Multispecific FAB fusion proteins and use thereof

Abstract

The present invention provides multispecific Fab fusion proteins (MSFP) that specifically bind to CD3 and EpCAM. The present invention further provides uses of the MSFPs for the preparation of pharmaceutical compositions, methods of treating cancer, and kits comprising the MSFPs. Also provided are anti-EpCAM antibodies or antigen-binding fragments thereof.

Description

MULTISPECIFIC FAB FUSION PROTEINS AND USE THEREOF FIELD OF THE INVENTION

[0001] The present invention relates to multi-specific Fab fusion proteins (MSFP) that specifically bind to CD3 and EpCAM. Further provided herein are pharmaceutical compositions comprising the MSFPs, methods of treating cancer using the MSFPs, and kits comprising the MSFPs. BACKGROUND OF THE INVENTION

[0002] Some antigens are over-expressed, mutagenized, or selectively mutagenized in tumor tissues. Therefore, antibodies targeting specific antigens on the surface of cancer cells can be used as cancer therapeutics. Epithelial cell adhesion molecule (EpCAM, CD326), also known as 17-1A, ESA, AUA1, EGP40, etc., is a 40 kD transmembrane glycoprotein composed of 314 amino acid. EpCAM is specifically expressed in various types of epithelial cells, and major types of human malignancies. For example, EpCAM is highly expressed in colon cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, breast cancer and ovarian cancer. Thus, EpCAM has become a hot target in cancer therapy, including vaccines, murine or human monoclonal antibodies, and antibodies conjugated to bacterial toxins or chemotherapy drugs, such as EpCAM specific antibodies ING-1, adecatumumab, edrecolomab, etc.

[0003] CD3, comprising three different polypeptide chains (<com:Math><semantics>ϵ<annotation encoding="application / x-tex">\epsilon< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>δ<annotation encoding="application / x-tex">\delta< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math> chains), is an antigen expressed by T cells. The three CD3 polypeptide chains associate with the T-cell receptor (TCR) and the <com:Math><semantics>ζ<annotation encoding="application / x-tex">\zeta< / annotation>< / semantics>< / com:Math>-chain to form the TCR complex, which has the function of activating signaling cascades in T cells. Currently, many therapeutic strategies target the TCR signal transduction to treat diseases using anti-human CD3 monoclonal antibodies. The CD3 specific antibody OKT3 is the first monoclonal antibody approved for human therapeutic use, and is clinically used as an immunomodulator for the treatment of allogenic transplant rejections.

[0004] During the past twenty years, efforts in the field of bispecific antibodies have gradually yielded clinical success. In 2009, Catumaxomab (anti-CD3, anti-EpCAM trifunctional antibody) was approved in the European Union for the treatment of symptomatic malignant ascites. However, although bispecific antibodies have been shown to have potential in effectively killing cancer cells, severe adverse effects, including systemic immune activation, immunogenicity (anti-drug antibody effect), and the generally poor manufacturability of these molecules, have greatly limited the widespread application of this type of drugs. Another drawback of CD19×CD3 bispecific scFv-scFv (single-chain variable fragment) fusion protein (Blinatumomab) is that this drug needs to be administered intravenously (i.v.) on a daily basis due to its short half-life and incompatibility with subcutaneous administration; yet, neurological effects such as disorientation, confusion, speech and language impairment, tremor or convulsion still occurred during clinical trials (Science 321:974-977, 2008). To better control these unwanted adverse effects, bispecific single-chain antibodies (BiTE) were administered intravenously with continuous infusion over a longer period of time. US20120244161 disclosed the stage I clinical trial of EpCAM×CD3 bispecific scFv-scFv fusion protein (MT110), in which low dosage (1-12 μg / kg / 24hr) continuous intravenous infusion was applied over a long period of time, with glucocorticoid administered prior to MT110 infusion or dosage increment. Furthermore, scFv-scFv fusion protein has a tendency for aggregation.

[0005] The drawbacks of current formats of bispecific antibodies remain great challenges for their widespread application in the treatment of cancer patients with good efficacy and safety. Therefore, there is an urgent need in the field for the development of new bispecific antibodies or treatment regimen with improved efficacy, stability, safety and manufacturability. BRIEF SUMMARY OF THE INVENTION

[0006] The present invention provides multi-specific Fab fusion proteins (such as bispecific Fab fusion proteins, or BSFP) that specifically bind to CD3 and EpCAM, and methods of treating cancer using the MSFPs.

[0007] In one aspect of the present invention, there is provided a method of treating a cancer in an individual (such as a human individual), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg to about 5 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg, about 0.1 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg to about 30 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the binding domain is an scFv. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment; and wherein the second scFv is fused to the N- terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence.

[0008] In some embodiments according to any of the methods described above, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency (such as any of less than about once per 1, 2, 3, 4, 6, 9, or 12 months, such as a single dose). In some embodiments, the multispecific Fab fusion protein is administered at a dose equivalent to about 0.1 µg / kg to about 100 μg / kg (such as about 0.3 μg / kg to about 5 μg / kg, or about 5 μg / kg to about 20 μg / kg) for a cynomolgus monkey. In some embodiments, the multispecific Fab fusion protein is administered at a dose that does not induce cytokine storm. In some embodiments, the multispecific Fab fusion protein is administered at a dose equivalent to no higher than about 30 μg / kg (such as no higher than about 20 μg / kg, 10 μg / kg, or 1 μg / kg) for a cynomolgus monkey. In some embodiments, the individual is a human individual.

[0009] In some embodiments according to any of the methods described above, the multispecific Fab fusion protein is administered at a first dose for a first period of time to the individual, and subsequently, the multispecific Fab fusion protein is administered at a second dose for a second period of time to the individual, and wherein the second dose exceeds the first dose. In some embodiments, the second period of time exceeds the first period of time. In some embodiments, the first period of time is at least about 7 days. In some embodiments, the second period of time is at least about 2 weeks. In some embodiments, the first dose is no more than about 1 μg / kg. In some embodiments, the second dose is about 0.1 μg / kg to about 10 μg / kg.

[0010] In some embodiments according to any of the methods described above, the method further comprises administering a glucocorticoid to the individual. In some embodiments, the glucocorticoid is dexamethasone. In some embodiments, the glucocorticoid is administered prior to the first dose of the multispecific Fab fusion protein. In some embodiments, the glucocorticoid is administered at a dose of about 0.1 mg / kg to about 5 mg / kg.

[0011] In some embodiments according to any of the methods described above, the Fab fragment specifically binds to the N-terminus of CD3 epsilon, such as an epitope within amino acids 1-27 of CD3 epsilon. In some embodiments, the VH of the Fab fragment comprises: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, the VL of the Fab fragment comprises a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH of the Fab fragment comprises an amino acid sequence having at least about 85% (such as about 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 7 and 39-43.. In some embodiments, the VL of the Fab fragment comprises an amino acid sequence having at least about 85% (such as about 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 8 and 44-47. In some embodiments, the Fab fragment comprises a human immunoglobulin heavy chain constant region 1 (CH1) comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the Fab fragment comprises a human lambda light chain constant region comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the CH1 and the CL of the Fab fragment are connected by one or more disulfide bonds. In some embodiments, the Fab fragment comprises a first polypeptide comprising an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the Fab fragment comprises a second polypeptide comprising an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 12.

[0012] In some embodiments according to any of the methods described above, the cancer is an EpCAM-positive solid cancer. In some embodiments, the EpCAM-positive solid cancer is a carcinoma or adenocarcinoma. In some embodiments, the EpCAM-positive solid cancer is selected from the group consisting of small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer, renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer. In one embodiment, the cancer is colorectal adenocarcinoma. In one embodiment, the cancer is lung adenocarcinoma.

[0013] In some embodiments according to any of the methods described above, the binding domain (such as scFv) comprises an N-VH-VL-C fusion polypeptide. In some embodiments, the VH of the scFv comprises a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15. In some embodiments, the VL of the scFv comprises a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the VH of the scFv comprises an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO:19. In some embodiments, the VL of the scFv comprises an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO:20. In some embodiments, the scFv comprises an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO:21.

[0014] In some embodiments according to any of the methods described above, the multispecific Fab fusion protein comprises a first polypeptide comprising an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO:22. In some embodiments, the multispecific Fab fusion protein comprises a second polypeptide comprising an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO:23.

[0015] The present invention further provides an anti-EpCAM antibody. In some embodiments, there is provided an anti-EpCAM antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising: (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and a light chain variable region comprising: (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and (3) a HVR- L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the heavy chain variable domain sequence comprises a VH comprising an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO:19.In some embodiments, the light chain variable domain sequence comprises a VL comprising an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO:20.

[0016] In some embodiments according to any one of the anti-EpCAM antibodies described above, the anti-EpCAM antibody comprises an Fc sequence of a human IgG. In some embodiments, the anti-EpCAM antibody is a multispecific antibody, such as a bispecific antibody.

[0017] In some embodiments according to any one of the anti-EpCAM antigen-binding fragments described above, the antigen-binding fragment is a single-chain Fv (scFv). In some embodiments, the scFv comprises an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 21.

[0018] In some embodiments, there is provided a multispecific (such as bispecific) Fab fusion protein comprising any one of the anti-EpCAM antigen-binding fragments described above. In some embodiments, the multispecific Fab fusion protein comprises a Fab fragment that specifically binds to CD3, a first copy of the anti-EpCAM antigen-binding fragment, and a second copy of the anti-EpCAM antigen binding fragment; wherein the first copy of the anti- EpCAM antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment; and wherein the second copy of the anti-EpCAM antigen binding fragment is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the Fab fragment specifically binds to the N-terminus of CD3 epsilon, such as an epitope within amino acids 1-27 of CD3 epsilon. In some embodiments, the VH of the Fab fragment comprises a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3. In some embodiments, the VL of the Fab fragment comprises: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the VH of the Fab fragment comprises an amino acid sequence having at least about 85% (such as about 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:7 and 39-43. In some embodiments, the VL of the Fab fragment comprises an amino acid sequence having at least about 85% (such as about 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 44-47. In some embodiments, the Fab fragment comprises a human immunoglobulin heavy chain constant region 1 (CH1) comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the Fab fragment comprises a human lambda light chain constant region comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the CH1 and the CL of the Fab fragment are connected by one or more disulfide bonds. In some embodiments, the Fab fragment comprises a first polypeptide comprising an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO:11. In some embodiments, the Fab fragment comprises a second polypeptide comprising an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO:12. In some embodiments, the multispecific (such as bispecific) Fab fusion protein comprising a first polypeptide comprising an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO:22, and a second polypeptide comprising an amino acid sequence having at least about 85% (such as about 100%) sequence identity to the amino acid sequence of SEQ ID NO:23.

[0019] In some embodiments, there is provided a composition comprising any of the anti- EpCAM antibodies or antigen-binding fragments thereof or the multispecific Fab fusion proteins described above and a pharmaceutically acceptable carrier. In some embodiments, there is provided a method of treating cancer in an individual, comprising administering an effective amount of the composition to the individual.

[0020] Further provided are isolated nucleic acid molecules that encode the MSFPs, anti- EpCAM antibodies or antigen-binding fragments thereof described above, expression vectors encoding the isolated nucleic acid molecules, isolated host cells comprising the expression vectors, and methods of producing the MSFPs, anti-EpCAM antibodies or antigen-binding fragments thereof, comprising culturing the isolated host cells and recovering the MSFPs, anti- EpCAM antibodies or antigen-binding fragments thereof from the cell culture.

[0021] Also provided herein are uses, compositions (such as pharmaceutical compositions), kits and articles of manufactures comprising any of the multispecific Fab fusion proteins or the anti-EpCAM antibodies or antigen-binding fragments thereof described above.

[0022] Use of any of the multispecific Fab fusion proteins described above or any of the anti- EpCAM antibodies or antigen-binding fragmetrs thereof described above in the preparation of a medicament for treating a cancer is further provided herein.

[0023] These and other aspects and advantages of the present invention will become apparent from the subsequent detailed description and the appended claims. It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 depicts the structure of an exemplary EpCAM×CD3 Fab fusion protein.

[0025] FIG. 2A depicts an SDS-PAGE gel of a purified exemplary EpCAM×CD3 Fab fusion protein (referred herein after as ITAB1002) under non-reducing conditions. The non-reducing SDS-PAGE shows a molecular weight of about 100 kD for the purified protein, similar to the theoretical molecular weight of the EpCAM×CD3 Fab fusion protein.

[0026] FIG. 2B depicts an SDS-PAGE gel of purified ITAB1002 under reducing conditions. The reducing SDS-PAGE shows that the apparent molecular weight of the purified protein is between about 45 kD and about 66 kD.

[0027] FIG. 3A depicts CE-SDS analysis results of purified ITAB1002 under non-reducing conditions. The non-reducing CE-SDS analysis shows a single protein peak at a migration time of about 21.59 min.

[0028] FIG. 3B depicts CE-SDS analysis results of purified ITAB1002 under reducing conditions. The reducing CE-SDS analysis shows two single protein peaks at migration times of about 18.37 min and about 18.84 min, corresponding to the light chain and the heavy chain of the Fab fusion protein, respectively.

[0029] FIG. 4A depicts the binding affinity of ITAB1002 to human PBMC expressing cell surface antigen CD3.

[0030] FIG. 4B depicts the binding affinity of ITAB1002 to cynomolgus monkey PBMC expressing cell surface antigen CD3.

[0031] FIG. 5 depicts the binding affinity of ITAB1002 to SW480 cells and CyEpCAM-CHO cells expressing cell surface antigen EpCAM.

[0032] FIG. 6A shows the ability of ITAB1002 and OKT3 in activating CD4<com:Sup>+< / com:Sup> human PBMC under various conditions.

[0033] FIG. 6B shows the ability of ITAB1002 and OKT3 in activating CD8<com:Sup>+< / com:Sup> human PBMC under various conditions.

[0034] FIG. 6C shows the ability of ITAB1002 in stimulating proliferation of CD4<com:Sup>+< / com:Sup> human PBMC under various conditions.

[0035] FIG. 6D shows the ability of ITAB1002 in stimulating proliferation of CD8<com:Sup>+< / com:Sup> human PBMC under various conditions.

[0036] FIG. 7 depicts ITAB1002-mediated human or cynomolgus monkey PBMC cytotoxicity against SW480 tumor cells.

[0037] FIG. 8 depicts ITAB1002-mediated human PBMC cytotoxicity against various representative cancer cell lines.

