonate coating buffer: Dissolve the contents of one carbonate-bicarbonate buffer capsule in 100 mL of sterile water.

[0440] pH ELISA incubation buffer: Add 0.5 g sodium bicarbonate and 2 g BSA to 200 mL 1X PBS buffer. Adjust the pH to 6.0 or 7.4 using 1N HCl.

[0441] pH ELISA Wash Buffer: Add 0.5 g sodium bicarbonate and 0.1% Tween-20 to 200 mL 1X PBS buffer. Adjust the pH to 6.0 or 7.4 using 1N HCl.

[0442] Termination solution: 1N HCl, add 83.3 mL to 1L of distilled water.

[0443] Results. BA3182 was first diluted to 100 nM in PBS, and then diluted to 2.5 nM in pH 6.0 or pH 7.4 incubation buffer for use in related family ELISA and 1.5 nM for use in unrelated antigen ELISA.

[0444] Affinity ELISA plates were coated with 100 µL of carbonate-bicarbonate coating buffer containing 1 μg / mL of recombinant human EpCAM or Trop2 antigen.

[0445] Spread 100 µL of distilled water containing 1 µg / mL human recombinant CD3 ε and δ complex antigen onto a sandwich ELISA plate.

[0446] Cover the plate with a sealing film and incubate overnight at 4°C.

[0447] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0448] Wash each well twice by dispensing 200 µL of pH 6.0 or pH 7.4 culture buffer into each well and completely aspirating the contents.

[0449] Add 200 µL of pH 6.0 or pH 7.4 incubation buffer to each well. Cover the plate with a sealing film and place it on a plate shaker (set to 200 rpm) at room temperature for 60 minutes.

[0450] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0451] The test substance was serially diluted to 2.5 nM, 1.5 nM, or 0.5 nM in culture buffer at pH 6.0 or pH 7.4.

[0452] Add diluted BA3182 to the plate at a rate of 100 μL / well.

[0453] Cover the board with a sealing film and place it on a board oscillator (set to 200 rpm) at room temperature for 60 minutes.

[0454] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0455] Each well was washed three times by dispensing 200 µL of pH 6.0 or pH 7.4 washing buffer into each well and completely aspirating the contents.

[0456] For affinity ELISA, dilute the anti-human IgG HRP secondary antibody 1:2500 in incubation buffer at pH 6.0 or pH 7.4. Add 100 µL of the anti-human IgG HRP secondary antibody diluted in incubation buffer at various pH values ​​to each well.

[0457] For sandwich ELISA, dilute human EpCAM-mFc, human Trop2-mFc, or unrelated human antigen-mFc to 1 μg / mL or 2 μg / mL in culture buffer at pH 6.0 or pH 7.4. Add 100 μL / well of the above-diluted human EpCAM-mFc, human Trop2-mFc, or unrelated human antigen-mFc to each well.

[0458] Cover the board with a sealing film and place it on a board oscillator (set to 200 rpm) at room temperature for 60 minutes.

[0459] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0460] Each well was washed three times by dispensing 200 µL of pH 6.0 or pH 7.4 washing buffer into each well and completely aspirating the contents.

[0461] For sandwich ELISA, the anti-mouse IgG HRP secondary antibody was diluted 1:2500 in culture buffer at pH 6.0 or pH 7.4.

[0462] Add 100 µL of anti-mouse IgG HRP secondary antibody diluted in incubation buffers of various pH values ​​to each well.

[0463] Cover the board with a sealing film and place it on a board oscillator (set to 200 rpm) at room temperature for 60 minutes.

[0464] Pour out the contents of the plate and tap out any remaining liquid onto a stack of paper towels.

[0465] Each well was washed three times by dispensing 200 µL of pH 6.0 or pH 7.4 washing buffer into each well and completely aspirating the contents.

[0466] Dispense the TMB substrate solution into all wells of the plate at a rate of 50 μL / well. Incubate at room temperature for 5 minutes.

[0467] Add 1N HCl to all wells of the plate at a rate of 50 μL / well. Read the plate at 450 nm using a microplate reader.

[0468] Figure 9A Three independent ELISA assays were performed using duplicate samples. Representative assays 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 presented in [the table / document / etc.]. Figure 10A and 9B The binding activity of BA3182 to recombinant human CD3 and human EpCAM or unrelated human antigens at pH 6.0 and pH 7.4 was demonstrated. Figure 11A and 10B The binding activity of BA3182 to recombinant human EpCAM or human Trop2 ECD at pH 6.0 and pH 7.4 was demonstrated. Figure 9A and 11B middle.

[0469] BA3182 exhibits high binding activity for human EpCAM at pH 6.0. Figure 10A , Figure 11A and Figure 9B Furthermore, its binding was lower at pH 7.4. BA3182 did not show binding with human Trop2 at either pH 6.0 or pH 7.4. Figure 11B and Figure 10B ) and unrelated human antigens ( Materials The data indicate that BA3182 has high specificity for human EpCAM and CD3.

[0470] Example 20. FACS analysis of the binding of BA3182 to cells expressing EpCAM or CD3 This example demonstrates the binding activity of the BA3182 bispecific antibody to cells expressing EpCAM or CD3. The binding activity of BA3182 to EpCAM-expressing cells was assessed using CHO cells expressing human EpCAM, CHO cells expressing cynomolgus monkey EpCAM, and HCT116 cells. The binding activity of BA3182 to CD3-expressing cells was assessed using Jurkat T cells and peripheral blood mononuclear cells (PBMCs) (human, cynomolgus monkey, rat, and canine). Cells were cultured with various concentrations of BA3182 at pH 6.0 (tumor microenvironment pH) and pH 7.4 (normal physiological pH). The amount of BA3182 bound to cells was quantified using an anti-human IgG antibody conjugated to Alexa Fluor488 (AF488). The stained cells were analyzed by fluorescence-activated cell sorting (FACS) to determine the median fluorescence intensity (MFI), which is proportional to the amount of BA3182 bound to the cells. MFI values ​​and corresponding BA3182 concentrations were analyzed using GraphPad Prism software at various pH values. EC50 values ​​of the binding activity of BA3182 to EpCAM and CD3 molecules expressed on cell surfaces at pH 6.0 and pH 7.4 were determined using variable-slope, nonlinear four-parameter curves. The mean EC50 values ​​of BA3182 binding to cells expressing EpCAM and CD3 are listed in Tables 6 and 7. Overall, at pH 6.0, BA3182 binds with high affinity to human and cynomolgus monkey EpCAM and CD3 antigens expressed on cell surfaces. Reduced affinity of BA3182 for these molecules, particularly CD3, was observed at pH 7.4. No binding of BA3182 to rat, mouse, or canine PBMCs was observed. Low homology (less than 60%) was observed between human and rat, mouse, or canine CD3 molecules. This result indicates that the CD3 epitope of BA3182 is absent in rat, mouse, and canine CD3 antigens.

[0471] Methods BA3182 1.03 mg / mL.

[0472] The isotype control was produced by Evectel (Zurich, Switzerland), lot number 10229, 3 mg / mL.

[0473] CHO-hEpCAM: CHO-S cells, Thermo Fisher Scientific, catalog number R80007, transfected to stably express human EpCAM (synthesized and constructed by Bioyara), clone #32.

[0474] CHO-cynomolgus macaque EpCAM: CHO-S cells, Thermo Fisher Scientific, catalog number R80007, transfected to stably express cynomolgus macaque EPCAM (synthesized and constructed by Bioatra), clone #3.

[0475] HCT116, human colon cancer, ATCC, catalog number CCL-247™.

[0476] Jurkat, human T lymphocytes, ATCC, catalog number TIB-152, clone E6-1.

[0477] Human PBMC: Precision for Medicine, catalog number 39000, batch number 201013292.

[0478] Crab-eating macaque PBMC: Worldwide Primates, catalog number CA-10, batch number C0767-22.