[0038] FIG. 9A depicts growth inhibitory effects of various dosages of ITAB1002 against subcutaneous SW480 xenograft in mice that were co-inoculated with human PBMC.

[0039] FIG. 9B shows pictures of tumors from mice in various treatment groups at the end of the experiment.

[0040] FIG. 10A depicts growth inhibitory effects of various dosages of ITAB1002 against SW480 xenograft in immunodeficient mice having their immune system reconstructed with human PBMC.

[0041] FIG. 10B shows pictures of tumors from mice in various treatment groups at the end of the experiment.

[0042] FIG. 10C show percentages of human CD3<com:Sup>+< / com:Sup> cells in white blood cell samples of mice at the end of the experiment.

[0043] FIG. 11 depicts growth inhibitory effects of ITAB1002 against NCI-H1975 xenograft in immunodeficient mice having their immune system reconstructed with human PBMC.

[0044] FIG. 12A depicts change of the CD4<com:Sup>+< / com:Sup> T cell number in the blood of cynomolgus monkeys over time after intravenous administration of ITAB1002 at various dosages. X-axis: H=hours; D=days.

[0045] FIG. 12B depicts change of the CD8<com:Sup>+< / com:Sup> T cell number in the blood of cynomolgus monkeys over time after intravenous administration of ITAB1002 at various dosages. X-axis: H=hours; D=days.

[0046] FIG. 13A depicts change of the concentration of IL-2 in the serum of cynomolgus monkeys over time after intravenous administration of ITAB1002 at various dosages.

[0047] FIG. 13B depicts change of the concentration of IL-4 in the serum of cynomolgus monkeys over time after intravenous administration of ITAB1002 at various dosages.

[0048] FIG. 13C depicts change of the concentration of IL-5 in the serum of cynomolgus monkeys over time after intravenous administration of ITAB1002 at various dosages.

[0049] FIG. 13D depicts change of the concentration of IL-6 in the serum of cynomolgus monkeys over time after intravenous administration of ITAB1002 at various dosages.

[0050] FIG. 13E depicts change of the concentration of TNF in the serum of cynomolgus monkeys over time after intravenous administration of ITAB1002 at various dosages.

[0051] FIG. 13F depicts change of the concentration of IFN-γ in the serum of cynomolgus monkeys over time after intravenous administration of ITAB1002 at various dosages.

[0052] FIG. 14 depicts change of the concentration of ITAB1002 in the serum of cynomolgus monkeys after a single intravenous administration of ITAB1002 at various dosages.

[0053] FIG. 15 compares ITAB1002 vs. ITAB1012-mediated human PBMC cytotoxicity against SW480 tumor cells.

[0054] FIG. 16 depicts MSFP-mediated SW480 cell killing activity in the presence or absence of dexamethasone (DXM).

[0055] FIG. 17 depicts MSFP-mediated IL-6 release by human T cells in the presence or absence of dexamethasone (DXM).

[0056] FIG. 18A shows serum levels of alanine aminotransferase (ALT) in monkeys treated with ITAB1002 only.

[0057] FIG. 18B shows serum levels of alanine aminotransferase (ALT) in monkeys treated with ITAB1002 with dexamethasone (DXM) pre-treatment.

[0058] FIG. 19A shows serum levels of total bilirubin(TBil) in monkeys treated with ITAB1002 only.

[0059] FIG. 19B shows serum levels of total bilirubin(TBil) in monkeys treated with ITAB1002 with dexamethasone (DXM) pre-treatment.

[0060] FIG. 20A shows serum levels of alkaline phosphatase (ALP) in monkeys treated with ITAB1002 only.

[0061] FIG. 20B shows serum levels of alkaline phosphatase (ALP) in monkeys treated with ITAB1002 with dexamethasone (DXM) pre-treatment.

[0062] FIG. 21A shows IL-6 levels in monkeys treated with ITAB1002 only.

[0063] FIG. 21B shows IL-6 levels in monkeys treated with ITAB1002 with dexamethasone (DXM) pre-treatment. DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention provides methods of treating cancer using a multispecific Fab fusion protein (MSFP) comprising a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM. In some embodiments, the MSFP comprises an anti-CD3 Fab fragment having the N-termini of its heavy chain and light chain polypeptides each fused to an anti-EpCAM scFv. Unlike current anti-cancer bispecific antibodies in the art, which suffer from poor manufacturability, aggregation, short half-life, severe adverse effects, and long infusion time, the multispecific Fab fusion proteins described herein have improved stability and safety profiles, which enabled methods of treatment using lower dosage and decreased dosing frequency, avoiding undesirable adverse effects, such as induction of cytokine storm. Decreased dosing frequency and shortened infusion time facilitate the treatment of patients, conducive to improving the quality of life of patients. For example, it was surprisingly found that the MSFPs described herein can be administered at a dose of about 0.01 μg / kg to about 250 μg / kg, such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg. Further provided are novel anti-EpCAM antibodies or antigen-binding fragments thereof.

[0065] The MSFPs described herein have the following advantages compared to other multispecific Fab fusion proteins known in the art. The MSFPs have higher stability, and enhanced efficacy in killing cancer cells. The extended half-life of the present MSFPs enable lower dosing frequency and shorter infusion time, providing more convenience to the patients. The cross-reactivity of the present MSFPs with primates, such as cynomolgus monkeys, facilitates toxicology studies. The present MSFPs have fewer adverse effects, including diminished neural effects, good safety and tolerance in cynomolgus monkeys. .

[0066] Accordingly, in one aspect, the present invention provides a method of treating a cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM, wherein the binding domain is fused to an N-terminus of the Fab fragment, and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 µg / kg).

[0067] In another aspect, the present invention provides an anti-EpCAM antibody or antigen- binding fragment thereof, comprising a heavy chain variable region comprising (1) a HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (2) a HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14; and (3) a HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15; and a light chain variable region comprising (1) a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; (2) a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and (3) a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18.

[0068] Also provided are kits and articles manufacture useful for the methods described herein. I. Definitions

[0069] The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y.(2009); Ausubel et al, Short Protocols in Molecular Biology, 3<com:Sup>rd< / com:Sup> ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other like references.

[0070] As used herein, the term "treatment" refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are mitigated or eliminated, including, but are not limited to, reducing the proliferation of (or destroying) cancerous cells, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and / or prolonging survival of individuals.

[0071] As used herein, an "effective amount" refers to an amount of an agent or drug effective to treat a disease or disorder in a subject. In the case of cancer, the effective amount of the agent may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0072] As used herein, an "individual" or a "subject" refers to a mammal, including, but not limited to, human, bovine, horse, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0073] The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or function. As used herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0074] The terms "native antibody", "full length antibody," "intact antibody" and "whole antibody" are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region. Native antibodies are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V<com:Sub>H< / com:Sub>) followed by a number of constant domains. Each light chain has a variable domain at one end (V<com:Sub>L< / com:Sub>) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0075] The term "constant domain" refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the C<com:Sub>H< / com:Sub>1, C<com:Sub>H< / com:Sub>2 and C<com:Sub>H< / com:Sub>3 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0076] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0077] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs, also referred to as CDRs) both in the light-chain and the heavy- chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0078] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>") and lambda ("<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>"), based on the amino acid sequences of their constant domains.

[0079] The term IgG "isotype" or "subclass" as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.

[0800] Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called <com:Math><semantics>α<annotation encoding="application / x-tex">\alpha< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>ϵ<annotation encoding="application / x-tex">\epsilon< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0081] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0082] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')<com:Sub>2< / com:Sub> fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0083] "Fv" is the minimum antibody fragment which contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light- chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0084] The Fab fragment has two polypeptide chains, containing the heavy- and light-chain variable domains, and also containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0085] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pluckthün, The Pharmacology of Monoclonal Antibodies. Springer Berlin Heidelberg, 1994. 269-315.

[0086] The "Fc" fragment comprises the carboxy-terminal portions of both H chains held together by di-sulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[0087] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. In some embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[8800] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature 256:495-97 (1975); Hongo et al., Hybridoma 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004)), and technologies for producing human or human- like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995)).

[0089] The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATTZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.

[0090] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0091] A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and / or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage- display libraries. Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 77 (1985); Boerner et al., J. Immunol. 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

[0092] The term "hypervariable region," "HVR," or "HV," when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). In native antibodies, H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of light chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). HVR is also referred to as "CDR" or "complementarity determining region".

[0093] The structures and locations of immunoglobulin variable regions may be determined by reference to Kabat, E. A. et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (immuno.bme.nwu.edu).

[0094] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0095] As use herein, the term "binds", "specifically binds to" or is "specific for" refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that binds to or specifically binds to a target (which can be an epitope) is an antibody that binds this target with greater affinity, avidity, more readily, and / or with greater duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of <com:Math><semantics>≤1μM<annotation encoding="application / x-tex">\leq 1 \mu M< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>≤100 nM<annotation encoding="application / x-tex">\leq 100 \text{ nM}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>≤10 nM<annotation encoding="application / x-tex">\leq 10 \text{ nM}< / annotation>< / semantics>< / com:Math>, or <com:Math><semantics>≤0.1 nM<annotation encoding="application / x-tex">\leq 0.1 \text{ nM}< / annotation>< / semantics>< / com:Math>. In some embodiments, an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding.

[0096] As used herein, "Percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN<com:Sup>TM< / com:Sup> (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0097] An amino acid substitution may include but are not limited to the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table A. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. TABLE A [Image disponible dans le document PDF, Image available in the PDF document]

[0098] Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0099] As used herein, a "Fab fusion protein" refers to a protein having a Fab fragment covalently linked to one or more binding domains that have different characteristics compared to the Fab fragment. The characteristics may be biological characteristics, such as in vitro or in vivo activity. The characteristics may also be simple chemical or physical properties, such as binding to a target molecule, catalytic reactions, and the like. The Fab fragment and the one or more binding domains may be directly connected by a single peptide bond, or connected via a peptide linker, but to each other in an in-frame manner.

[0100] The term "multispecific" as used in conjunction with an antibody (such as a Fab fusion protein) refers to an antibody (such as a Fab fusion protein) having polyepitopic specificity (i.e., is capable of specifically binding to two, three, or more, different epitopes on one biological molecule or is capable of specifically binding to epitopes on two, three, or more, different biological molecules).

[0101] The term "bispecific" as used in conjunction with an antibody (such as a Fab fusion protein) refers to an antibody (such as a Fab fusion protein) capable of specifically binding to two different epitopes on one biological molecule, or capable of specifically binding to epitopes on two different biological molecules. Unless otherwise indicated, the order in which the antigens bound by a bispecific antibody are listed in a bispecific antibody or Fab fusion protein name is arbitrary. That is, the terms "anti-CD3 / EpCAM," "anti-EpCAM / CD3," "EpCAM×CD3" and "CD3×EpCAM" may be used interchangeably to refer to bispecific antibodies (such as bispecific Fab fusion proteins) that specifically bind to both CD3 and EpCAM.

[0102] The terms "multispecific Fab fusion protein" and "MSFP" are used herein interchangeably to refer to a Fab fusion protein that has polyepitopic specificity.

[0103] As used herein, the "C terminus" of a polypeptide refers to the last amino acid residue of the polypeptide which donates its amine group to form a peptide bond with the carboxyl group of its adjacent amino acid residue. "N terminus" of a polypeptide as used herein refers to the first amino acid of the polypeptide which donates its carboxyl group to form a peptide bond with the amine group of its adjacent amino acid residue.

[0104] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0105] The term "cell" includes the primary subject cell and its progeny.

[0106] The term "cytokine storm," also known as a "cytokine cascade" or "hypercytokinemia," is a potentially fatal immune reaction typically consisting of a positive feedback loop between cytokines and immune cells, with highly elevated levels of various cytokines (e.g. INF-<com:Math><semantics>γ<annotation encoding="application / x-tex">\gamma< / annotation>< / semantics>< / com:Math>, IL-10, IL-6, CCL2, etc.).

[0107] It is understood that embodiments of the invention described herein include "consisting" and / or "consisting essentially of" embodiments.

[0108] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about" X" includes description of "X".

[0109] As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

[0110] The term "about X-Y" used herein has the same meaning as "about X to about Y."

[0111] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. II. Methods of treating cancer

[0112] One aspect of the present application provides a method of treating a cancer in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain (such as scFv) is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 250 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about <com:Math><semantics>0.1μg / kg<annotation encoding="application / x-tex">0.1 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method does not induce cytokine storm.

[0113] In some embodiments, there is provided a method of killing cancer cells in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain (such as scFv) is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the tumor cell death rate mediated by the MSFP is at least about any of 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method does not induce cytokine storm. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0114] In some embodiments, there is provided a method of inhibiting proliferation of cancer cells in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain (such as scFv) is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01~\mu g / kg< / annotation>< / semantics>< / com:Math> to about <com:Math><semantics>250μg / kg<annotation encoding="application / x-tex">250~\mu g / kg< / annotation>< / semantics>< / com:Math> (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01~\mu g / kg< / annotation>< / semantics>< / com:Math> to about <com:Math><semantics>5μg / kg<annotation encoding="application / x-tex">5~\mu g / kg< / annotation>< / semantics>< / com:Math>, about <com:Math><semantics>0.1μg / kg<annotation encoding="application / x-tex">0.1~\mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method does not induce cytokine storm. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0115] In some embodiments, there is provided a method of inducing redistribution of peripheral T cells in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method does not induce cytokine storm. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0116] In some embodiments, the MSFP is a bispecific Fab fusion protein that specifically binds to CD3 and EpCAM.

[0117] In some embodiments, the MSFP specifically binds to more than two (such as 3 or more) epitopes. In some embodiments, the MSFP is a trispecific Fab fusion protein further comprising a binding domain (such as an scFv) that specifically binds to a cell surface protein, such as a tumor antigen, that is not EpCAM. In some embodiments, the MSFP comprises a multispecific (such as bispecific) binding domain.

[0118] In some embodiments, the MSFP comprise a single binding domain that specifically binds to EpCAM. In some embodiments, the binding domain comprises a single polypeptide chain. In some embodiments, the binding domain is an scFv. In some embodiments, the single binding domain is fused to the N-terminus of the heavy chain polypeptide of the Fab fragment. In some embodiments, the single binding domain is fused to the N-terminus of the light chain polypeptide of the Fab fragment.

[0119] In some embodiments, the MSFP comprises two binding domains that specifically bind to EpCAM. In some embodiments, the two binding domains target the same epitope in EpCAM. In some embodiments, the two binding domains have the same amino acid sequence. In some embodiments, the two binding domains have different amino acid sequences. In some embodiments, the two binding domains target different epitopes in EpCAM. In some embodiments, each of the two binding domains comprises a single polypeptide chain. In some embodiments, each of the two binding domains is an scFv. In some embodiments, one binding domain is fused to the N-terminus of the heavy chain polypeptide of the Fab fragment, and the other binding domain is fused to the N-terminus of the light chain polypeptide of the Fab fragment.

[0120] Thus, in some embodiments, there is provided a method of treating a cancer in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds to CD3; (2) a first binding domain (such as an scFv) that specifically binds to EpCAM; (3) and a second binding domain (such as an scFv) that specifically binds to EpCAM, wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment, wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment, and wherein the multispecific (such as bispecific) Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the first binding domain (such as scFv) and the second binding domain (such as scFv) have the same sequence. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0121] In some embodiments, there is provided a method of killing cancer cells in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds to CD3; (2) a first binding domain (such as an scFv) that specifically binds to EpCAM; (3) and a second binding domain (such as an scFv) that specifically binds to EpCAM, wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment, wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment, and wherein the multispecific (such as bispecific) Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the first binding domain (such as scFv) and the second binding domain (such as scFv) have the same sequence. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0122] In some embodiments, there is provided a method of inhibiting proliferation of cancer cells in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds to CD3; (2) a first binding domain (such as an scFv) that specifically binds to EpCAM; (3) and a second binding domain (such as an scFv) that specifically binds to EpCAM, wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment, wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment, and wherein the multispecific (such as bispecific) Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the first binding domain (such as scFv) and the second binding domain (such as scFv) have the same sequence. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0123] In some embodiments, there is provided a method of inducing redistribution of peripheral T cells in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds to CD3; (2) a first binding domain (such as an scFv) that specifically binds to EpCAM; (3) and a second binding domain (such as an scFv) that specifically binds to EpCAM, wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment, wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment, and wherein the multispecific (such as bispecific) Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the first binding domain (such as scFv) and the second binding domain (such as scFv) have the same sequence. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0124] The Fab fragment may be derived from any suitable anti-CD3 antibodies known in the art. In some embodiments, the Fab fragment specifically binds to the N-terminus of CD3 epsilon. In some embodiments, the Fab fragment specifically binds to an epitope within amino acids 1-27 of CD3 epsilon. In some embodiments, the Fab fragment is derived from SP34. In some embodiments, the Fab fragment comprises any one, two, or three HVRs (or CDRs) of the heavy chain variable region of SP34, such as HVRs comprising the amino acid sequences of SEQ ID NOs: 1-3. In some embodiments, the Fab fragment comprises any one, two, or three HVRs (or CDRs) of the light chain variable region of SP34, such as HVRs comprising the amino acid sequences of SEQ ID NOs: 4-6. In some embodiments, the Fab fragment comprises the VH of SP34, such as the VH comprising an amino acid sequence selected from SEQ ID NOs: 7 and 39- 43. In some embodiments, the Fab fragment comprises the VL of SP34, such as the VL comprising an amino acid sequence selected from SEQ ID NOs:8 and 44-47. In some embodiments, the Fab fragment comprises a human immunoglobulin heavy chain constant region 1 (CH1), such as the CH1 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the CH1 and the CL of the Fab fragment are connected by one or more disulfide bonds. In some embodiments, the Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the Fab fragment comprises a second polypeptide comprising the amino acid sequence of SEQ ID NO: 12.

[0125] Thus, in some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain (such as scFv) is fused to an N-terminus of the Fab fragment comprising a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from SEQ ID NOs: 7 and 39-43, and / or a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NOs: 8 and 44-47. In some embodiments, the Fab fragment comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and / or a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0126] In some embodiments, there is provided a method of treating a cancer in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds to CD3; wherein the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6; (2) a first binding domain (such as an scFv) that specifically binds to EpCAM; (3) and a second binding domain (such as an scFv) that specifically binds to EpCAM, wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment, wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment, and wherein the multispecific (such as bispecific) Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the first binding domain (such as scFv) and the second binding domain (such as scFv) have the same sequence. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from SEQ ID NOs: 7 and 39- 43, and / or a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NOs: 8 and 44-47. In some embodiments, the Fab fragment comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and / or a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0127] In some embodiments, the binding domain that specifically binds to EpCAM (also referred herein as EpCAM binding domain) is an antigen-binding fragment of an anti-EpCAM antibody. In some embodiments, the EpCAM binding domain is a single chain antigen-binding fragment of an anti-EpCAM antibody. In some embodiments, the EpCAM binding domain is a scFv. In some embodiments, the scFv comprises an N-VH-VL-C fusion polypeptide. In some embodiments, the EpCAM binding domain is derived from the EpCAM antibody of the present invention, comprising any one, two, or three of the HVRs of the heavy chain variable region comprising the amino acid sequences of SEQ ID NOs: 13-15.In some embodiments, the EpCAM binding domain comprises any one, two, or three of the HVRs of the light chain variable region comprising the amino acid sequences of SEQ ID NOs: 16-18. In some embodiments, the EpCAM binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the EpCAM binding domain comprises a VL comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the EpCAM binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0128] Thus, in some embodiments, there is provided a method of treating a cancer in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds to CD3 (such as the N-terminal 1-27 amino acids of CD3 epsilon); (2) a first scFv that specifically binds to EpCAM; (3) and a second scFv that specifically binds to EpCAM, wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment, wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment, wherein the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18; and wherein the multispecific (such as bispecific) Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg, or about 2.5 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg to about 100 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg). In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0129] In some embodiments, there is provided a method of treating a cancer in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds to CD3; wherein the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6; (2) a first scFv that specifically binds to EpCAM; (3) and a second scFv that specifically binds to EpCAM, wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment, wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment, wherein the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR- L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18; and wherein the multispecific (such as bispecific) Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about <com:Math><semantics>0.1μg / kg<annotation encoding="application / x-tex">0.1 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>). In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from SEQ ID NOs: 7 and 39-43, and / or a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NOs: 8 and 44-47. In some embodiments, the Fab fragment comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and / or a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0130] The EpCAM binding domain may be fused to the N-terminus of the heavy chain polypeptide and / or the N-terminus of the light chain polypeptide of the Fab fragment via a linker, such as a flexible peptide linker, for example, a peptide linker comprising glycines and serines. In some embodiments, the multispecific (such as bispecific) Fab fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the multispecific (such as bispecific) Fab fusion protein comprises a second polypeptide comprising the amino acid sequence of SEQ ID NO: 23.

[0131] Thus, in some embodiments, there is provided a method of treating a cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:23, wherein the multispecific Fab fusion protein is administered at a dose of about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 250 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about <com:Math><semantics>0.1μg / kg<annotation encoding="application / x-tex">0.1 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0132] The methods provided herein may be practiced in an adjuvant setting. In some embodiments, the method is practiced in a neoadjuvant setting, i.e., the method may be carried out before the primary / definitive therapy. In some embodiments, the method is used to treat an individual who has previously been treated. Any of the methods of treatment provided herein may be used to treat an individual who has not previously been treated. In some embodiments, the method is used as a first line therapy. In some embodiments, the method is used as a second line therapy. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0133] The methods described herein are useful for various aspects of cancer treatment. In some embodiments, there is provided a method of inhibiting cell proliferation (such as tumor growth) in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 µg / kg). In some embodiments, at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) cell proliferation is inhibited. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment; and wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N- terminus (such as N-terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0134] In some embodiments, there is provided a method of inhibiting tumor metastasis in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) metastasis is inhibited. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first scFv is fused to the N- terminus of the VH of the Fab fragment; and wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0135] In some embodiments, there is provided a method of reducing (such as eradicating) pre- existing tumor metastasis (such as metastasis to the lymph node) in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N- terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) metastasis is reduced. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment; and wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N-terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0136] In some embodiments, there is provided a method of reducing incidence or burden of preexisting tumor metastasis (such as metastasis to the lymph node) in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N- terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment; and wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N-terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0137] In some embodiments, there is provided a method of reducing tumor size in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about 0.1 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 μg / kg). In some embodiments, the tumor size is reduced at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment; and wherein the second scFv is fused to the N- terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N-terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0138] In some embodiments, there is provided a method of prolonging time to disease progression of cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg to about 100 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg). In some embodiments, the method prolongs the time to disease progression by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment; and wherein the second scFv is fused to the N- terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N-terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0139] In some embodiments, there is provided a method of prolonging survival of an individual having cancer, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 µg / kg). In some embodiments, the method prolongs the survival of the individual by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 month. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment; and wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N-terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0140] In some embodiments, there is provided a method of alleviating one or more symptoms in an individual having cancer, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment; and wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N-terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0141] The methods described herein are suitable for treating a variety of cancers, including both solid cancer and liquid cancer. The methods are applicable to cancers of all stages, including early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission. The methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy with other types of cancer therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting. In some embodiments, the cancer has been refractory to prior therapy.

[0142] Examples of cancers that may be treated by the methods of the invention include, but are not limited to, adenocortical carcinoma, AIDS-related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma, craniopharyngioma, haemangioblastomas, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, and glioblastoma), breast cancer, bronchial adenomas / carcinoids, carcinoid tumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknown primary, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing's family of tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumor, head and neck cancer, hepatocellular (liver) cancer (e.g., hepatic carcinoma and heptoma), hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), lymphoid neoplasm (e.g., lymphoma), medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneal, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma, lymphoma, primary central nervous system lymphoma (microglioma), rectal cancer, renal cancer, renal pelvis and ureter cancer (transitional cell cancer), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell cancer, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0143] In some embodiments, the method is suitable for treating cancers that overexpress EpCAM on the surface of the cancer cells, such as EpCAM-positive solid cancers. In some embodiments, the cancer cells in the individual express at least about any of more than 2, 5, 10, 20, 50, 100, 200, 500, 1000 or more fold of EpCAM compared to normal cells. In some embodiments, the EpCAM-positive solid cancer is a carcinoma or adenocarcinoma. In some embodiments, the EpCAM-positive solid cancer is selected from the group consisting of small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer, renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

[0144] Thus, in some embodiments, there is provided a method of treating an EpCAM-positive solid cancer (such as carcinoma or adenocarcinoma) in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 250 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about <com:Math><semantics>0.1μg / kg<annotation encoding="application / x-tex">0.1 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg, or about 2.5 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg to about 100 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the EpCAM-positive solid cancer is selected from the group consisting of small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer, renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

[0145] In some embodiments, there is provided a method of treating an EpCAM-positive solid cancer (such as carcinoma or adenocarcinoma) in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds to CD3; (2) a first binding domain (such as an scFv) that specifically binds to EpCAM; (3) and a second binding domain (such as an scFv) that specifically binds to EpCAM, wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment, wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment, and wherein the multispecific (such as bispecific) Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about <com:Math><semantics>0.1μg / kg<annotation encoding="application / x-tex">0.1 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>). In some embodiments, the first binding domain (such as scFv) and the second binding domain (such as scFv) have the same sequence. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the EpCAM-positive solid cancer is selected from the group consisting of small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer, renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

[0146] In some embodiments, there is provided a method of treating an EpCAM-positive solid cancer (such as carcinoma or adenocarcinoma) in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain (such as scFv) is fused to an N- terminus of the Fab fragment comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from SEQ ID NOs: 7 and 39-43, and / or a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NOs: 8 and 44-47. In some embodiments, the Fab fragment comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and / or a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the EpCAM- positive solid cancer is selected from the group consisting of small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer, renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

[0147] In some embodiments, there is provided a method of treating an EpCAM-positive solid cancer (such as carcinoma or adenocarcinoma) in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds to CD3 (such as the N- terminal 1-27 amino acids of CD3 epsilon); (2) a first scFv that specifically binds to EpCAM; (3) and a second scFv that specifically binds to EpCAM, wherein the first scFv is fused to the N- terminus of the VH of the Fab fragment, wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment, wherein the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18; and wherein the multispecific (such as bispecific) Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti- EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the EpCAM-positive solid cancer is selected from the group consisting of small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer, renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

[0148] In some embodiments, there is provided a method of treating an EpCAM-positive solid cancer (such as carcinoma or adenocarcinoma) in an individual (such as a human), comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds to CD3; wherein the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6; (2) a first scFv that specifically binds to EpCAM; (3) and a second scFv that specifically binds to EpCAM, wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment, wherein the second scFv is fused to the N- terminus of the VL of the Fab fragment, wherein the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18; and wherein the multispecific (such as bispecific) Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from SEQ ID NOs: 7 and 39-43, and / or a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NOs: 8 and 44-47. In some embodiments, the Fab fragment comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and / or a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the EpCAM-positive solid cancer is selected from the group consisting of small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer, renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

[0149] In some embodiments, there is provided a method of treating an EpCAM-positive solid cancer (such as carcinoma or adenocarcinoma) in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22 and a second polypeptide comprising the amino acid sequence of SEQ ID NO:23, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the EpCAM-positive solid cancer is selected from the group consisting of small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer, renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

[0150] In some embodiments, there is provided a method of treating small intestine cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about 0.1 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first scFv is fused to the N-terminus of the VH of the Fab fragment; and wherein the second scFv is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N-terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR- L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0151] In some embodiments, there is provided a method of treating colorectal cancer (such as colorectal adenocarcinoma) in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 μg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N-terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the colorectal cancer is adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, leiomysarcoma, melanoma, or squamous cell carcinoma.

[0152] In some embodiments, there is provided a method of treating lung cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about 0.1 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the lung cancer is a non-small cell lung cancer (NSCLC). Examples of NSCLC include, but are not limited to, large-cell carcinoma, adenocarcinoma, neuroendocrine lung tumors, and squamous cell carcinoma.

[0153] In some embodiments, there is provided a method of treating cervical cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual.

[0154] In some embodiments, there is provided a method of treating liver cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the liver cancer is liver cell carcinoma, fibrolamellar variants of hepatocellular carcinoma, or mixed hepatocellular cholangiocarcinoma.

[0155] In some embodiments, there is provided a method of treating gastric cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the gastric cancer is an adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumor, squamous cell carcinoma, small cell carcinoma, or leiomyosarcoma.