[0479] SD rat PBMCs: IQ Biosciences, catalog number IQB-RPB101, batch number P20K0105.

[0480] Balb / c mouse PBMCs: IQ Biosciences, catalog number IQB-MPB101, batch number P21A0603.

[0481] Beagle Canine PBMC: IQ Biosciences, catalog number IQB-CPB102, batch number P21C2303.

[0482] PE-conjugated mouse anti-hEpCAM: BioLegend, catalog number 324206, clone 9C4, batch number B222943.

[0483] Mouse IgG2b isotype conjugated with PE: BioLand, catalog number 400314, clone MPC-11, batch number B214529.

[0484] Goat anti-human IgG AF488 antibody: Thermo Fisher Scientific, catalog number A11013, batch number 2110842.

[0485] PBS: 10X, Thermo Fisher Scientific, catalog number 14190-144, lot number 2152877.

[0486] Cell culture grade water: Cytiva, catalog number SH30529.03, batch number AH30009593.

[0487] Trypan blue dye: Thermo Fisher Scientific, catalog number 15250-061, lot number 1861515.

[0488] BSA: Sigma Corporation, catalog number A9647, batch number SLBV4996.

[0489] TrypLE™ expression enzyme (1X): Thermo Fisher Scientific, catalog number 12605-10, lot number 2193025 FBS: Thermo Fisher Scientific, catalog number 16140-011, batch number 2372673RP.

[0490] McCoy's 5A: Thermo Fisher Scientific, catalog number 16600-082, lot number 232363.

[0491] DMEM: Thermo Fisher Scientific, catalog number 11965-084, batch number 2346179.

[0492] RPMI-1640: Thermo Fisher Scientific, catalog number 11875-085, lot number 2463433.

[0493] 100x NEAA: Thermo Fisher Scientific, catalog number 11140-050, lot #2337217.

[0494] 100 mM Sodium Pyruvate: Thermo Fisher Scientific, Catalog No. 11360-070, Lot No. 2323639.

[0495] G418: Invivogen, catalog number ant-gn-5, batch number GNL-38-06A.

[0496] Sodium bicarbonate: Thermo Fisher Scientific, catalog number 25080-094, batch number 2039755.

[0497] 12N HCl: VWR, catalog number 87003-251, batch number 4118020.

[0498] T-150 tissue culture flask: Corning Incorporated, catalog number 430825.

[0499] 50 mL conical tube: BioPioneer, catalog number CNT-50, batch number 10272021.

[0500] 96-well U-shaped base plate: Greiner bio-one, catalog number 650101, batch number B17033C7.

[0501] pH meter: ORION STAR A111, Thermo Fisher Scientific, serial number J17162 Oscillator: MaxQ 2000.

[0502] Flow cytometer: ACEA NovoCyte 20602.

[0503] PBS buffer: Add 100 mL of 10X PBS buffer to 900 mL of sterile water.

[0504] 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.

[0505] 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.

[0506] 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.

[0507] 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 6N or 0.6N HCl.

[0508] CHO-hEpCAM / CHO-cynoEpCAM medium: DMEM + 1×NEAA (0.1 mM non-essential amino acid solution) + 1×sodium pyruvate (1 mM) + 10% FBS + 1 mg / mL G418.

[0509] HCT116 medium: McCoy's 5A + 10% FBS.

[0510] Jurkat medium: RPMI-1640 + 10% FBS.

[0511] Cell Culture. Cell StainingCHO-hEpCAM, CHO-cynoEpCAM, HCT116, and Jurkat cells were maintained in the specified medium and routinely passaged twice weekly. For FACS analysis, cells were collected during the exponential growth phase. Frozen human, cynomolgus macaque, rat, mouse, and canine PBMCs were freshly thawed in RPMI-1640 / 10% FBS medium and analyzed by FACS.

[0512] Binding analysis of BA3182 to EpCAM expressed on CHO-hEpCAM, CHO-cynoEpCAM, and HCT116 cells On the same day as the FACS analysis, the culture medium is removed and discarded.

[0513] Simply rinse the cell layer with PBS solution.

[0514] Add 3 mL of TrypLE™ expression enzyme solution to each T-150 flask. Wait until the cell layers disperse.

[0515] Add 7 mL of culture medium to the bottle and resuspend the cells by gentle pipetting.

[0516] Collect the cells and transfer the cell suspension to a 50 mL conical tube.

[0517] Cells stained with trypan blue were counted and then centrifuged at 2000 rpm for 3 minutes at 4°C.

[0518] Wash the cells once with PBS and then add 3×10 5 The cells were transferred into the Eppendorf tube.

[0519] Add 2 µL of mouse anti-hEpCAM (PE-conjugated mouse IgG) or PE-isotype mouse IgG to 100 µL of PBS solution containing 1% BSA. Add 100 μL of diluted PE antibody to each tube and incubate on ice with shaking at 100 RPM for one hour in the dark.

[0520] Wash the cells three times with 150 µL PBS solution.

[0521] Cells were fixed with 4% PFA for 10 minutes at room temperature, followed by washing with PBS once.

[0522] Cells were resuspended in 100 µL PBS and analyzed using a NovoCyte flow cytometer.

[0523] The number of EpCAM molecules on the cell surface was estimated using BD Quantibrite™ PE bead standards.

[0524] Binding analysis of BA3182 to CD3 expressed on Jurkat T cells, human, cynomolgus monkey, rat, mouse, and canine PBMC. Collect cells as described in the cell staining section above and wash them once with PBS.

[0525] Adjust the cell concentration to 3 × 10⁻⁶ cells in a wash buffer at pH 6.0 or pH 7.4. 6 Cells / mL

[0526] Will contain 3×10 5 100 µL of pH 6.0 or pH 7.4 washing buffer per cell was aliquoted into 96-well U-shaped plates.

[0527] Briefly centrifuge the cells and discard the buffer solution.

[0528] Prepare 3-fold serial dilutions of the antibody starting at 1500 nM (for CHO-cynoEpCAM) or 500 nM (for CHO-hEpCAM and HCT116) in FACS buffer at pH 6.0 or pH 7.4.

[0529] Add the diluted test substance to the cells at 100 μL / well, mix gently and incubate on ice with shaking (100 rpm) for one hour.

[0530] 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 washing buffer.

[0531] Goat anti-human IgG AF488 antibody was diluted 1:300 in FACS buffer at pH 6.0 or pH 7.4.

[0532] Add 100 µL of the diluted antibody from the above steps to the cells and incubate on ice in the dark for 45 minutes.

[0533] Aggregate the cells and wash three times with 150 µL of pH 6.0 or pH 7.4 washing buffer.

[0534] Cells were fixed at room temperature for 10 minutes with 4% paraformaldehyde (PFA) diluted in 1X PBS, followed by washing with 1X PBS.

[0535] The cells were resuspended in 100 µL of 1X PBS.

[0536] Cells were analyzed using a NovoCyte flow cytometer and software. 20,000 events were acquired for each sample.

[0537] Data Analysis. Results.

[0538] Prepare the cell suspension as described in the cell culture section above. Wash the cells once with PBS.

[0539] Adjust the cell concentration to 3 × 10⁻⁶ cells in a wash buffer at pH 6.0 or pH 7.4. 6 per ml.

[0540] Will contain 3×10 5 100 µL of pH 6.0 or pH 7.4 washing buffer per cell was aliquoted into 96-well U-shaped plates.

[0541] Briefly centrifuge the cells and discard the buffer solution.

[0542] Prepare 3-fold serial dilutions of the antibody starting at 2500 nM in FACS buffer at pH 6.0 or pH 7.4.

[0543] Add the diluted antibody to the cells at 100 μL / well, mix gently, and incubate on ice with shaking at 100 rpm for one hour.

[0544] 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 washing buffer.