[0156] In some embodiments, there is provided a method of treating pancreatic cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about 0.1 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the pancreatic cancer is a serous cystic neoplasm, mucinous cystic neoplasm, intraductal papillary mucinous neoplasm, pancreatic adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, signet ring cell carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with giant cells, solid pseudopapillary neoplasm, ampullary cancer, or pancreatic neuroendocrine tumor. In some embodiments, the pancreatic cancer is a pancreatic adenocarcinoma.

[0157] In some embodiments, there is provided a method of treating skin cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about 0.1 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the skin cancer is melanoma. In some embodiments, the melanoma is superficial spreading melanoma, lentigo maligna melanoma, nodular melanoma, mucosal melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft-tissue melanoma, or acral lentiginous melanoma.

[0158] In some embodiments, there is provided a method of treating renal cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about 0.1 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the renal cell carcinoma is an adenocarcinoma. In some embodiments, the renal cell carcinoma is a clear cell renal cell carcinoma, papillary renal cell carcinoma (also called chromophilic renal cell carcinoma), chromophobe renal cell carcinoma, collecting duct renal cell carcinoma, granular renal cell carcinoma, mixed granular renal cell carcinoma, renal angiomyolipomas, or spindle renal cell carcinoma.

[0159] In some embodiments, there is provided a method of treating bladder cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the bladder cancer is a low grade bladder cancer. In some embodiments, the bladder cancer is a high grade bladder cancer. In some embodiments, the bladder cancer is muscle invasive (e.g., T2, T3 or T4). In some embodiments, the bladder cancer is non-invasive (e.g., Ta, T1 Cis, Cis with Ta and / or T1). In some embodiments, the bladder cancer is transitional cell carcinoma or urothelial carcinoma (such as metastatic urothelial carcinoma), including, but not limited to, papillary tumors and flat carcinomas. In some embodiments, the bladder cancer is squamous cell carcinoma, non-squamous cell carcinoma, adenocarcinoma, or small cell carcinoma.

[0160] In some embodiments, there is provided a method of treating thyroid cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the thyroid cancer is papillary carcinoma, follicular carcinoma, Hurthle cell carcinoma, medullary thyroid carcinoma, anaplastic carcinoma, thyroid lymphoma, thyroid sarcoma, or parathyroid cancer.

[0161] In some embodiments, there is provided a method of treating prostate cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about 0.1 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the prostate cancer is an adenocarcinoma. In some embodiments, the prostate cancer is a sarcoma, neuroendocrine tumor, small cell cancer, ductal cancer, or a lymphoma.

[0162] In some embodiments, there is provided a method of treating ovarian cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about 0.1 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method does not induce cytokine storm. In some embodiments, the ovarian cancer is ovarian epithelial cancer.

[0163] In some embodiments, there is provided a method of treating endometrial cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about 0.1 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the endometrial cancer is adenocarcinoma, carcinosarcoma, squamous cell carcinoma, undifferentiated carcinoma, small cell carcinoma, or transitional carcinoma.

[0164] In some embodiments, there is provided a method of treating breast cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μg / kg to about 250 μg / kg (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about 0.1 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the breast cancer is early stage breast cancer, non-metastatic breast cancer, advanced breast cancer, stage IV breast cancer, locally advanced breast cancer, metastatic breast cancer, breast cancer in remission, breast cancer in an adjuvant setting, or breast cancer in a neoadjuvant setting. In some embodiments, the breast cancer is fibroadenoma, or intraductal papilloma. In some embodiments, the breast cancer is HER2 positive or HER2 negative. In some embodiments, the breast cancer is a triple negative breast cancer.

[0165] In some embodiments, there is provided a method of treating bile duct cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math> to about 5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, about 0.1 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, or about 2.5 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math> to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the bile duct cancer is an intrahepatic bile duct cancer, a perihilar bile duct cancer, a distal bile duct cancer. In some embodiments, the bile duct cancer is a cholangiocarcinoma, sarcoma, lymphoma, or small cell cancer.

[0166] In some embodiments, there is provided a method of treating head and neck cancer in an individual, comprising administering to the individual an effective amount of a multispecific (such as bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds to CD3, and a binding domain (such as scFv) that specifically binds to EpCAM; wherein the binding domain is fused to an N-terminus of the Fab fragment; and wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 µg / kg to about 250 µg / kg (such as about 0.01 μg / kg to about 5 μg / kg, about 0.1 μg / kg to about 30 μg / kg, or about 2.5 μg / kg to about 100 μg / kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibits proliferation of cancer cells; and (3) induces redistribution of peripheral T cells. In some embodiments, the multispecific Fab fusion protein comprises a first scFv that specifically binds to EpCAM, and a second scFv that specifically binds to EpCAM; wherein the first binding domain (such as scFv) is fused to the N-terminus of the VH of the Fab fragment; and wherein the second binding domain (such as scFv) is fused to the N-terminus of the VL of the Fab fragment. In some embodiments, the first scFv and the second scFv have the same sequence. In some embodiments, the Fab fragment binds to the N-terminus (such as N- terminal 1-27 amino acids) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the first scFv and / or the second scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein is administered intravenously. In some embodiments, the multispecific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering a glucocorticoid (such as dexamethasone) to the individual. In some embodiments, the head and neck cancer is a squamous cell carcinoma in the head and neck. In some embodiments, the head and neck cancer is a hypopharyngeal cancer, laryngeal cancer, lip and oral cavity cancer, metastatic squamous neck cancer with occult primary, nasopharyngeal cancer, or opharyngeal cancer, paranasal sinus and nasal cavity cancer, or salivary gland cancer.

[0167] Exemplary routes of administration of the multispecific Fab fusion protein (MSFP) include, but are not limited to, oral, intravenous, intracavitary, intratumoral, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, ocular, topical, intraperitoneal, intracranial, intrapleural, and epidermal routes, or be delivered into lymph glands, body spaces, organs or tissues known to contain cancer cells. In some embodiments, the MSFP is administered intravenously. In some embodiments, the MSFP is administered by infusion. In some embodiments, the MSFP is administered subcutaneously. In some embodiments, the MSFP is administered by injection.

[0168] In some embodiments, the MSFP is administered by intravenous infusion. In some embodiments, the MSFP is infused to the individual over a period of time no more than about any of 24 hours, 20 hours, 15 hours, 10 hours, 8 hours, 6 hours, 3 hours, 2 hours, 1 hours, 30 minutes, or less. In some embodiments, the MSFP is infused to the individual over a period of time of any one of about 30 minutes to about 1 hour, about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to about 10 hours, about 10 hours to about 12 hours, about 12 hours to about 18 hours, about 18 hours to about 24 hours, about 30 minutes to about 2 hours, about 2 hours to about 5 hours, about 5 hours to about 10 hours, about 10 hours to about 20 hours, about 30 minutes to about 10 hours, or about 30 minutes to about 20 hours. The MSFP may be infused to the individual at any suitable rate. In some embodiments, the MSFP may be infused at a rate more than about any of 0.01 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg / hr, 0.02 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg / hr, 0.05 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg / hr, 0.1 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg / hr, 0.2 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg / hr, 0.5 μg / kg / hr, 0.6 μg / kg / hr, 0.7 μg / kg / hr, 0.8 μg / kg / hr, 0.9 μg / kg / hr, 1 μg / kg / hr, 1.5 μg / kg / hr, 2 μg / kg / hr, 3 μg / kg / hr, 4 μg / kg / hr, 5 μg / kg / hr, 10 μg / kg / hr, 15 μg / kg / hr, 20 μg / kg / hr, 25 μg / kg / hr, 50 μg / kg / hr, 75 μg / kg / hr, 100 μg / kg / hr or more.

[0169] The dosing regimen of the MSFPs administered to the individual may vary with the particular MSFP composition, the method of administration, and the particular type and stage of cancer being treated. In some embodiments, that effective amount of the MSFP is below the level that induces a toxicological effect (i.e., an effect above a clinically acceptable level of toxicity) or is at a level where a potential side effect can be controlled or tolerated when the composition is administered to the individual.

[0170] In some embodiments, the effective amount of the MSFP is below the level that induces an adverse effect in the central nervous system. For example, an adverse effect observed in antibody therapy is the occurrence of infusion-related side effects, such as the cytokine release syndrome ("CRS"), the severe cases of which are known as "cytokine storms". When a "cytokine storm" is induced, the healthy individual's immune system is activated and releases large amounts of the pro-inflammatory cytokines, such as INF-γ, CCL2, IIL-10, IL-6, etc. It is a potentially fatal immune reaction typically consisting of a positive feedback loop between cytokines and immune cells, with highly elevated levels of various cytokines. Other adverse side effects described to be associated with CRS are fatigue, vomiting, tachycardia, hypertension, back pain, but also central nervous system reactions (CNS reactions), such as seizures, encephalopathy, cerebral edema, aseptic meningitis, and headache. In some embodiments, the MSFP is administered at a dose that does not induce cytokine release syndrome, such as cytokine storm. In some embodiments, the MSFP is administered at a dose that does not induce significant release of one or more cytokines selected from the group consisting of IL-2, IL-4, IL-5, IL-6, TNF, and INF-γ. In some embodiments, a significant release of a cytokine is sustained release of a cytokine over the course of at least about any of 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, or more. In some embodiments, a significant release of a cytokine is a serum or blood level of a cytokine at a concentration of at least about any of 1, 5, 10, 20, 50, 100, 200, 500, 1000 or more pg / mL. Without being bound by any theory, the MSFPs described herein require binding to EpCAM on the target tumor cell in order to recruit and activate T cells. Such requirement can greatly reduce unwanted cytokine storms, and unwanted activation of T cells in the absence of the desired target tumor cell.

[0171] In some embodiments, the MSFP is administered at a dose of no more than about any one of 0.01 μg / kg, 0.05 μg / kg, 0.1 μg / kg, 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 5 μg / kg, 10 μg / kg, 15 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, 20 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, 25 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, 30 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, 50 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, 100 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, 150 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, 200 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, 250 <com:Math><semantics>μg / kg<annotation encoding="application / x-tex">\mu g / kg< / annotation>< / semantics>< / com:Math>, 300 μg / kg, 400 μg / kg, 500 μg / kg, 600 μg / kg, 700 μg / kg, 800 μg / kg, 900 μg / kg, or 1 mg / kg. In some embodiments, the dose of the MSFP is within any of the following range, wherein the ranges have an upper limit of any of: 0.05 µg / kg, 0.1 µg / kg, 0.5 µg / kg, 1 µg / kg, 2 µg / kg, 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg , 30 μg / kg, 50 μg / kg, 100 μg / kg, 150 μg / kg, 200 μg / kg, 250 μg / kg, 300 μg / kg, 400 μg / kg, 500 μg / kg, 600 μg / kg, 700 μg / kg, 800 μg / kg, 900 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg, or 1 mg / kg, and an independently selected lower limit of any of 0.01 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg, 0.05 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg, 0.1 μg / kg, 0.5 μg / kg, 1 μg / kg, 2 μg / kg, 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 50 μg / kg, 100 μg / kg, 150 μg / kg, 200 μg / kg, 250 μg / kg, 300 μg / kg, 400 μg / kg, 500 μg / kg, <com:Math><semantics>600μg / kg<annotation encoding="application / x-tex">600 \mu g / kg< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>700μg / kg<annotation encoding="application / x-tex">700 \mu g / kg< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>800μg / kg<annotation encoding="application / x-tex">800 \mu g / kg< / annotation>< / semantics>< / com:Math>, or <com:Math><semantics>900μg / kg<annotation encoding="application / x-tex">900 \mu g / kg< / annotation>< / semantics>< / com:Math>, and wherein the lower limit is less than the upper limit. In some embodiments, the MSFP is administered at a dose of any one of about 0.01 μg / kg to about 0.05 μg / kg, about 0.05 μg / kg to about 0.1 μg / kg, about 0.1 μg / kg to about 0.5 μg / kg, about 0.5 μg / kg to about 1 μg / kg, about 0.01 μg / kg to about 0.1 μg / kg, about 0.1 μg / kg to about 1 μg / kg, about 1 μg / kg to about 5 μg / kg, about 5 μg / kg to about 10 μg / kg, about 10 μg / kg to about 15 μg / kg, about 15 μg / kg to about 20 μg / kg, about 20 μg / kg to about 25 μg / kg, about 25 μg / kg to about 30 μg / kg, about 5 μg / kg to about 15 μg / kg, about 10 μg / kg to about 30 μg / kg, about 30 μg / kg to about 50 μg / kg, about 50 μg / kg to about 100 μg / kg, about 0.01 μg / kg to about 1 μg / kg, about 0.01 μg / kg to about 5 μg / kg, about 0.01 μg / kg to about 30 μg / kg, about 0.01 μg / kg to about 250 μg / kg, about 0.1 μg / kg to about 10 μg / kg, about 0.1 μg / kg to about 30 μg / kg, about 0.1 μg / kg to about 250 μg / kg, about 1 μg / kg to about 10 μg / kg, about 1 μg / kg to about 20 μg / kg, about 1 μg / kg to about 30 μg / kg, about 1 μg / kg to about 250 μg / kg, about 100 μg / kg to about 250 μg / kg, about 5 μg / kg to about 250 μg / kg, about 250 μg / kg to about 500 μg / kg, about 500 μg / kg to about 1000 μg / kg, or about 0.01 μg / kg to about 1000 μg / kg. The doses described herein may refer to a suitable dose for cynomolgus monkeys, a human equivalent dose thereof, or an equivalent dose for the specific species of the individual. In some embodiments, the MSFP is administered at a dose equivalent to about 0.1 μg / kg to about 100 μg / kg (such as such as about 0.3 μg / kg to about 5 μg / kg, or about 5 μg / kg to about 20 μg / kg) for a cynomolgus monkey. In some embodiments, the MSFP is administered at a dose equivalent to no more than about 30 μg / kg (such as no more than about 20, 15, or 10 μg / kg) for a cynomolgus monkey.

[0172] In some embodiments, the MSFP is administered at a dose of about 0.1 µg / kg to about <com:Math><semantics>10μg / kg<annotation encoding="application / x-tex">10 \mu g / kg< / annotation>< / semantics>< / com:Math>, such as about any one of 0.3, 0.5, 0.6, 1, 1.2, 2, 2.4, 3.6, or <com:Math><semantics>4μg / kg<annotation encoding="application / x-tex">4 \mu g / kg< / annotation>< / semantics>< / com:Math>.