[0545] Goat anti-human IgG AF488 antibody was diluted 1:300 in FACS buffer at pH 6.0 or pH 7.4.

[0546] Add 100 µL of diluted secondary antibody to the cells and incubate on ice in the dark for 45 minutes.

[0547] Aggregate the cells and wash three times with 150 µL of pH 6.0 or pH 7.4 washing buffer.

[0548] Cells were fixed at room temperature for 10 minutes with 4% PFA diluted in 1X PBS. Cells were washed with 1X PBS and centrifuged at 2000 rpm for 3 minutes at 4°C.

[0549] Cells were resuspended in 100 µL of 1X PBS. Cells were analyzed using a NovoCyte flow cytometer and software. 20,000 events were acquired for each sample.

[0550] Figures 12A-12C Using GraphPad Prism software, version 9.2.0, variable slope, 4-parameter nonlinear regression curves were generated using median fluorescence intensity (MFI) and antibody concentration.

[0551] Figures 13A-13CPerform at least three independent FACS experiments on each test cell line or PBMC. The gate selection strategy for identifying positive cells is shown in [the diagram]. Figures 14A-14C The binding activity of BA3182 to CHO-hEpCAM, CHO-cynoEpCAM, and HCT116 cells was demonstrated in [the study]. Figures 15A-15B The binding activity of BA3182 to Jurkat cells, human and cynomolgus monkey PBMCs was demonstrated in [the study / test]. Figures 13A-13C The expression level of EpCAM molecules on the cell surface is shown in [the figure]. Figures 14A-14C middle.

[0552] BA3182 binds well to cells expressing EpCAM at pH 6.0 and pH 7.4, producing favorable dose-response curves. Materials The mean EC50 of BA3182 binding to EpCAM at the tumor microenvironment pH (pH 6.0) was 3.4 nM, compared to 11.0 nM for human and cynomolgus monkey EpCAM expressed in CHO cells and 5.9 nM for hEpCAM expressed on HCT116 cells (Table 6). The mean EC50 of BA3182 binding to EpCAM at the normal physiological pH (pH 7.4) was 5.6 nM, compared to 28.6 nM for human and cynomolgus monkey EpCAM expressed in CHO cells and 8.3 nM for hEpCAM expressed on HCT116 cells (Table 6). BA3182 binds to the EpCAM antigen expressed on the cell surface with low affinity at pH 7.4. No binding with native CHO cells was observed (data not shown).

[0553] The dose-response curve of BA3182 binding to CD3-expressing cells is shown in Methods BA3182 binds to the CD3 antigen with a higher affinity at pH 6.0. BA3182 binds to CD3-expressing cells with a significantly lower affinity at pH 7.4. The mean EC50 values ​​for BA3182 binding to CD3 expressed on the cell surface were 808.6 nM, 460.3 nM, and 161.6 nM for human PBMCs, cynomolgus monkey 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, respectively (Table 7). No binding of BA3182 to rat, mouse, or canine PBMCs was observed (data not shown).

[0554] Table 6: Binding affinity of BA3182 to cells expressing EpCAM

[0555] Table 7: Binding of BA3182 to CD3-expressing cells

[0556] Example 21. Bioanalytical Method for Functional T Cell Activation This example demonstrates the functional activity of the BA3182 bispecific antibody using Promega's 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 conjugated with the BA3182 bispecific antibody in the presence of EpCAM-expressing cells, the TCR / CD3 effector cells activated the NFAT pathway, resulting in an NFAT-RE-mediated cryoluminescence signal, which was detected by adding Bio-Glo™ reagent and quantified using a photometer. To determine the BA3182 EC50 value, the relative cryoluminescence unit (RLU) value was analyzed using a nonlinear fitting model (variable slope, four parameters) built into GraphPadPrism software. The mean EC50 of BA3182 added to CHO cells expressing hEpCAM was 0.049 nM at pH 6.0 and 0.383 nM at pH 7.4. The mean EC50 of BA3182 added to CHO cells expressing cynoEpCAM was 0.576 nM at pH 6.0 and 9.647 nM at pH 7.4. The mean EC50 of BA3182 added to HCT116 cells was 0.273 nM at pH 6.0 and 1.988 nM at pH 7.4. These results indicate that BA3182 mediates TCR / CD3 binding of T cells extremely potently at pH 6.0 (tumor microenvironment), while its potency is significantly lower at physiological pH (pH 7.4).

[0557] Data Analysis. BA3182 RPMI-1640: Thermo Fisher Scientific / Gibco, Catalog No. 11875-085, Lot No. 2463433 DMEM: Thermo Fisher Scientific / Gibco, Catalog No. 11965-084, Lot No. 2346179 FBS: Thermo Fisher Scientific / Gibco, Catalog No. 16140-011, Lot No. 2372673RP 100x NEAA: Gibbs Corporation, Catalog No. 11140-050, Lot #2337217 100 mM Sodium Pyruvate: Gibberel, Catalog No. 11360-070, Lot No. 2323639 Bio-Glo™ Buffer: Prometheus, Catalog No. G719A, Lot No. 0000473817 Bio-Glo™ Analytical Substrate: Prometheus, Catalog No. G720A, Lot No. 0000481126 Cell culture grade water: Sitopan Company, catalog number SH30529.03, batch number AH30009593 12N HCl: VWR, Catalog No. 87003-251, Batch No. 4118020 50 mL conical tube: Bio-Pharmaceutical Co., Ltd., catalog number CNT-50, batch number 10272021 96-well analytical plate: Corning Incorporated, catalog number 3917, lot number 32021006 Syringe filter: Celltreat, catalog number 229747, lot number 211004-052-1A pH meter: ORION STAR A111, Thermo Fisher Scientific, serial number J17162 Sealing film for microplates: E&K Scientific, sealing plate, catalog number T396100, lot number SG262G Oscillator: MaxQ 2000 Sample dilution blocking solution: Corning Incorporated, catalog number 3956, lot number 07718000 Board reader: Molecular Devices, SpectraMax i3X pH 6.0 analysis buffer: DMEM + 1×NEAA (0.1 mM non-essential amino acid solution) + 1×sodium pyruvate (1 mM) + 10% FBS, adjusted to pH 6.0 with 6N HCl, and sterilized with a 0.2 μm syringe filter.

[0558] pH 7.4 analysis buffer: DMEM + 1×NEAA (0.1 mM non-essential amino acid solution) + 1×sodium pyruvate (1 mM) + 10% FBS, adjusted to pH 7.4 with 6N HCl, and sterilized with a 0.2 μm syringe filter.

[0559] CHO-hEpCAM / CHO-cynoEpCAM medium: DMEM + 1×NEAA (0.1 mM non-essential amino acid solution) + 1×sodium pyruvate (1 mM) + 10% FBS + 1 mg / mL G418 HCT116 medium: McCoy's 5A + 10% FBS CHO hEpCAM clone #9, Bioatra Corporation, 11th generation.

[0560] CHO cynoEpCAM clone #3, Bioatra Corporation, 13th generation.

[0561] HCT116, human colon cancer, ATCC, catalog number CCL-247™, 10th generation.

[0562] TCR / CD3 effector cells, Promega, T cell activation bioassay (NFAT), propagation model, catalog number J1601, expanded / cryopreserved, 7th generation.

[0563] Results. Prepare 2x working solutions of the test samples in analysis buffer at pH 6.0 or pH 7.4, at 500 nM for CHO hEpCAM and HCT116 cells and 2500 nM for cynoEpCAM cells. Then, perform 5-fold serial dilutions for a total of 11 data points.

[0564] Dispense 25 µL of serially diluted 2x test solution into each well according to the plate layout. The final starting concentration is 250 nM or 1250 nM, depending on the target cells.

[0565] The day before the assay, cells expressing EpCAM were seeded into 96-well analytical plates. 100 µL of EpCAM-containing cells were added to each plate. 5 Add culture medium at a concentration of 10 cells / mL to the plate and inoculate with 4 × 10⁶ cells / mL. 4 Cells / well. Incubate overnight at 37°C and 5% CO2. On the day of assay, prepare 5-fold serial dilutions of antibody at pH 6.0 and pH 7.4 for a total of 11 data points in assay buffer at 2x concentrations. 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 sealant and place the plate on ice.

[0566] Thaw two vials of frozen TCR / CD3 effector cells in a 37°C water bath for 3 minutes. Gently mix the cell suspension and transfer the contents to two 1.7 mL sterile Ependorf tubes. Briefly centrifuge at 6000 rpm for 30 seconds using a benchtop centrifuge to aggregate the cells. Aspirate the supernatant and resuspend the cells in one vial at pH 6.0 with 4 mL of analytical medium, and in the other vial at pH 7.4 with 4 mL of analytical medium.

[0567] Carefully remove the culture medium from the analysis plate without disturbing the cells.

[0568] Add 2x diluted antibody per well at 25 μL, depending on the layout.

[0569] Add TCR / CD3 effector cell suspension at 25 μL / well according to the layout.

[0570] The plate was covered and incubated at 37°C and 5% CO2 for 6 hours.

[0571] Preheat the Bio-Glo™ buffer and the substrate from Promega to room temperature.

[0572] When the 6-hour incubation period is over, add Bio-Glo™ buffer to the substrate and mix thoroughly until the substrate is completely dissolved.

[0573] Add Bio-Glo™ substrate to the analysis plate at 50 μL / well according to the layout, and incubate in the dark at 100 rpm on a shaker for 5 minutes at room temperature.

[0574] The cold light signal was recorded using a SpectraMax i3X reader from Molecular Instruments.

[0575] Figure 16A The functional activity of the analyte at pH 6.0 and pH 7.4 was determined by mapping bioluminescent units (RLU) against antibody concentration. EC50 values ​​were calculated using a nonlinear fitting (variable slope, four-parameter) model built into Graph Pad Prism software, version 9.2.0.

[0576] Materials Three independent experiments were performed on each cell type. The functional activity of BA3182 was determined using Promega's T-cell activation bioassay. The results showed that BA3182 exhibited higher activity at an acidic pH (pH 6.0) mimicking the tumor microenvironment. Conversely, BA3182 showed lower activity at a normal physiological pH (neutral pH 7.4). Methods , 16BThe mean EC50 of BA3182 against CHO hEpCAM cells was 0.049 nM at pH 6.0 and 0.383 nM at pH 7.4 (Table 8). For CHO cynoEpCAM cells, the mean EC50 was 0.576 nM and 9.647 nM at pH 6.0 and pH 7.4, respectively (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).

[0577] Table 8: Efficacy of BA3182 in mediating T cell activation in CHO-hEpCAM cells at pH 6.0 and pH 7.4

[0578] Table 9: Efficacy of BA3182 in mediating T cell activation in CHO-cynoEpCAM cells at pH 6.0 and pH 7.4.

[0579] Table 10: Efficacy of BA3182 in mediating T cell activation in HCT116 cells at pH 6.0 and pH 7.4

[0580] Example 22. Cytotoxicity assays using human and cynomolgus monkey PBMCs This example demonstrates the functional activity of the BA3182 bispecific antibody using an in vitro cytotoxicity assay, where human or cynomolgus macaque peripheral blood mononuclear cells (PBMCs) are activated upon in vitro stimulation with the BA3182 antibody to kill cells expressing EpCAM. For this purpose, HCT116 cells or CHO cells expressing cynoEpCAM were treated with serially diluted BA3182 antibody, followed by the addition of human or cynomolgus macaque PBMCs. Co-cultures were incubated in a medium at pH 6.5, simulating a tumor microenvironment, or at pH 7.4, simulating normal physiological pH. BA3182-mediated cell lysis was monitored in real-time using Agilent xCELLigence Real-Time Cell Analysis (RTCA). The cell lysis rate of the target cells was calculated by referencing the growth of untreated target cells at predetermined time points. The percentage of cell lysis obtained was plotted against the concentration of BA3182. A variable-slope, four-parameter nonlinear regression model was used to fit the data to determine EC50 and EC20 values. The mean EC50 of BA3182-mediated HCT116 cell lysis was 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 of BA3182-mediated HCT116 cell lysis was 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 of BA3182-mediated cynoEpCAM-expressing CHO cell lysis induced by cynomolgus macaque PBMCs was 17.39 pM at pH 6.5 and 184.73 pM at pH 7.4.

[0581] Cell Culture and Plating. The BA3182 isotype control bispecific antibody was manufactured by Evecter (Zurich, Switzerland), batch number 10229, 3 mg / mL.

[0582] RPMI-1640: Thermo Fisher Scientific / Gibco, Catalog No. 11875-085, Lot No. 2463433 DMEM: Thermo Fisher Scientific / Gibco, Catalog No. 11965-084, Lot No. 2346179 FBS: Sigma Corporation, Catalog No. 12306C-500mL, Lot No. 16J367 100x NEAA: Gibbs Corporation, Catalog No. 11140-050, Lot No. 2337217 100 mM Sodium Pyruvate: Gibberel, Catalog No. 11360-070, Lot No. 2323639 Cell culture grade water: Corning Incorporated, catalog number 25-055-CM, batch number 17216006 12N HCl: VWR, Catalog No. 87003-251, Batch No. 4118020 50 mL conical tube: Bio-Pharmaceutical Co., Ltd., catalog number CNT-50, batch number 10272021 E-Plate View 96: Agilent Technologies, Catalog No. 300601010, Lot No. 20211132 Syringe filter: Celltreat, catalog number 229747, lot number 211004-052-1A pH meter: ORION STAR A111, Thermo Fisher Scientific, serial number J17162 Trypanosome Blue Dye: Gibberel, Catalogue No. 15250-061, Lot No. 1861515 Adhesive plate sealing film for microplates: E&K Technologies, sealing plate, catalog number T396100, lot number SG262G Sample dilution blocking solution: Corning Incorporated, catalog number 3956, lot number 07718000 Agilent xCELLigence Real-Time Cell Analysis (RTCA) MP Analyzer Plate reader: SpectraMax i3X, Molecular Instruments PBMC pH 6.5 analysis medium: RPMI-1640 + 10% FBS, adjusted to pH 6.5 with 6N HCl, and sterilized using a 0.2 μm syringe filter.

[0583] PBMC pH 7.4 analysis medium: RPMI-1640 + 10% FBS, adjusted to pH 7.4 with 6N HCl, and sterilized using a 0.2 μm syringe filter.

[0584] CHO cynoEpCAM medium: DMEM + 1×NEAA (0.1 mM non-essential amino acid solution) + 1×sodium pyruvate (1 mM) + 10% FBS + 1 mg / mL G418 HCT116 medium: McCoy's 5A + 10% FBS CHO cynoEpCAM clone #3, Bioatra Corporation, 13th generation HCT116, human colon cancer, ATCC, catalog number CCL-247™, 11th generation Human PBMC: Precision Medicine Company, Catalog No. 93000-10M, Batch No. 13143; 2010113397; 2010113371; 2010113411; 2010113292; 2010113290; 201675377; 201877535; 201885639; 201897529 Crab-eating macaque PBMC: Wardbury Pharmaceuticals, Catalog No. CA-10, Lot No. 207117-1 PBMC Resting Test Antibody Preparation and Assay Setup

[0585] The day before the analysis, the target cells were isolated and resuspended in the culture medium as a single-cell solution.

[0586] The cells were stained with trypan blue and counted using a hemocytometer.

[0587] The target cell concentration was adjusted to 4 × 10⁻⁶. 4 Add 100 µL of the solution to a 96-well analytical plate and seed 4 × 10⁶ cells / mL in cell culture medium. 3 Cells / well

[0588] The target cells were cultured overnight at 37°C and 5% CO2.