[0173] The effective amount of the MSFP may be administered in a single dose or in multiple doses. For methods that comprises administration of the MSFP in multiple doses, exemplary dosing frequencies include, but are not limited to, daily, daily without break, weekly, weekly without break, weekly for two out of three weeks, weekly for three out of four weeks, once every three weeks, once every two weeks, monthly, every six months, yearly, etc. In some embodiments, the MSFP is administered about once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 8 weeks. In some embodiments, the MSFP is administered at least about any of 1x, 2x, 3x, 4x, 5x, 6x, or 7x (i.e., daily) a week. In some embodiments, the intervals between each administration are less than about any of 3 years, 2 years, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the intervals between each administration are more than about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years. In some embodiments, there is no break in the dosing schedule.

[0174] In some embodiments, the MSFP is administered at a first dose for a first period of time, and consecutively the MSFP is administered at a second dose for a second period of time, wherein the second dose exceeds the first dose. The first period of time and the second period of time may be of any suitable length, including for example about any one of 1, 2, 3, 4, 5, 6, or more weeks. In some embodiments, the second period of time exceeds the first period of time. N some embodiments, the first period of time is at least about 7 days. In some embodiments, the second period of time is at least about any one of 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or more. The first dose and the second dose can be of any suitable dose as described above. In some embodiments, the first dose is no more than about any one of 2, 1.5, 1, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 µg / kg or less. In some embodiments, the second dose is about 0.1 µg / kg to about 10 μg / kg, such as about 0.3 μg / kg to about 5 μg / kg. In some embodiments, the second dose is about any one of 0.3, 0.6, 1.2, 2.4, or 3.6 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

[0175] In some embodiments, the MSFP is administered at a low frequency, for example, any one of no more frequent than once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, once per 7 months, once per 8 months, once per 9 months, once per 10 months, once per 11 months, once per year, once per 18 months, once per two years, once per three years, or less. In some embodiments, the MSFP is administered in a single dose. In some embodiments, the MSFP is administered twice weekly.

[0176] The administration of the MSFP can be extended over an extended period of time, such as from 1 day to about a week, from about a week to about a month, from about a month to about a year, from about a year to about several years. In some embodiments, the MSFP is administered over a period of at least any of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or more. [01 / 7 / 7] In some embodiments, the method further comprises administration of one or more glucocorticoids. Glucocorticoids (GC) are a class of steroid hormones that bind to the glucocorticoid receptor (GR), which is present in almost every vertebrate animal cell, including humans. These compounds are potent anti-inflammatory agents, regardless of the inflammation's cause. In some embodiments, the glucocorticoid suppresses release of one or more cytokines selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8 and IFN-γ. Suitable glucocorticoids include, but are not limited to, cortisone, hydrocortisone, cortisol, cloprednol, prednisone, prednisolone, methylprednisolone, deflazacort, fludrocortisone, triamcinolone, dexamethasone, and betamethasone. In some embodiments, the glucocorticoid is selected from the group consisting of cortisone, hydrocortisone, cortisol, cloprednol, prednisone, prednisolone, methylprednisolone, deflazacort, fludrocortisone, triamcinolone, dexamethasone, betamethasone and a pharmaceutically acceptable ester, salt, or complex of thereof. In some embodiments, the glucocorticoid is dexamethasone. In some embodiments, the glucocorticoid is a pharmaceutically acceptable ester, salt, or complex of dexamethasone.

[0178] The glucocorticoid may be administered to the individual simultaneously with the MSFP or prior to the administration of the MSFP. The glucocorticoid may be administered at a time no longer than about any one of 3 hours, 2 hours, 1 hours, 30 minutes, or less from the administration of the MSFP. In some embodiments, the glucocorticoid is administered simultaneously or prior to each dose of the MSFP. In some embodiments, the glucocorticoid is administered simultaneously or prior to the first dose of the MSFP. In some embodiments, wherein the MSFP is administered at a first dose for a first period of time, and consecutively the MSFP is administered at a second dose for a second period of time, the glucocorticoid is administered prior (such as about 1 hour prior) to the first administration of the MSFP for the first period of time, and the glucocorticoid is administered prior (such as about 1 hour prior) to the first administration of the MSFP for the second period of time. In some embodiments, the glucocorticoid is administered prior to the administration of the MSFP when the individual has an elevated liver enzyme level (such as ALT, TBil, and / or ALP) and / or an elevated cytokine level (such as IL-6). The glucocorticoid may be administered at any suitable dosage, including, for example, at least about any one of 0.1, 0.5, 1, 2, 3, 4, 5 mg / kg or more. In some embodiments, the glucocorticoid is administered at a dose of at least about any one of 1, 2, 5, 10, 15, 20, 25 mg or more.

[0179] In some embodiments, the glucocorticoid is dexamethasone. In some embodiments, the method comprises administering dexamethasone to the individual prior to the administration of the first dose of the MSFP. In some embodiments, the dexamethasone is administered at a dose of about 0.1 mg / kg to about 5 mg / kg. Multispecific Fab Fusion Proteins

[0180] The multispecific Fab fusion proteins (MSFP) used in the methods described herein comprise an anti-CD3 Fab fragment fused to a first EpCAM binding domain at the N-terminal end of the VH of the Fab fragment and / or a second EpCAM binding domain at the N-terminal end of the VL of the Fab fragment. In some embodiments, the EpCAM binding domain is an anti-EpCAM scFv. In some embodiments, the EpCAM binding domain is connected to the VH or VL of the anti-CD3 Fab fragment via a linker. An exemplary bispecific Fab fusion protein useful for methods of the present invention is shown in FIG. 1. Fab fragment

[0181] The Fab fragment of the MSFPs described herein specifically binds to CD3, such as human CD3. "CD3" is known in the art as a multi-protein complex of six chains (see, Abbas and Lichtman, 2003; Janeway et al., p172 and 178, 1999). In mammals, the complex comprises a CD3 gamma chain, a CD3 delta chain, two CD3 epsilon chains, and a homodimer of CD3 zeta chains. The CD3 gamma, CD3 delta, and CD3 epsilon chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3 gamma, CD3 delta, and CD3 epsilon chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3 gamma, CD3 delta, and CD3 epsilon chains each contain a single conserved motif known as an immunoreceptor tyrosine- based activation motif or ITAM, whereas each CD3 zeta chain has three. Without being bound by theory, it is believed the ITAMs are important for the signaling capacity of a TCR complex. CD3 as used herein may be from various animal species, including human, primate, mouse, rat, or other mammals.

[0182] In some embodiments, the Fab fragment of the MSFP specifically binds to an individual CD3 chain, such as CD3 gamma chain, CD3 delta chain, or CD3 epsilon chain. In some embodiments, the Fab fragment specifically binds to a complex formed from two or more individual CD3 chains (e.g., a complex of more than one CD3 epsilon chains, a complex of a CD3 gamma and CD3 epsilon chain, a complex of a CD3 delta and CD3 epsilon chain). In some embodiments, the Fab fragment specifically binds to a CD3 epsilon chain. In some embodiments, the Fab fragment specifically binds to the N-terminus of CD3 epsilon. In some embodiments, the Fab fragment specifically binds to amino acids 1-27 of CD3 epsilon.

[0183] The Fab fragments can be generated by a variety of methods known in the art (see, e.g., U.S. Pat. Nos. 6,291,161; 6,291,158). Sources of Fabs include monoclonal antibody or antigen- binding fragments thereof from various species, including human, camelid (from camels, dromedaries, or llamas; Hamers-Casterman et al. (1993) Nature, 363:446 and Nguyen et al. (1998) J. Mol. Biol., 275:413), shark (Roux et al. (1998) Proc. Nat'l. Acad. Sci. (USA) 95:11804), fish (Nguyen et al. (2002) Immunogenetics, 54:39), rodent, avian, or ovine. In some embodiments, the Fab fragment is derived from a human or humanized antibody.

[0184] In some embodiments, the Fab fragment specifically binds to both human and non- human primates (such as cynomolgus monkey) CD3. Exemplary anti-human CD3 antibody with cross reactivity to monkey CD3 include, but are not limited to, SP34 mouse monoclonal antibody, (see, for example, Pressano, S. The EMBO J. 4:337-344, 1985; Alarcon, B. EMBO J. 10:903-912, 1991; Salmeron A. et al., J. Immunol. 147:3047-52, 1991; Yoshino N. et al., Exp. Anim 49:97-110, 2000; Conrad M L. et al., Cytometry 71A:925-33, 2007; and Yang et al., J. Immunol. 137:1097-1100: 1986). MSFPS having anti-CD3 Fab fragments with cross-reactivity to monkey CD3 may facilitate toxicity studies in non-human primates, which can provide more relevant safety assessments for human clinical trial candidates, without having to perform toxicity studies in chimpanzees or using surrogate molecules.

[0185] In some embodiments, the Fab fragment is derived from an anti-CD3 antibody that does not have cross-reactivity to non-human primates. Exemplary anti-CD3 antibodies include the Cris-7 monoclonal antibody (Reinherz, E. L. et al. (eds.), Leukocyte typing II, Springer Verlag, New York, (1986)), BC3 monoclonal antibody (Anasetti et al. (1990) J. Exp. Med. 172:1691), OKT3 (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med. 313:337) and derivatives thereof such as OKT3 ala-ala (Herold et al. (2003) J. Clin. Invest. 11:409), visilizumab (Carpenter et al. (2002) Blood 99:2712), and 145-2C11 monoclonal antibody (Hirsch et al. (1988) J. Immunol. 140: 3766). Further CD3 binding molecules contemplated herein include UCHT-1 (Beverley, P C and Callard, R. E. (1981) Eur. J. Immunol. 11: 329-334) and CD3 binding molecules described in WO2004 / 106380; WO2010 / 037838; WO2008 / 119567; WO2007 / 042261; WO2010 / 0150918.

[0186] In some embodiments, the Fab fragment comprises one constant and one variable region of an immunoglobulin heavy, and one constant and one variable region of an immunoglobulin light chain. In some embodiments, the heavy chain constant and variable regions heterodimerize with the light chain variable and constant regions, and are covalently linked by a disulfide bond between the heavy and light chain constant regions. In some embodiments, the Fab fragment has the basic structure NH<com:Sub>2< / com:Sub>-VL-CL-S-S-CH<com:Sub>1< / com:Sub>-VH-NH<com:Sub>2< / com:Sub>. In some embodiments, the CH1 and the CL of the Fab fragment are connected by one or more disulfide bonds. In some embodiments, the number of disulfide bonds between the first constant region of heavy chain (CH1) and the light constant (CL) of the Fab is at least one, such as 2, 3, 4, or more. In some embodiments, cysteine residues are engineered in the Fab fragment (such as in the CH1 and CL regions) to introduce disulfide bonds.

[0187] In some embodiments, the Fab fragment of the MSFP does not comprise a disulfide bond. For example, the heavy and light chains may be engineered in such a way so as to stably interact without the need for disulfide bonds. In some embodiments, the heavy chain or light chain can be engineered to remove a cysteine residue, and wherein the heavy and light chains still stably interact and function as a Fab. In some embodiments, mutations are made to facilitate stable interactions between the heavy and light chains. For example, a "knobs into holes" engineering strategy can be used to facilitate dimerization between the heavy and light chains of a Fab (see e.g., 1996 Protein Engineering, 9:617 - 621). Also contemplated for use herein are variant Fab fragments designed for a particular purpose, for example, amino acid changes in the constant domains of CH1 and / or CL, and removal of a disulfide bond or addition of tags for purification, etc.

[0188] In some embodiments, the configuration of the variable and constant regions within the Fab fragment may be different from what is found in a native Fab. In some embodiments, the orientation of the variable and constant regions may be VH-CL in one chain, and VL-CH1 in another chain (see, for example, Shaefer et al. (2011), PNAS, 108:111870-92).

[0189] In some embodiments, the Fab fragments of the MSFP are derived from monoclonal antibodies. Suitable monoclonal antibodies may be of any type, including IgA, IgM, IgD, IgG, IgE and subtypes thereof, such as IgG1, IgG2, IgG3, and IgG4. The light chain domains may be derived from the kappa or lambda chain. In some embodiments, the Fab fragment is designed recombinantly.

[0190] In some embodiments, the Fab fragment comprises a human immunoglobulin CH1. In some embodiments, the human immunoglobulin CH1 comprises the amino acid sequence of SEQ ID NO: 9. In some embodiment, the Fab fragment comprises a human lambda light chain constant region. In one embodiment, the human lambda light chain constant region comprises the amino acid sequence of SEQ ID NO:10. SEQ ID NO: 9 (human CH1) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS SEQ ID NO: 10 (human lambda CL) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTE

[0191] The Fab fragment of the MSFP specifically binds to CD3 via an antigen-binding site formed between the heavy chain variable region and the light chain variable region (VH and VL). The antigen-binding site comprises at least one (such as 1, 2, or 3) HVR of an immunoglobulin heavy chain and / or at least one (such as 1, 2, or 3) HVR of an immunoglobulin light chain. In some embodiments, the MSFP comprises 1, 2, 3, 4, 5, or all 6 HVRs of a VH and VL sequence of a full-length antibody that specifically binds to CD3.

[0100] In some embodiments, the Fab fragment is derived from SP34. In some embodiments, the Fab fragment is a CD3 Fab fragment described in U.S. Patent No. 8,846,042. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising one, two or three HVRs (or CDRs) from SEQ ID NO: 7, and / or a light chain variable region (VL) comprising one, two or three HVRs (or CDRs) from SEQ ID NO: 8. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising three HVRs from SEQ ID NO: 7, and / or a light chain variable region (VL) comprising three HVRs from SEQ ID NO: 8. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising one, two or three HVRs selected from SED ID Nos: 1-3, and / or a light chain variable region (VL) comprising one, two or three HVRs selected from SED ID NOs: 4-6. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a sequence selected from the group consisting of SEQ ID NOs:7 and 39-43. In some embodiments, the Fab fragment comprises a light chain variable region (VL) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to a sequence selected from the group consisting of SEQ ID NO: 8 and 44-47. In some embodiments, a V<com:Sub>H< / com:Sub> or VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the Fab fragment comprising that sequence retains the ability to bind to CD3. In some embodiments, one or two amino acids have been substituted, inserted and / or deleted in any one or more of the HVRs. In some embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from SEQ ID NOs: 7 and 39-43. In some embodiments, the Fab fragment comprises a light chain variable region (VL) comprising an amino acid sequence selected from SEQ ID NOs: 8 and 44-47. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7, and / or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the Fab fragment comprises a heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and / or a light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 12.