[0589] Results. Thaw PBMCs in a water bath at 37°C for 3 minutes and add them dropwise to 10 mL of warm PBMC analysis medium (pH 7.4), continuously rotating the conical tube to help the cells adapt to the medium temperature.

[0590] Use a 10 mL pipette to move the liquid up and down until no visible clots remain.

[0591] Collect cells using a centrifuge at 1400 rpm for 10 minutes, and discard the culture medium supernatant.

[0592] The cell aggregates were resuspended in 10 mL of fresh, warm culture medium and pipetted to separate the cells.

[0593] Incubate overnight (between 18 and 20 hours) in a 37°C, 5% CO2 incubator.

[0594] Before coating, the cells are counted and their viability is determined.

[0595] Collect cells using a centrifuge at 1400 rpm for 10 minutes, and discard the culture medium supernatant.

[0596] The cells were resuspended together with a specified volume of culture medium at pH value to reach 4 × 10⁻⁶. 5 Density of cells per milliliter.

[0597] Figures 17A-17C For HCT116 / human PBMC cells, starting at 10 nM and for CHO cynomolgus monkey EpCAM / cynomolgus monkey PBMC cells, starting at 200 nM, the test substance was serially diluted to 2x starting concentration in analytical medium at pH 6.5 or pH 7.4 to prepare stock solutions. Five-fold dilutions were performed for a total of 11 data points. The serially diluted test substance was dispensed at 100 μL / well, and PBMC suspension was added at 100 μL / well. The final volume was 200 μL / well.

[0598] To ensure complete dissolution, the co-culture of PBMCs and target cells was treated with 1% SDS.

[0599] The cells were incubated at 37°C and 5% CO2 for 100 hours. Data were collected every 15 minutes, resulting in a total of 400 readings.

[0600] Materials The functional activity of BA3182 was determined using CD3-mediated killing of target cells by PBMCs. Human PBMCs from six healthy subjects were co-cultured with HCT116 and various concentrations of BA3182 for 120 hours. Cell growth was determined using XCELLigence real-time cell analysis technology. Cytotoxicity was determined using the following functions:

[0601] The results showed that BA3182 exhibited higher activity at tumor-environment pH (acidic pH, pH 6.5). Alternatively, BA3182 showed lower activity at normal physiological pH (neutral pH, pH 7.4). Methods The mean EC50 of BA3182 against HCT116 cells was 1.41 pM in the tumor microenvironment (pH 6.5) and 5.61 pM at the normal physiological pH (pH 7.4). The mean EC20 of BA3182 against HCT116 cells was 0.54 pM in the tumor microenvironment (pH 6.5) and 4.09 pM at pH 7.4 (Table 11).

[0602] Table 11: Efficacy of BA3182 in inducing cell lysis in cells expressing EpCAM A.

[0603] B.

[0604] Example 23. PBMC Cytokine Release Assay In this study, the release of IL-2, IL-6, IL-10, INFγ, and TNFα cytokines from human peripheral monocytes (PBMCs) stimulated with various concentrations of BA3182 in the presence of EpCAM-expressing cells was investigated. HCT116 cancer cells were seeded into tissue culture plates and incubated overnight at 37°C and 5% CO2. The next day, the growth medium was removed, and human PBMCs from nine donors were added to generate co-cultures at pH 6.5, representing the tumor microenvironment. Serial dilutions of BA3182 or isotype control antibodies were added to the plates, and the cultures were maintained at 37°C and 5% CO2. After 48 hours, the plates were centrifuged to aggregate the cells, the supernatant was collected, transferred to new plates, and stored at -80°C until cytokine levels were analyzed. Cytokine concentrations were determined using the Quantikine ELISA kit from Andy Biotech, and the EC50 values ​​of BA3182 were calculated using GraphPad Prism software. BA3182 induced the release of IL-2 and INF-γ cytokines in nine donors at pH 6.5. Baseline levels of IL-6, IL-10, and TNFα cytokines were detected, and their levels varied across donors. No effect of BA3182 or its isoforms on these cytokines was observed.

[0605] Cell Culture. Table 12: Antibodies

[0606] Homotype: Non-CAB human IgG anti-egg white lysozyme (HEL) containing anti-hCD3 scFv.

[0607] Table 13: Reagents and Consumables

[0608] Table 14: Equipment

[0609] Cell Staining. Incubation with Antibodies.HCT116 cells were maintained in McCoy's 5A medium supplemented with 10% FBS. Cells were routinely passaged twice a week. Cells were harvested and counted during the exponential growth phase for coating and determination of EpCAM surface expression.

[0610] Measurement of Cytokines. EpCAM surface expression was confirmed by flow cytometry before plating target cells for analysis. For this purpose, the culture medium was removed from T75 HCT116 culture flasks, and the cell layers were simply washed with PBS pH 7.4. One mL of 0.25% trypsin-EDTA was added to each flask, and the cells were allowed to return to 37°C until the cell layers dispersed. The trypsin reaction was blocked with 9 mL of culture medium, and the cells were resuspended by gentle pipetting. For FACS staining, 2 × 10⁶ cells were plated. 5 HCT116 cells were added to 1.5 mL Eppendorf tubes and washed with PBS pH 7.4. The cells were rotated to remove the supernatant, and then resuspended in 100 µL PBS + 1% BSA containing 2 µL of PE-anti-human EpCAM 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 one hour. After incubation, the cells were washed three times with 1 mL PBS pH 7.4. The cells were then resuspended in 120 µL PBS + 1% BSA and analyzed using a NovoCyte flow cytometer.

[0611] Results. HCT116 cells were seeded at a density of 6000 cells / well in McCoy's 5A medium supplemented with 10% FBS in 96-well tissue culture plates and incubated overnight at 37°C and 5% CO2. After overnight incubation, the growth medium was removed, and RPMI 1640 + 10% FBS medium containing 30,000 human PBMCs was added to each well at pH 6.5 (reflecting the tumor microenvironment). PBMCs were added at a PBMC:target cell ratio of 5:1. BA3182 or isotype control antibody was serially diluted from 150 nM to 0.008 nM (3-fold dilution) in RPMI 1640 + 10% FBS at pH 6.5 and added to the plates. The culture was maintained at 37°C and 5% CO2. After incubating with the test antibody for 48 hours, the plate was centrifuged at 2000 rpm for 3 minutes at ambient temperature to aggregate the cells, and the supernatant was transferred to a new 96-well plate and stored at -80°C until the cytokine levels were analyzed.

[0612] Figures 18A-18BCytokine concentrations were determined using the Quanikine™ ELISA assay according to the manufacturer's protocol. The supernatant was diluted in analytical diluent as needed to obtain values ​​within the linear range of the standard curve for the assay. EC50 values ​​were calculated from nonlinear 4-parameter regression curves of the cytokine standards using Graph Pad Prism software, version 9.0.

[0613] Figures 19A-19B At pH 6.5 (tumor microenvironment pH), BA3182 induced the release of IL-2 and INFg cytokines from human PBMCs co-cultured with the human colon cancer cell line HCT116. In the absence of HCT116 cells, stimulation of human PBMCs with BA3182 did not induce a cytokine response (data not shown). The isotype control antibody, a bispecific antibody against egg white lysozyme / CD3, did not induce the release of IL-2 or INFg. Figures 20A-20B and Materials Neither BA3182 nor its isotype antibody had any measurable effect beyond background levels on the release of IL-6, IL-10, or TNFα. Antibodies (21A-21B and 22A-22B). Low nanomolar EC50 values ​​of BA3182 were determined for IL-2 and IFNγ induction from PBMCs from nine human donors, tested at pH 6.5. PBMCs from a tenth donor did not respond to any stimulation (including a positive control of CD3 / CD28-fixed microbeads) when co-cultured with HCT116 cancer cells (data not shown), and this donor was excluded from the study. EC50 values ​​for IL-2 and IFNγ induction by BA3182 at pH 6.5 varied by donor and are presented in Table 15. These data indicate that, in vitro, in the presence of EpCAM antigen, the BA3182 antibody is a potent inducer of IL-2 and IFNγ production in human PBMCs. BA3182 does not induce the production of pro-inflammatory cytokines (such as IL-6 and TFN-α) or the anti-inflammatory cytokine IL-10 in human PBMCs.