[0192] SEQ ID NO: 1 (CD3 HVR-H1) TYAMN SEQ ID NO: 2 (CD3 HVR-H2) RIRSKYNNYATYYADSVKD SEQ ID NO: 3 (CD3 HVR-H3) HGNFGNSYVSWFAY SEQ ID NO: 4 (CD3 HVR-L1) RSSTGAVTTSNYAN SEQ ID NO: 5 (CD3 HVR-L2) GTNKRAP SEQ ID NO: 6 (CD3 HVR-L3) ALWYSNLWV SEQ ID NO: 7 (CD3 VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN YATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAY WGQGTMVTVSS SEQ ID NO: 8 (CD3 VL) QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAP GTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL SEQ ID NO: 11 (CD3 Heavy chain) EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNN YATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAY WGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS SEQ ID NO: 12 (CD3 Light chain) QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAP GTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAP SVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKY AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTE SEQ ID NO: 39 (CD3 VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNY ATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYW GQGTMVTVSS SEQ ID NO: 40 (CD3 VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVSRIRSKYNN YATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAY WGQGTMVTVSS SEQ ID NO: 41 (CD3 VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNN YATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAY WGQGTMVTVSS SEQ ID NO: 42 (CD3 VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNN YATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAY WGQGTMVTVSS SEQ ID NO: 43 (CD3 VH) EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNN YATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAY WGQGTLVTVSS SEQ ID NO: 44 (CD3 VL) QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRGLIGGTNKRAPG TPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL SEQ ID NO: 45 (CD3 VL) QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAP WTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL SEQ ID NO: 46 (CD3 VL) QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRGLIGGTNKRAP WTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL SEQ ID NO: 47 (CD3 VL) QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWYQQKPGQAPRGLIGGTNKRAP WTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

[0193] In some embodiments, a specific VH and / or VL of an anti-CD3 Fab fragment may be used to screen a library of the complementary variable region to identify VH / VL with desirable properties, such as increased affinity for CD3. Such methods are described, for example, in Portolano et al., J. Immunol. (1993) 150:880-887; Clarkson et al., Nature (1991) 352:624-628; and Klimka et al., British Journal of Cancer (2000) 83:252-260; Beiboer et al., J. Mol. Biol. (2000) 296:833-849; and Rader et al., PNAS (1998) 95:8910-8915. EpCAM binding domain

[0194] The MSFPs described herein comprises one or two binding domains that specifically bind to EpCAM. Epithelial cell adhesion molecule (EpCAM, CD326), also known as 17-1A, ESA, AUA1, EGP40, etc., is a 40 kD transmembrane glycoprotein composed of 314 amino acid. EpCAM is involved in cell signaling, migration, proliferation, and differentiation. EpCAM is specifically expressed in various types of epithelial cells and main types of human malignancies. For example, EpCAM is highly expressed in colon cancer, lung cancer, prostate cancer, liver cancer, pancreatic cancer, breast cancer and ovarian cancer, thus can be used as diagnostic marker for various cancers. EpCAM is also a potential target for immunotherapeutic strategies, including vaccines, murinized or humanized monoclonal antibodies, and antibodies conjugated with bacterial toxins or chemotherapy drugs, such as EpCAM specific antibodies ING-1, adecatumumab, edrecolomab, etc.

[0195] The binding domains, including the EpCAM binding domains, in the MSFP not only provide additional binding specificities and enhanced properties (e.g., increased serum half-life, or activation of immune activation cascades), but also create steric hindrance to significantly reduce the binding affinity of the Fab fragment to CD3 due to fusion to the N terminus of the VH and / or VL chains. This is in direct contrast to other Fab fusion proteins, such as TRIBODIES<com:Sup>TM< / com:Sup>, which fuses additional binding domains at the C terminus of the Fab fragment (see, e.g., Journal of Immunology, 2000, 165: 7050-7057). The binding domains are not intended to dimerize, unlike other known fusion proteins, such as those described in WO2008 / 024188 and WO2009 / 149185. A further distinguishing characteristic of the MSFP is that the binding domains reduce the binding affinity of the Fab to CD3 when the MSFP is not bound to cell surface targets, such as EpCAM, on tumor cells.

[0196] In some embodiments, the MSFP has a prolonged in vivo half-life compared to the anti- CD3 Fab alone. In some embodiments, the half-life of the MSFP is at least about any of 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times the half-life of the anti-CD3 Fab fragment alone.

[0197] The binding domains, including EpCAM, may be selected from antigen-binding domains, such as scFv or scTCR, extracellular domains of receptors, ligands for cell surface molecules / receptors, or receptor binding domains thereof, and tumor binding proteins. In some embodiments, the antigen binding domain is selected from an scFv, a VH, a VL, a domain antibody variant (dAb), a camelid antibody (VHH), a fibronectin 3 domain variant, an ankyrin repeat variant, and other antigen-specific binding domains derived from other protein scaffolds.

[0198] In some embodiments, the EpCAM binding domain is an scFv that specifically binds to EpCAM (also referred herein as anti-EpCAM scFv). In some embodiments, the VH and VL of the anti-EpCAM scFv are connected to each other via a peptide linker, such as a flexible linker comprising glycines and / or serines. In some embodiments, the VH and VL of the anti-EpCAM scFv are connected to each other directly. In some embodiments, the anti-EpCAM scFv comprises an N-VH-VL-C fusion polypeptide. In some embodiments, the anti-EpCAM scFv comprises an N-VL-VH-C fusion polypeptide.

[0199] The EpCAM binding domain (such as scFv) may be derived from any suitable anti- EpCAM antibody. In some embodiments, the anti-EpCAM antibody is a human, humanized, or chimeric antibody. In some embodiments, the EpCAM binding domain specifically binds to both human and non-human primates (such as cynomolgus monkey). In some embodiments, the EpCAM binding domain specifically recognizes human EpCAM, but does not have cross- reactivity to non-human primates. Exemplary anti-EpCAM antibodies are known in the art, for example, see, U.S. Patent NO. 8,884,602. The anti-EpCAM binding domain may comprise at least one (such as 1, 2, or 3) HVR of an immunoglobulin heavy chain and / or at least one (such as 1, 2, or 3) HVR of an immunoglobulin light chain. In some embodiments, the anti-EpCAM binding domain comprises 1, 2, 3, 4, 5, or all 6 HVRs of a VH and VL sequence of a full-length antibody that specifically binds to EpCAM.

[0200] In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising one, two or three HVRs (or CDRs) from SEQ ID NO: 19, and / or a light chain variable region (VL) comprising one, two or three HVRs (or CDRs) from SEQ ID NO: 20. In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising three HVRs from SEQ ID NO: 19, and / or a light chain variable region (VL) comprising three HVRs from SEQ ID NO: 20. In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising one, two or three HVRs selected from SED ID Nos: 13-15, and / or a light chain variable region (VL) comprising one, two or three HVRs selected from SED ID Nos: 16-18. In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:19, and / or a light chain variable region (VL) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:20. In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti- EpCAM scFv comprises the amino acid sequence of SEQ ID NO:21. SEQ ID NO: 13 (EpCAM HVR-H1) NYWMS SEQ ID NO: 14 (EpCAM HVR-H2) NIKQDGSEKFYADSVKG SEQ ID NO: 15 (EpCAM HVR-H3) VGPSWEQDY SEQ ID NO: 16 (EpCAM HVR-L1) TGSSSNIGSYYGVH SEQ ID NO: 17 (EpCAM HVR-L2) SDTNRPS SEQ ID NO: 18 (EpCAM HVR-L3) QSYDKGFGHRV SEQ ID NO: 19 (EpCAM VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQDGSE KFYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGPSWEQDYWGQGTLV TVSA SEQ ID NO: 20 (EpCAM VL) GAQSVLTQPPSVSGAPGQRVTISCTGSSSNIGSYYGVHWYQQLPGTAPKLLIYSDTNRPS GVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDKGFGHRVFGGGTKLTVL SEQ ID NO: 21 (EpCAM scFv) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQDGSE KFYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGPSWEQDYWGQGTLV TVSAGGGGGGGGGGGGGGAQSVLTQPPSVSGAPGQRVTISCTGSSSNIGSYYGVHWY QQLPGTAPKLLIYSDTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDKGFG HRVFGGGTKLTVL Linkers

[0201] The MSFPs described herein may comprise a linker (such as a peptide linker) situated between the VH or the VL of the Fab fragment and the binding domains. In some embodiments, the linker between the VH of the Fab fragment and the first binding domain is the same as the linker between the VL of the Fab fragment and the first binding domain. In some embodiments, the linker between the VH of the Fab fragment and the first binding domain is different from the linker between the VL of the Fab fragment and the first binding domain. In some embodiments, the anti-EpCAM scFv comprises a linker (such as peptide linker) situated between the VH and VL of the scFv, which may be the same or different from any of the linkers between the VH and VL of the Fab fragment and the binding domains.

[0202] The linkers can be peptide linkers of any length. In some embodiments, the peptide linker is from 1 amino acids to 10 amino acids long, from 2 amino acids to 15 amino acids long, from 3 amino acids to 12 amino acids long, from 4 amino acids to 10 amino acids long, from 5 amino acids to 9 amino acids long, from 6 amino acids to 8 amino acids long, or from 1 amino acids to 20 amino acids long. In some embodiments, the peptide linker is any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids long. In some embodiments, the peptide linker is any of 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long. In some embodiments, the N-terminus of the peptide linker is covalently linked to the C-terminal of the binding domain, and the C terminus of the peptide linker is covalently linked to the N-terminus of the VH or VL of the Fab fragment.

[0203] In some embodiments, the linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G)<com:Sub>n< / com:Sub>, glycine-serine polymers (including, for example, (GS)<com:Sub>n< / com:Sub>, (GSGGS)<com:Sub>n< / com:Sub> and (GGGS)<com:Sub>n< / com:Sub>, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and therefore may be able to serve as a neutral tether between components. Glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Exemplary flexible linkers include, but are not limited to Gly-Gly, Gly-Gly- Ser-Gly (SEQ ID NO: 24), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 25), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 26), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 27), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 28), Gly-Ser-Ser-Gly (SEQ ID NO: 29), and the like. In some embodiments, the linker between the VH of the anti-CD3 Fab fragment and the EpCAM binding domain (such as scFv) is Gly- Gly. In some embodiments, the linker between the VL of the anti-CD3 Fab fragment and the EpCAM binding domain (such as scFv) is Gly-Gly. The ordinarily skilled artisan will recognize that design of an MSFP can include linkers that are all or partially flexible, such that the linker can include a flexible linker portion as well as one or more portions that confer less flexible structure to provide a desired MSFP structure.

[0204] In some embodiments, the linker between the Fab and the binding domains is a stable linker (not cleavable by protease, especially MMPs).

[0205] In some embodiments, the linker is a cleavable linker. In some embodiments, the linker between the Fab VH or VL and the binding domain comprises a protease substrate cleavage sequence, for example, an MMP substrate cleavage sequence. A well-known peptide sequence of PLGLAG (SEQ ID NO: 30) in a substrate can be cleaved by most MMPs. Substrate sequences that can be cleaved by MMPs have been extensively studied. For example, the sequence of PLGLAG (SEQ ID NO: 30) can be cleaved by most MMPs. In some embodiments, the protease cleavage site is recognized by MMP-2, MMP-9 or a combination thereof.

[0206] In some embodiments, the MSFP comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the MSFP comprises a second polypeptide comprising the amino acid sequence of SEQ ID NO:23. In some embodiments, the MSFP comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 23. Further provided are MSFPs and compositions (such as pharmaceutical compositions) thereof comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 23. SEQ ID NO: 22 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQDGSE KFYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGPSWEQDYWGQGTLV TVSAGGGGGGGGGGGGGGAQSVLTQPPSVSGAPGQRVTISCTGSSSNIGSYYGVHWY QQLPGTAPKLLIYSDTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDKGFG HRVFGGGTKLTVLGGEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPG KGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCV RHGNFGNSYVSWFAYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCPPCS SEQ ID NO: 23 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQDGSE KFYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGPSWEQDYWGQGTLV TVSAGGGGGGGGGGGGGGAQSVLTQPPSVSGAPGQRVTISCTGSSSNIGSYYGVHWY QQLPGTAPKLLIYSDTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDKGFG HRVFGGGTKLTVLGGQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPG QAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFG GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECPPCS III. EpCAM Antibodies

[0207] Further provided by the present application are novel anti-EpCAM antibodies or antigen-binding fragments thereof, including multispecific (such as bispecific) Fab fusion proteins comprising an antigen-binding fragment derived from the anti-EpCAM antibody. Compared to anti-EpCAM antibodies and fragments known in the art, the anti-EpCAM antibodies and antigen-binding fragments thereof described herein have enhanced stability and ability to be expressed. Additionally, the anti-EpCAM antibodies and antigen-binding fragments thereof of the present invention have cross-reactivity towards EpCAM from both human and non-human primates (such as cynomolgus monkeys), which is beneficial for extrapolating results from toxicity and efficacy studies in monkeys to human clinical studies for evaluating the EpCAM antibodies or derivatives thereof (such as MSFPs).

[0208] The antibodies of the present invention bind to an EpCAM epitope with an equilibrium binding constant (K<com:Sub>d< / com:Sub>) of <com:Math><semantics>≤1μM<annotation encoding="application / x-tex">\leq 1 \mu M< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>e.g.<annotation encoding="application / x-tex">e.g.< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>≤100 nM<annotation encoding="application / x-tex">\leq 100 \text{ nM}< / annotation>< / semantics>< / com:Math>, preferably <com:Math><semantics>≤10 nM<annotation encoding="application / x-tex">\leq 10 \text{ nM}< / annotation>< / semantics>< / com:Math>, and more preferably <com:Math><semantics>≤1<annotation encoding="application / x-tex">\leq 1< / annotation>< / semantics>< / com:Math> nM. For example, the anti-EpCAM antibodies provided herein exhibit a K<com:Sub>d< / com:Sub> in the range approximately between <com:Math><semantics>≤1<annotation encoding="application / x-tex">\leq 1< / annotation>< / semantics>< / com:Math> nM to about 1 pM.