[0614] Table 15: EC50 values ​​of IL-2 and INF-γ cytokine production induced by BA3182 in human PBMCs co-cultured with HCT116 cancer cells in the presence of BA3182.

[0615] ND = Not determined by curve fitting.

[0616] Example 24. C1q Affinity ELISA In this example, the binding of the human complement protein C1q to BA3182 was determined using a C1q affinity ELISA assay. ELISA plates were coated with either BA3182 or a positive control antibody (B12). Purified human C1q protein was serially diluted and added to the plates. Binding of the C1q protein to the antibody was detected by adding a sheep anti-human C1q-HRP conjugated antibody. A staining product was generated by adding a TMB colorimetric substrate. The OD absorbance in each well was determined using a microplate reader. Data were analyzed, and nonlinear 4-parameter regression curves were generated using Graph Pad Prism software. The results indicated a weak binding of C1q to BA3182. This result was expected to be due to a mutation at position 297 of the glycosylation site, changing from asparagine (N) to glutamine (Q). This mutation has been shown to successfully eliminate effector functions, such as complement-dependent cytotoxicity (CDC).

[0617] Reagents and Consumables Methods. Results.

[0619] Figure 23 Spread ELISA plates with 100 μL / well of carbonate-bicarbonate buffer containing 3 µg / mL BA3182 or B12 antibody and incubate overnight at 4°C. The next day, block each plate with 300 μL / well of casein buffer and incubate at room temperature for 1 hour. After this, wash each plate three times with 300 μL / well of PBS containing 0.05% Tween 20. Perform semi-logarithmic titration of C1q in casein buffer (600, 189.75, 60.01, 18.98, 6.00, 1.90, 0.60, 0.19, 0.06, and 0.02 μg / mL); add 100 μL of each dilution to each well and incubate at room temperature for 2 hours. Then, wash the plates and subsequently incubate at room temperature for 1 hour with 100 µL / well of secondary antibody sheep anti-human C1q Ab-HRP (1:200 dilution). After washing, add 100 µL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate to each well. Incubate the plate in the dark at room temperature for 5–20 minutes, and terminate the enzymatic reaction with 50 µL of 2M HCl. Read the absorbance at 450 nm using a Molecular DeviceSpectraMax i3x microplate reader. Analyze the data and generate nonlinear 4-parameter regression curves using Graph PadPrism software, version 9.0.

[0620] Figure 23 The molar concentration of C1q was calculated using a molecular weight of 410 kDa, and a four-parameter regression curve was generated using absorbance at 450 nm to calculate the EC50 value. The C1q protein binds strongly to the positive control antibody B12, producing a favorable dose-response curve. Materials B12 is human IgG. 1,k The antibody targets the gp120 glycoprotein, and C1q is expected to bind to it. C1q binds weakly to BA3182. Antibodies This indicates that C1q has a low affinity for the BA3182 antibody. The calculated EC50 values ​​of the binding affinity of C1q for BA3182 are 12.89 times higher than those of C1q for B12, at 613.1 nM and 47.55 nM, respectively (Table 16). The BA3182 antibody is a human IgG antibody containing the N297Q mutation. 1,k Deglycosylation mutants have been shown to reduce the binding affinity of human IgG1 to C1q protein and inhibit complement fixation and activation.

[0621] Table 16: Binding affinity of C1q for BA3182

[0622] Example 25. Combination kinetics analysis of SPR via SPR The objective of this study was to determine the binding kinetics of BA3182 with human and cynomolgus macaque EpCAM and CD3ε / δ heterodimers at pH 6.0, pH 6.5, and pH 7.4 using surface plasmon resonance (SPR). The ligand was immobilized on the surface of a flat amine sensor chip, followed by the injection of titrated concentrations of BA3182. Binding interactions between BA3182, the ligand, and the control surface were monitored in real time. Binding kinetics (association rate kd, dissociation rate ka, affinity K) were calculated using a 1:1 Langmuir model built into the analytical software. D ).

[0623] 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 decreases 5.5-fold to 6.7 nM. Binding to EpCAM in cynomolgus monkeys shows a similar trend: the binding affinity is approximately 2-3 nM at acidic pH and decreases 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 CD3 in cynomolgus monkeys shows a similar trend: the binding affinity is approximately 11 nM at acidic pH and decreases 3-fold to approximately 30 nM at normal physiological pH.

[0624] Equipment and Reagents Buffers. Use BA3182 at a stock solution concentration of 1.03 mg / mL.

[0625] Methods Results. Before use, prepare SPR operating buffers containing sodium bicarbonate at pH 6.0, pH 6.5, and pH 7.4 as described below, because the pH of the buffer may change during storage.

[0627] PBST-SB-75 pH 6.0 1. Add 16.6 mL of 7.5% sodium bicarbonate solution to 483.4 mL of 1x PBS.

[0628] 2. Add 250 µL of Tween-20.

[0629] 3. Add 2.19 g NaCl.

[0630] 4. Adjust the pH to 5.9 using 6N HCl.

[0631] 5. Filter using a 0.22 µM PES bottle filter.

[0632] 6. Use an ultrasonic generator and vacuum degassing for 5 minutes.

[0633] 7. Remove 50 mL of the aliquot for sample preparation.

[0634] PBST-SB-75 pH 6.5 1. Add 16.6 mL of 7.5% sodium bicarbonate solution to 483.4 mL of 1x PBS.

[0635] 2. Add 250 µL of Tween-20.

[0636] 3. Add 2.19 g NaCl.

[0637] 4. Adjust the pH to 6.4 using 6N HCl.

[0638] 5. Filter using a 0.22 µM PES bottle filter.

[0639] 6. Use an ultrasonic generator and vacuum degassing for 5 minutes.

[0640] 7. Remove 50 mL of the aliquot for sample preparation.

[0641] PBST-SB-75 pH 7.4 1. Add 16.6 mL of 7.5% sodium bicarbonate solution to 483.4 mL of PBS.

[0642] 2. Add 250 µL of Tween-20.

[0643] 3. Add 2.19 g NaCl.

[0644] 4. Adjust the pH to 7.35 using 6N HCl.

[0645] 5. Filter using a 0.22 µM PES bottle filter.

[0646] 6. Use an ultrasonic generator and vacuum degassing for 5 minutes.

[0647] 7. Remove 50 mL of the aliquot for sample preparation.

[0648] Sensor regeneration solution 10 mM glycine, pH 2.0 Figure 24 . The SPR32 Pro instrument features eight flow channels (1-8), each with four detection points (A, B, C, D). The 32 detection points can be processed independently or in groups. Before immobilizing the ligands, the surfaces of each new planaramine sensor chip are pre-conditioned using a built-in method according to the manufacturer's recommendations. PBST pH 7.4 is used as the operating buffer for pre-conditioning and immobilizing EpCAM or CD3.