[0209] The anti- EpCAM antibodies of the invention serve to completely or partially modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with the functional activity of the widely distributed EpCAM. The EpCAM antibodies are considered to completely modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with EpCAM functional activity when the level of EpCAM functional activity in the presence of EpCAM antibody is decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level of EpCAM functional activity in the absence of binding with an anti-EpCAM antibody described herein. The anti-EpCAM antibodies are considered to significantly block, inhibit, reduce, antagonize, neutralize or otherwise interfere with EpCAM functional activity when the level of EpCAM activity in the presence of the anti-EpCAM antibody is decreased by at least 50%, e.g., 55%, 60%, 75%, 80%, 85% or 90% as compared to the level of EpCAM activity in the absence of binding with an anti- EpCAM antibody described herein. The anti-EpCAM antibodies are considered to partially modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with EpCAM functional activity when the level of EpCAM activity in the presence of the anti- EpCAM antibody is decreased by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of EpCAM activity in the absence of binding with an anti-EpCAM antibody described herein.

[0210] In some embodiments, the anti-EpCAM antibody moiety specifically binds to an EpCAM present on the surface of a cell. In some embodiments, the cell presents on its surface abnormally high levels of EpCAM. In some embodiments, the cell is a cancer cell. In some embodiments, the cancer cell is in a solid tumor. In some embodiments, the cancer cell is a metastatic cancer cell.

[0211] The anti-EpCAM antibody moieties in some embodiments comprise specific sequences or certain variants of such sequences. In some embodiments, the amino acid substitutions in the variant sequences do not substantially reduce the ability of the anti-EpCAM antibody moiety to bind the EpCAM. For example, alterations that do not substantially reduce EpCAM binding affinity may be made. Alterations that substantially improve EpCAM binding affinity or affect some other property, such as specificity, immunogenicity, ADCC or CDC, and / or cross- reactivity with related variants of the EpCAM, are also contemplated.

[0212] In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof (such as an scFv) comprising a heavy chain variable region (VH) comprising one, two or three HVRs (or CDRs) from SEQ ID NO: 19, and / or a light chain variable region (VL) comprising one, two or three HVRs (or CDRs) from SEQ ID NO: 20. In some embodiments, the anti- EpCAM antibody or antigen-binding fragment thereof (such as an scFv) comprises a heavy chain variable region (VH) comprising three HVRs from SEQ ID NO: 19, and / or a light chain variable region (VL) comprising three HVRs from SEQ ID NO: 20.

[0213] In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof (such as an scFv) comprises a heavy chain variable region (VH) comprising a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18.

[0214] In some embodiments, the anti-EpCAM antibody or antigen-binding fragment comprises a VH comprising: (1) a HVR-H1 having one or two amino acid substitutions of SEQ ID NO: 13; (2) a HVR-H2 having one or two amino acid substitutions of SEQ ID NO: 14; and / or (3) a HVR-H3 having one or two amino acid substitutions of SEQ ID NO: 15. In some embodiments, the anti-EpCAM antibody or antigen-binding fragment comprises a VL comprising: (1) a HVR-L1 having one or two amino acid substitutions of SEQ ID NO: 16; (2) a HVR-L2 having one or two amino acid substitutions of SEQ ID NO: 17; and / or (3) a HVR-L3 having one or two amino acid substitutions of SEQ ID NO: 18.

[0215] In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof (such as an scFv) comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18.

[0216] In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof (such as an scFv) comprises a heavy chain variable region (VH) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:19, and / or a light chain variable region (VL) comprising an amino acid sequence at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the sequence of SEQ ID NO:20. In some embodiments, a V<com:Sub>H< / com:Sub> or VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the Fab fragment comprising that sequence retains the ability to bind to EpCAM. In some embodiments, one or two amino acids have been substituted, inserted and / or deleted in any one or more of the HVRs. In some embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof (such as an scFv) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 20.

[0217] In some embodiments, the anti-EpCAM antibody is a full-length antibody. In some embodiments, the full-length anti-EpCAM antibody comprises an Fc sequence from an immunoglobulin, such as IgA, IgD, IgE, IgG, and IgM. In some embodiments, the full-length anti-EpCAM antibody comprises an Fc sequence of IgG, such as any of IgG1, IgG2, IgG3, or IgG4. In some embodiments, the full-length anti-EpCAM antibody comprises an Fc sequence of a human immunoglobulin. In some embodiments, the full-length anti-EpCAM antibody comprises an Fc sequence that has been altered or otherwise changed so that it has enhanced antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) effector function.

[0218] Also provided is an isolated antibody or an antigen-binding fragment thereof which competes with any of the anti-EpCAM antibodies described herein for binding with EpCAM. In some embodiments, there is provided an isolated antibody or an antigen-binding fragment thereof which binds to the same epitope as any of the anti-EpCAM antibodies described herein. Methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.

[0219] Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a monoclonal antibody has the same specificity as a monoclonal antibody of the invention (e.g., the anti-EpCAM antibody having a variable heavy chain of SEQ ID NO:19, and a variable light chain of SEQ ID NO:20) by ascertaining whether the former prevents the latter from binding to EpCAM. If the monoclonal antibody being tested competes with the monoclonal antibody of the invention, as shown by a decrease in binding by the monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same, or a closely related, epitope.

[0220] An alternative method for determining whether a monoclonal antibody has the specificity of monoclonal antibody of the invention is to pre-incubate the monoclonal antibody of the invention with soluble EpCAM protein (with which it is normally reactive), and then add the monoclonal antibody being tested to determine if the monoclonal antibody being tested is inhibited in its ability to bind EpCAM. If the monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the invention.

[0221] In some embodiments, the anti-EpCAM antibody is a monoclonal antibody, such as a monovalent antibody. In some embodiments, the anti-EpCAM antigen-binding fragment is in the form of a Fab, Fab', a F(ab)'2, single-chain Fv (scFv), an Fv fragment, a diabody, or a linear antibody.

[0222] In some embodiments, there is provided an anti-EpCAM scFv comprising a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the anti-EpCAM scFv comprises a VH comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-EpCAM scFv comprises a VL comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 21.

[0223] In some embodiments, the anti-EpCAM antibody is a multispecific antibody that binds to EpCAM, but also binds one or more other targets and optionally inhibits their function. Multispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for two or more different antigens (e.g., bispecific antibodies have binding specificities for at least two antigens). For example, one of the binding specificities can be for the EpCAM protein, the other one can be for any other antigen. In some embodiments, the other antigen is a cell-surface protein or receptor or receptor subunit. For example, the cell-surface protein can be CD3, such as CD3 epsilon. Thus, according to one embodiment, a bispecific antibody of this invention can bind both EpCAM and, e.g., a second cell surface receptor.

[0224] In some embodiments, the multi-specific anti-EpCAM molecule is, for example, a diabody (Db), a single-chain diabody (scDb), a tandem scDb (Tandab), a linear dimeric scDb (LD-scDb), a circular dimeric scDb (CD-scDb), a di-diabody, a tandem scFv, a tandem di-scFv (e.g., a bispecific T cell engager), a tandem tri-scFv, a tri(a)body, a bispecific Fab<com:Sub>2< / com:Sub>, a di- miniantibody, a tetrabody, an scFv-Fc-scFv fusion, a dual-affinity retargeting (DART) antibody, a dual variable domain (DVD) antibody, an IgG-scFab, an scFab-ds-scFv, an Fv2-Fc, an IgG- scFv fusion, a dock and lock (DNL) antibody, a knob-into-hole (KiH) antibody (bispecific IgG prepared by the KiH technology), a DuoBody (bispecific IgG prepared by the Duobody technology), a heteromultimeric antibody, or a heteroconjugate antibody. In some embodiments, the multi-specific anti-EpCAM molecule is a tandem scFv (e.g., a tandem di-scFv, such as a bispecific T cell engager).

[0225] Further provided are fusion proteins, conjugates, or isolated cells comprising any of the anti-EpCAM antibodies or antigen-binding fragments thereof described above.

[0226] In some embodiments, there is provided a multispecific (such as bispecific) Fab fusion protein comprising an anti-EpCAM antigen-binding fragment comprising a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18.

[0227] In some embodiments, the multispecific Fab fusion protein comprises a single anti- EpCAM antigen-binding fragment, wherein the anti-EpCAM antigen-binding fragment is fused to the N-terminus of the heavy chain polypeptide of the Fab fragment or the light chain polypeptide of the Fab fragment. In some embodiments, the multispecific Fab fusion protein comprises two anti-EpCAM antigen-binding fragment or two copies of anti-EpCAM antigen- binding fragments.

[0228] In some embodiments, there is provided a multispecific (such as bispecific) Fab fusion protein comprising a Fab fragment and an anti-EpCAM antigen-binding fragment, wherein the N-terminus of the heavy chain polypeptide or the N-terminus of the light chain polypeptide of the Fab fragment is fused to the anti-EpCAM antigen-binding fragment, and wherein the anti- EpCAM antigen-binding fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:13; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:14; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:15; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:16; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:17; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:18. In some embodiments, the multispecific Fab fusion protein comprises a Fab fragment, a first anti- EpCAM antigen-binding fragment, and a second anti-EpCAM antigen-binding fragment, wherein the N-terminus of the heavy chain polypeptide of the Fab fragment is fused to the first anti-EpCAM antigen-binding fragment, and wherein the N-terminus of the light chain polypeptide of the Fab fragment is fused to the second anti-EpCAM antigen-binding fragment. In some embodiments, the first anti-EpCAM antigen-binding fragment has the same sequence as the second anti-EpCAM antigen-binding fragment. In some embodiments, the anti-EpCAM antigen-binding fragment comprises a VH comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-EpCAM antigen-binding fragment comprises a VL comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti- EpCAM antigen-binding fragment is an scFv, such as an scFv comprising the amino acid sequence of SEQ ID NO: 21.

[0229] The multispecific Fab fusion proteins comprising one or more anti-EpCAM antigen- binding fragments described herein may further comprise one or more features of the multispecific Fab fusion proteins described in subsection "Multispecific Fab fusion protein" of section II "Methods of treating cancer" described above.

[0230] In some embodiments, the multispecific Fab fusion protein comprises a Fab fragment that specifically binds to an immune effector molecule. In some embodiments, the Fab fragment binds to a T cell receptor. In some embodiments, the Fab fragment binds to CD3\varepsilon chain. In some embodiments, the Fab fragment binds to a cell surface target selected from FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa, FcyRIIIa, NKG2D, CD25, CD28, CD137, CTLA-4, FAS, FGFR1, FGFR2, FGFR3, FGFR4, GITR, LTβR, TLR, TRAIL receptor 1, TRAIL receptor 2, EGFR, Her2 / neu, and ErbB3.

[0231] In some embodiments, the Fab fragment specifically binds to CD3, such as the N- terminus of CD3 epsilon, for example, the N-terminal 1-27 amino acids of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: a HVR-H1 comprising the amino acid sequence of SEQ ID NO:1; a HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and a HVR-H3 comprising the amino acid sequence of SEQ ID NO:3; and / or a light chain variable region (VL) comprising: a HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; a HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and a HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In some embodiments, the Fab fragment comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:7 and 39-43, and / or a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 44-47. In some of the embodiments, the multispecific Fab fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO:22, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:23.

[0232] In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent (e.g., cytotoxic agent, a radioisotope and a chemotherapeutic agent) or a label for detecting EpCAM in patient samples or in vivo by imaging (e.g., radioisotope, fluorescent dye and enzyme). In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof is conjugated to a toxin.

[0233] In some embodiments, there is provided an anti-EpCAM chimeric antigen receptor (CAR) comprising: a) an extracellular domain comprising any of the anti-EpCAM antibodies or antigen-binding fragments described herein; and b) an intracellular signaling domain. A transmembrane domain may be present between the extracellular domain and the intracellular domain. Between the extracellular domain and the transmembrane domain of the anti-EpCAM CAR, or between the intracellular domain and the transmembrane domain of the anti-EpCAM CAR, there may be a spacer domain, such as peptide linker (e.g., a flexible peptide linker). Examples of intracellular signaling domains for use in the anti-EpCAM CAR of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. Any method for producing a CAR may be used herein. See, for example, US6,410,319, US7,446, 191, US7,514,537, WO 2002 / 077029, US2010 / 065818, US 2010 / 025177, US 2007 / 059298, and Berger C. et al., J. Clinical Investigation 118: 1 294-308 (2008).

[0234] In some embodiments, there is provided an anti-EpCAM recombinant T cell receptor (TCR) comprising an extracellular domain comprising any of the anti-EpCAM antibodies or antigen-binding fragments described herein. Methods of engineering TCRs have been described, for example, in see Stone J.D. et al. T Cell receptor engineering, Methods Enzymol. (2012) 503:189-222.

[0235] Also provided are isolated cells expressing the anti-EpCAM CAR or TCR, such as CAR-T or TCR-T cells, and methods of treating a disease (such as cancer) using the anti- EpCAM CAR or TCR, or cells expressing the ant-EpCAM or TCR thereof.

[0236] The anti-EpCAM antibodies or antigen-binding fragments described herein can be used in a variety of therapeutic and diagnostic methods. Further provided are methods of treating cancer in an individual, comprising administering an effective amount of the anti-EpCAM antibody or antigen-binding fragment thereof described above or pharmaceutical compositions thereof to the individual. For example, the anti-EpCAM antibodies (or antigen-binding fragments thereof) can be used alone or in combination with other agents in treating a disease characterized by abnormal EpCAM expression, including, but not limited to, head and neck cancer, pancreatic cancer, colorectal cancer, and lung cancer. The antibodies provided herein can also be used for detecting EpCAM protein in patients or patient samples. Monoclonal antibodies

[0237] Screening of monoclonal antibodies of the invention, can be also carried out, e.g., by measuring EpCAM-mediated signaling, and determining whether the test monoclonal antibody is able to modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with EpCAM-mediated signaling. These assays can include competitive binding assays. Additionally, these assays can measure a biologic readout.

[0238] Various procedures known within the art may be used for the production of monoclonal antibodies directed against EpCAM, or against derivatives, fragments, analogs homologs or orthologs thereof. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies" or "fully human antibodies" herein. Human monoclonal antibodies are prepared, for example, using the procedures described in the Examples provided below. Human monoclonal antibodies can be also prepared by using the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0239] Antibodies are purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0240] The EpCAM antibodies of the invention are monoclonal antibodies. Monoclonal antibodies that modulate, block, inhibit, reduce, antagonize, neutralize or otherwise interfere with EpCAM-mediated cell signaling are generated, e.g., by immunizing an animal with membrane bound and / or soluble EpCAM, such as, for example, human EpCAM or an immunogenic fragment, derivative or variant thereof. Alternatively, the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding EpCAM such that EpCAM is expressed and associated with the surface of the transfected cells. Alternatively, the antibodies are obtained by screening a library that contains antibody or antigen binding domain sequences for binding to EpCAM. This library is prepared, e.g., in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles (i.e., "phage displayed library"). Hybridomas resulting from myeloma / B cell fusions are then screened for reactivity to EpCAM.