[0649] Test sites A, B, and C were activated by injecting a mixture of EDC / NHS (250 mM / 50 mM) at a flow rate of 25 μL / min for 240 seconds. No protein was immobilized on the control surface (test sites 1A-8A). Human EpCAM-His was diluted to 0.5 μg / mL in 10 mM NaAc pH 5.0 and injected into test sites 1B-8B at a flow rate of 25 μL / min for 240 seconds. CynoEpCAM-His was diluted to 0.5 μg / mL in 10 mM NaAc pH 5.5 and injected into test sites 1C-8C at a flow rate of 25 μL / min for 240 seconds. Test sites A, B, and C were blocked by injecting ethanolamine (1M) at a flow rate of 25 μL / min for 240 seconds. Test sites A, B, C, and D were activated by injecting a mixture of EDC / NHS (250 mM / 50 mM) at a flow rate of 25 μL / min for 240 seconds. No protein was immobilized on the control surface (test sites 1A-8A). Human CD3-hFc fusion was diluted to 1 μg / mL in 10 mM NaAc pH 5.5 and injected into test sites 1D-8D at a flow rate of 25 μL / min for 240 seconds. CynoCD3-His was diluted to 0.05 μg / mL in 10 mM NaAc pH 5.5 and injected into test sites 1B-8B at a flow rate of 25 μL / min for 240 seconds. Test sites A, B, C, and D were blocked by injecting ethanolamine (1M) at a flow rate of 25 μL / min for 240 seconds.

[0650] BA3182 was buffer-exchanged with 1xPBS using an Amicon Ultra-15 rotary filter (150 kDa MWCO). 1 mL of BA3182 was added to the top reservoir, followed by approximately 14 mL of 1xPBS. The solution was centrifuged at 4000 RPM for 12 minutes. The flow-through was discarded, and the process was repeated three times. Protein concentration was determined using UV280. The buffer-exchanged BA3182 was diluted in working buffer to an initial concentration of 5 µg / mL. Then, serial dilutions of two-fold (from 5 µg / mL to 0.078 µg / mL) were performed for a total of seven dilution points.

[0651] Serial dilutions of BA3182 (highest concentration in channel 8, lowest concentration in channel 2, and operating buffer in channel 1) were injected into detection sites A, B, and C (flow rate 25 μL / min, contact time 120 s; dissociation rate measurement was performed for 120 s). The sensor surface was regenerated by injecting 10 mM glycine pH 2.0 (flow rate 25 μL / min, contact time 15 s). The analyte injection was repeated twice (a total of three analyte injections). Operating buffer was injected as a blank analyte before and after the BA3182 injection.

[0652] A control surface, A, where no protein was immobilized, was used to subtract the reference. Additionally, data from each operation using only buffer as the analyte (0 nM analyte) were subtracted. The data that underwent double subtraction were fitted using a 1:1 binding model with the provided analysis software, Sierra Analyzer R3 (Bruker). The molar concentration of the analyte was calculated using a molecular weight of 200 kDa.

[0653] Kinetic data from representative experiments at various tested pH conditions were input into the SPR simulation software (www.sprpages.nl\spr-simulation) to analyze the signal reduction from pH 6.0 to pH 7.4. The software input data included calculated association / dissociation rates and KL from actual SPR experiments. D The parameters include the molecular weight of the analyte and ligand, the amount of analyte immobilized on the sensor surface, association and dissociation times, the maximum analyte concentration used, and the number of analyte concentration and dilution steps. The program generates a sensor map assuming 100% immobilization of the ligands for binding. For random immobilization of ligands, this is typically in the 30-40% range. The actual fraction can be determined by reducing the activity concentration until the maximum signal in the simulation matches the maximum signal in the actual experiment. The activity concentration of the ligand (EpCAM or CD3) at pH 6.0 is set to 100%.

[0654] Figure 26 Three independent SPR experiments were conducted at various pH values. The same sensor chip was used for all pH conditions. The binding kinetics of BA3182 with recombinant human and cynomolgus monkey 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 value are shown in [Table 1]. Figure 25In the study, BA3182 bound 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 monkey 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).

[0655] Simulations of the resulting sensor maps indicate that the signal reduction at higher pH values ​​is caused by a decrease in active ligands on the wafer surface. Figure 25 This differential binding profile is expected to be due to the antibody being engineered to exhibit reduced binding under physiological conditions while maintaining full binding capacity in the tumor microenvironment. BA3182 binds to both humans and cynomolgus monkeys (EpCAM) with similar affinity (within a factor of two), indicating that cynomolgus monkeys are a suitable species for toxicological testing.

[0656] Three independent SPR experiments were conducted at various pH values. The same sensor chip was used for all pH conditions. The binding kinetics of BA3182 with recombinant human and cynomolgus monkey CD3 at pH 6.0, pH 6.5, and pH 7.4 are summarized in Table 18. Binding curves for representative experiments at each pH value are shown in [Table 18]. Figure 27 middle.

[0657] 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 monkey 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 of most analyte concentrations is within the noise level and cannot be used to calculate K. D The binding data shown in Table 18 at pH 7.4 are based only on the highest concentration used. (Based on a single concentration of K...) D The values ​​were within twice the values ​​calculated using the entire dilution series. Except for lower binding affinity at higher pH values, a maximum decrease in SPR signal occurred from pH 6.0 to pH 7.4. Materials Simulations of the resulting sensor maps indicate that the signal reduction at higher pH values ​​is caused by a decrease in active ligands on the wafer surface. Antibodies ).

[0658] Table 17: Affinity of BA3182 to EpCAM in humans and cynomolgus monkeys under different pH conditions. Results and averages from three independent experiments are listed.

[0659]

[0660] Table 18: Affinity of BA3182 to CD3 in humans and cynomolgus monkeys under different pH conditions. Results and averages from three independent experiments are listed. Data calculated from single analyte concentrations are highlighted in gray.

[0661]

[0662] Example 26. Fcγ and FcRn receptor binding assays performed by SPR This example uses surface plasmon resonance (SPR) technology to demonstrate the binding affinity (Kb) of BA3182 to human FcγRI, FcγRIIa, FcγIIb / c, FcγRIIIa, FcγRIIIb and FcRn receptors. D The extracellular domain of the Fc-γ receptor was captured on a wafer using an anti-His-tagged antibody. Serial dilutions of BA3182 and a control IgG1 antibody were injected at eight detection sites containing the Fc-γ receptor and on a control surface containing only the anti-His antibody. BA3182 possesses a non-glycosylated Fc domain (N297Q mutation) and, as expected, does not bind to the Fc-γ receptor. Binding to FcRn at pH 6.0 was measured by immobilizing BA3182 or control IgG1 on the wafer surface and injecting serial dilutions of FcRn. BA3182 exhibited similar binding affinity to FcRn to the IgG1 control antibody. Binding kinetics were calculated using the SICK Langmuir 1:1 model constructed into the analysis software.

[0663] Equipment and Reagents Methods. BA3182 IgG1-Ctrl (batch number 13578) manufactured by Evectel (Zurich, Switzerland) FcγRI (CD64) Capture Assay

[0664] FcγRIIa H131 (CD32a) Capture Assay The SPR32 Pro instrument features eight flow channels (1-8), each with four detection points (A, B, C, D). These 32 detection points can be processed independently or in groups. Before protein fixation, the surfaces of each new sensor chip are pre-conditioned using the built-in method according to the manufacturer's recommendations. PBST pH 7.4 is used as the operating buffer for pre-conditioning and fixing all antibodies.

[0665] All 32 spots on the pre-calibrated 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. The His-tagged antibody was diluted to 50 μg / mL in NaAc pH 4.5 and injected onto all 32 detection spots at a flow rate of 10 μL / min for 360 seconds. All 32 detection spots were blocked by injecting ethanolamine (1M) at a flow rate of 10 μL / min for 420 seconds.

[0666] For the BA3182 binding assay to the Fcγ receptor, a capture assay format was used. The Fcγ receptor was diluted in operating buffer and captured at points B, C, or D of the anti-His sensor chip. Then, BA3182 or IgG1-ctrl antibody (serially diluted in operating buffer) was injected into points AB, BC, or CD. Detection points without the captured protein were used as control surfaces. Operating buffer injections were used as blanks. The His capture surfaces were regenerated after each analyte injection. One IgG1-ctrl antibody injection and three BA3182 injections were performed for each assay.

[0667] ​ Operating buffer: Phosphate-buffered saline (PBST) containing Tween-20, pH 7.4.

[0668] FcγRI (CD64): 50 nM in PBST, pH 7.4, injected at point D (flow rate: 10 μL / min, contact time 15 seconds).

[0669] IgG1-ctrl: a two-fold serial dilution in PBST, pH 7.4, starting at 6 μg / mL, injected onto the CD site (flow rate: 20 μL / min, contact time 180 seconds; dissociation rate measured 400 seconds).

[0670] BA3182: A two-fold serial dilution of PBST, pH 7.4, starting at 6 μg / mL, injected onto the CD site (flow rate: 20 μL / min, contact time 180 s; dissociation rate measured 400 s).

[0671] ​ FcγRIIa H131 (CD32a): The concentration of the operating buffer in HBS-EP+ is 181.8 nM, injected at point C (flow rate: 10 μL / min, contact time 60 seconds).

[0672] IgG1-ctrl: a two-fold serial dilution of HBS-EP+, starting at 1.5 mg / mL, was injected at the BC site (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measured 60 seconds).

[0673] BA3182: A two-fold serial dilution of HBS-EP+, starting at 1.5 mg / mL, was injected at point BC (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measured 60 seconds).

[0674] Regeneration: 10 mM glycine, pH 2.0, injected into point BC (flow rate: 25 μL / min, contact time 15 seconds).

[0675] FcγRIIb / c (CD32b / c) capture analysis method Working buffer: HEPES / NaCl buffer containing EDTA and surfactant P20 (HBS-EP+). FcγRIIb / c (CD32b / c): 192.3 nM in HBS-EP+, injected at point C (flow rate: 10 μL / min, contact time: 60 seconds).

[0676] IgG1-ctrl: a two-fold serial dilution of HBS-EP+, starting at 1.5 mg / mL, was injected at the BC site (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measured 60 seconds).

[0677] BA3182: A two-fold serial dilution of HBS-EP+, starting at 1.5 mg / mL, was injected at point BC (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measured 60 seconds).

[0678] Regeneration: 10 mM glycine, pH 2.0, injected into point BC (flow rate: 25 μL / min, contact time 15 seconds).

[0679] FcγRIIIa F158 (CD16a) capture analysis method FcγRIIIa (CD16a): 171.7 nM in HBS-EP+, injected at point C (flow rate: 10 μL / min, contact time: 60 seconds).

[0680] IgG1-ctrl: a two-fold serial dilution of HBS-EP+, starting at 1.5 mg / mL, was injected at the BC site (flow rate: 20 μL / min, contact time 60 seconds; dissociation rate measured 60 seconds).

[0681] BA3182: A two-fold serial dilution of HBS-EP+, starting at 1.5 mg / mL, was i...

Claims

1. A conditionally active bispecific antibody comprising: an IgG antibody or antibody fragment binding to a 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; and at least one scFv antibody fragment binding to a T-lymphocyte protein, which is linked to the C-terminus of at least one light chain of said IgG antibody or antibody fragment; wherein The IgG light chain variable region is selected from a group consisting of light chain variable regions having L1, L2, and L3 complementarity-determining regions, and the L1, L2, and L3 complementarity-determining regions each have the following sequences: 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, SEG ID NO:2 and SEQ ID NO:23; and The complementary determinant regions H1, H2, and H3 of the variable region of the IgG heavy chain 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 binding to a 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; and at least one scFv antibody fragment binding to a T-lymphocyte protein, which is linked to the C-terminus of at least one light chain of said IgG antibody or antibody fragment; wherein The IgG heavy chain variable region is selected from a group of heavy chain variable regions having H1, H2, and H3 complementarity-determining regions, and the H1, H2, and H3 complementarity-determining regions each have the following sequences: 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; and The complementary determinant regions L1, L2, and L3 of the variable region of the IgG light chain have the following sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

3. The conditionally active bispecific antibody according to 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 NO:53-69.

4. The conditionally active bispecific antibody according to 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 NO:70-96.

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

6. The conditionally active bispecific antibody according to any one of claims 1 to 5, wherein the IgG antibody or antibody fragment is obtained from an unconditionally active parental anti-EpCAM antibody.

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

6.

8. The conditionally active bispecific antibody according to any one of claims 1 to 7, wherein the IgG antibody or antibody fragment has a higher binding affinity to the EpCAM protein in a tumor microenvironment at pH 5.0 to 6.9 than in a non-tumor microenvironment at pH 7.0 to 7.

6.

9. The conditionally active bispecific antibody according to any one of claims 1 to 7, wherein the ratio of the binding affinity of the IgG antibody or antibody fragment to the human EpCAM protein at pH 6.0 to the binding affinity to the human EpCAM protein at pH 7.4 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.

10. The conditionally active bispecific antibody of claim 9, wherein the ratio of the binding affinity of the IgG antibody or antibody fragment to the human EpCAM protein at pH 6.0 to the binding affinity to the human EpCAM protein at pH 7.4 is 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 the CD3 protein.

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

6.

13. The conditionally active bispecific antibody according to any one of claims 11 to 12, wherein the scFv antibody fragment is obtained from an unconditionally active parental anti-CD3 antibody.

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

6.

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

97.

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

99.

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

4.

18. The conditionally active bispecific antibody according to any one of claims 1 to 17, wherein the ratio of the binding affinity of the IgG antibody or antibody fragment to the EpCAM protein of the cynomolgus monkey at pH 6.0 to the binding affinity of the EpCAM protein of the cynomolgus monkey at pH 7.4 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.

19. The conditionally active bispecific antibody according to any one of claims 11 to 17, wherein the binding affinity of the IgG antibody or antibody fragment to the EpCAM protein of the cynomolgus monkey at pH 6.0 is 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 binding to a 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; wherein The light chain variable region is selected from a group of light chain variable regions having L1, L2, and L3 complementary determinant regions, and the L1, L2, and L3 complementary determinant regions have the following sequences: 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; and The heavy chain variable region includes three complementary determinant regions H1, H2 and H3, which have sequences of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

21. A conditionally active antibody or antibody fragment binding to a 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; wherein The heavy chain variable region is selected from a group of heavy chain variable regions having H1, H2, and H3 complementary determinant regions, and the H1, H2, and H3 complementary determinant regions have the following sequences: 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; and The complementary determinant regions L1, L2, and L3 of the light chain variable region have sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

22. 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 NO:53-69.

23. The conditionally active antibody or antibody fragment according to 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 NO:70-96.

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

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

6.

26. The conditionally active antibody or antibody fragment according to any one of claims 20 to 24, wherein the antibody or antibody fragment is obtained from an unconditionally active parental anti-EpCAM antibody.

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

6.

28. The conditionally active antibody or antibody fragment according to any one of claims 20 to 24, wherein the ratio of the binding affinity of the antibody or antibody fragment to the human EpCAM protein at pH 6.0 to the binding affinity of the human EpCAM protein at pH 7.4 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.

29. The conditionally active antibody or antibody fragment of claim 28, wherein the ratio of the binding affinity of the IgG antibody or antibody fragment to the human EpCAM protein at pH 6.0 to the binding affinity to the human EpCAM protein at pH 7.4 is 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. The conditionally active bispecific antibody or antibody fragment according to any one of claims 1 to 6, wherein the conditionally active bispecific antibody or antibody fragment has a binding affinity for human and cynomolgus monkey EpCAM proteins at least 5 times greater than the binding affinity of the same conditionally active bispecific antibody or antibody fragment for rat or mouse EpCAM proteins.

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 the cynomolgus macaque EpCAM protein is at least 50% of the binding affinity of the conditionally active antibody or antibody fragment to the human EpCAM protein.

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

Images