[0241] Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0242] The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

[0243] Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

[0244] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.

[0245] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0246] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0247] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as Chinese hamster ovary (CHO) cells, Human Embryonic Kidney (HEK) 293 cells, simian COS cells, PER.C6□, NS0 cells, SP2 / 0, YB2 / 0, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody. Human Antibodies and Humanization of Antibodies

[0248] Monoclonal antibodies of the invention include fully human antibodies or humanized antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin.

[0249] An anti-EpCAM antibody can be generated using any procedures known in the art. For example, anti-EpCAM antibodies can be identified using a modified RIMMS (Repetitive Immunization Multiple Sites) immunization strategy in mice and subsequent hybridoma generation. In other, alternative methods, an anti-EpCAM antibody is developed, for example, using phage-display methods using antibodies containing only human sequences. Such approaches are well-known in the art, e.g., in WO92 / 01047 and U.S. Pat. No. 6,521,404. In this approach, a combinatorial library of phage carrying random pairs of light and heavy chains are screened using natural or recombinant source of EpCAM or fragments thereof. In another approach, an anti-EpCAM antibody can be produced by a process wherein at least one step of the process includes immunizing a transgenic, non-human animal with human EpCAM protein. In this approach, some of the endogenous heavy and / or kappa light chain loci of this xenogenic non-human animal have been disabled and are incapable of the rearrangement required to generate genes encoding immunoglobulins in response to an antigen. In addition, at least one human heavy chain locus and at least one human light chain locus have been stably transfected into the animal. Thus, in response to an administered antigen, the human loci rearrange to provide genes encoding human variable regions immunospecific for the antigen. Upon immunization, therefore, the xenomouse produces B-cells that secrete fully human immunoglobulins.

[0250] A variety of techniques are well-known in the art for producing xenogenic non-human animals. For example, see U.S. Pat. No. 6,075,181 and No. 6,150,584. This general strategy was demonstrated in connection with generation of the first XenoMouse<com:Sup>TM< / com:Sup> strains as published in 1994. See Green et al. Nature Genetics 7:13-21 (1994. See also, U.S. Patent Nos. 6,162,963; 6,150,584; 6,114,598; 6,075,181; and 5,939,598 and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2 and European Patent No., EP 0 463 151 B1 and International Patent Applications No. WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, WO 00 / 76310 and related family members.

[0251] In an alternative approach, others have utilized a "minilocus" approach in which an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more VH genes, one or more D<com:Sub>H< / com:Sub> genes, one or more J<com:Sub>H< / com:Sub> genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. See e.g., U.S. Patent Nos. 5,545,806; 5,545,807; 5,591,669; 5,612,205;5,625,825; 5,625,126; 5,633,425; 5,643,763; 5,661,016; 5,721,367; 5,770,429; 5,789,215; 5,789,650; 5,814,318; 5,877; 397; 5,874,299; 6,023,010; and 6,255,458; and European Patent No. 0 546 073 B1; and International Patent Application Nos. WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO 94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852, and WO 98 / 24884 and related family members.

[0252] Generation of human antibodies from mice in which, through microcell fusion, large pieces of chromosomes, or entire chromosomes, have been introduced, has also been demonstrated. See European Patent Application Nos. 773 288 and 843 961.

[0253] Human anti-mouse antibody (HAMA) responses have led the industry to prepare chimeric or otherwise humanized antibodies. While chimeric antibodies have a human constant region and an immune variable region, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, particularly in chronic or multi-dose utilizations of the antibody. Thus, it would be desirable to provide fully human antibodies against EpCAM in order to vitiate or otherwise mitigate concerns and / or effects of HAMA or HACA response.

[0254] The production of antibodies with reduced immunogenicity is also accomplished via humanization, chimerization and display techniques using appropriate libraries. It will be appreciated that murine antibodies or antibodies from other species can be humanized or primatized using techniques well known in the art. See e.g., Winter and Harris Immunol Today 14:43 46 (1993) and Wright et al. Crit, Reviews in Immunol. 12125-168 (1992). The antibody of interest may be engineered by recombinant DNA techniques to substitute the CH1, CH2, CH3, hinge domains, and / or the framework domain with the corresponding human sequence (See WO 92102190 and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,792;, 5,714,350; and 5,777,085). Also, the use of Ig cDNA for construction of chimeric immunoglobulin genes is known in the art (Liu et al. P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521 (1987)). mRNA is isolated from a hybridoma or other cell producing the antibody and used to produce cDNA. The cDNA of interest may be amplified by the polymerase chain reaction using specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library is made and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to human constant region sequences. The sequences of human constant regions genes may be found in Kabat et al. (1991) Sequences of Proteins of immunological Interest, N.I.H. publication no. 91-3242. Human C region genes are readily available from known clones. The choice of isotype will be guided by the desired effecter functions, such as complement fixation, or activity in antibody-dependent cellular cytotoxicity. Preferred isotypes are IgGl, IgG2, IgG3, and IgG4. Either of the human light chain constant regions, kappa or lambda, may be used. The chimeric, humanized antibody is then expressed by conventional methods.

[0255] Antibody fragments, such as Fv, F(ab')<com:Sub>2< / com:Sub> and Fab may be prepared by cleavage of the intact protein, e.g., by protease or chemical cleavage. Alternatively, a truncated gene is designed. For example, a chimeric gene encoding a portion of the F(ab')<com:Sub>2< / com:Sub> fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule.

[0256] Consensus sequences of H and L J regions may be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments. C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.

[0257] Expression vectors include plasmids, retroviruses, YACs, EBV derived episomes, and the like. A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The resulting chimeric antibody may be joined to any strong promoter, including retroviral LTRs, e.g., SV-40 early promoter, (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcoma virus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloney murine leukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)). Also, as will be appreciated, native Ig promoters and the like may be used.

[0258] Further, human antibodies or antibodies from other species can be generated through display type technologies, including, without limitation, phage display, retroviral display, ribosomal display, and other techniques, using techniques well known in the art and the resulting molecules can be subjected to additional maturation, such as affinity maturation, as such techniques are well known in the art. Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes and Plückthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley and Smith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol. 17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russel et al. Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al. Immunol. Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A (1992), and U.S. Patent No. 5,733,743. If display technologies are utilized to produce antibodies that are not human, such antibodies can be humanized as described above.

[0259] Using these techniques, antibodies can be generated to EpCAM expressing cells, soluble forms of EpCAM, epitopes or peptides thereof, and expression libraries thereto (See e.g., U.S. Patent No. 5,703,057) which can thereafter be screened as described above for the activities described herein.

[0260] The EpCAM antibodies of the invention can be expressed by a vector containing a DNA segment encoding the single chain antibody described above.

[0261] These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. Vectors include chemical conjugates such as described in WO 93 / 64701, which has targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT / US95 / 02140 (WO 95 / 22618) which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal or synthetic.

[0262] Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include moloney murine leukemia viruses. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., et al., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (see LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) and Adeno-associated Virus Vectors (see Kaplitt, M. G., et al., Nat. Genet. 8:148 (1994).

[0263] Pox viral vectors introduce the gene into the cells cytoplasm. Avipox virus vectors result in only a short term expression of the nucleic acid. Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors are preferred for introducing the nucleic acid into neural cells. The adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The particular vector chosen will depend upon the target cell and the condition being treated. The introduction can be by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, CaPO<com:Sub>4< / com:Sub> precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0264] The vector can be employed to target essentially any desired target cell. For example, stereotaxic injection can be used to direct the vectors (e.g. adenovirus, HSV) to a desired location. Additionally, the particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as a SynchroMed Infusion System. A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and may be useful in delivering the vector to the target cell. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.

[0265] These vectors can be used to express large quantities of antibodies that can be used in a variety of ways. For example, to detect the presence of EpCAM in a sample. The antibody can also be used to try to bind to and disrupt EpCAM-mediated signaling.

[0266] Techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F(ab')<com:Sub>2< / com:Sub> fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F<com:Sub>(ab')2< / com:Sub> fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F<com:Sub>v< / com:Sub> fragments.

[0267] The invention also includes F<com:Sub>v< / com:Sub>, Fab, Fab' and F(ab')<com:Sub>2< / com:Sub> anti-EpCAM fragments, single chain EpCAM antibodies, single domain antibodies (e.g., nanobodies or VHHs), multispecific (such as bispecific) anti-EpCAM antibodies, and heteroconjugate anti-EpCAM antibodies.

[0268] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain / light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93 / 08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0269] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0270] According to another approach described in WO 96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0271] Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')<com:Sub>2< / com:Sub> bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab')<com:Sub>2< / com:Sub> fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0272] Additionally, Fab' fragments can be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')<com:Sub>2< / com:Sub> molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.

[0273] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (V<com:Sub>H< / com:Sub>) connected to a light-chain variable domain (V<com:Sub>L< / com:Sub>) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V<com:Sub>H< / com:Sub> and V<com:Sub>L< / com:Sub> domains of one fragment are forced to pair with the complementary V<com:Sub>L< / com:Sub> and V<com:Sub>H< / com:Sub> domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

[0274] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0275] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

[0276] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (see U.S. Patent No. 4,676,980), and for treatment of HIV infection (see WO 91 / 00360; WO 92 / 200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

[0277] It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating diseases and disorders associated with aberrant EpCAM signaling. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).

[0278] The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0279] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include <com:Sup>212< / com:Sup>Bi, <com:Sup>131< / com:Sup>I, <com:Sup>131< / com:Sup>In, <com:Sup>90< / com:Sup>Y, and <com:Sup>186< / com:Sup>Re.

[0280] Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See WO94 / 11026).

[0281] Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies of the invention. (See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds), Carger Press, New York, (1989)).

[0282] Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. The preferred binding is, however, covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).

[0283] Preferred linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N- hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)- toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

[0284] The linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.

[0285] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[0286] Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Use of antibodies against EpCAM

[0287] It will be appreciated that administration of therapeutic entities in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin<com:Sup>TM< / com:Sup>), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some e...

Claims

CLAIMS:

1. A use, for treating a cancer in an individual, of an effective amount of a multispecific Fab fusion protein, wherein the multispecific Fab fusion protein comprises a Fab fragment that specifically binds to CD3 (anti-CD3 Fab fragment), and a binding domain that specifically binds to EpCAM (anti-EpCAM binding domain); wherein the anti-EpCAM binding domain is fused to an N- terminus of the anti-CD3 Fab fragment; and wherein the anti-EpCAM binding domain comprises a heavy chain hypervariable region 1 (HVR-H1) comprising the amino acid sequence of SEQ ID NO: 13, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15, a light chain hypervariable region 1 (HVR-L1) comprising the amino acid sequence of SEQ ID NO: 16, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:

18.

2. The use of claim 1, wherein the anti-EpCAM binding domain comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

20.

3. The use of claim 1 or 2, wherein the anti-EpCAM binding domain is an anti-EpCAM single-chain Fv (scFv).

4. The use of claim 3, wherein the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO:

21.

5. The use of claim 3 or 4, wherein i) the C-terminus of the anti-EpCAM scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment; or ii) the C-terminus of the anti-EpCAM scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment.

6. The use of claim 3 or 4, wherein the multispecific Fab fusion protein comprises a first anti-EpCAM scFv and a second anti-EpCAM scFv; wherein the C-terminus of the first anti- EpCAM scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment; and wherein the C-terminus of the second anti-EpCAM scFv is fused to the N-terminus of the VL of the anti- CD3 Fab fragment.

7. The use of claim 6, wherein the first anti-EpCAM scFv and the second anti-EpCAM scFv have the same sequence.

8. The use of claim 6, wherein the first anti-EpCAM scFv and the second anti-EpCAM scFv have different sequences.

9. The use of any one of claims 1-8, wherein the multispecific Fab fusion protein is formulated for intravenous use.

10. The use of any one of claims 1-9, wherein the multispecific Fab fusion protein is for use at a low frequency.

11. The use of any one of claims 1-10, wherein the multispecific Fab fusion protein is for use twice weekly.

12. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math>.

13. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>0.05μg / kg<annotation encoding="application / x-tex">0.05 \mu g / kg< / annotation>< / semantics>< / com:Math>.

14. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>0.1μg / kg<annotation encoding="application / x-tex">0.1 \mu g / kg< / annotation>< / semantics>< / com:Math>.

15. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>0.3μg / kg<annotation encoding="application / x-tex">0.3 \mu g / kg< / annotation>< / semantics>< / com:Math>.

16. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>0.5μg / kg<annotation encoding="application / x-tex">0.5 \mu g / kg< / annotation>< / semantics>< / com:Math>.

17. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>0.6μg / kg<annotation encoding="application / x-tex">0.6 \mu g / kg< / annotation>< / semantics>< / com:Math>.

18. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at 1 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

19. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at 1.2 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

20. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>2μg / kg<annotation encoding="application / x-tex">2 \mu g / kg< / annotation>< / semantics>< / com:Math>.

21. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at 2.4 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

22. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>2.5μg / kg<annotation encoding="application / x-tex">2.5 \mu g / kg< / annotation>< / semantics>< / com:Math>.

23. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>3.6μg / kg<annotation encoding="application / x-tex">3.6 \mu g / kg< / annotation>< / semantics>< / com:Math>.

24. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at 4 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

25. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at 5 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

26. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>10μg / kg<annotation encoding="application / x-tex">10 \mu g / kg< / annotation>< / semantics>< / com:Math>.

27. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>15μg / kg<annotation encoding="application / x-tex">15 \mu g / kg< / annotation>< / semantics>< / com:Math>.

28. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>20μg / kg<annotation encoding="application / x-tex">20 \mu g / kg< / annotation>< / semantics>< / com:Math>.

29. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at 25 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

30. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at 30 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

31. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at 50 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

32. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>100μg / kg<annotation encoding="application / x-tex">100 \mu g / kg< / annotation>< / semantics>< / com:Math>.

33. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at 150 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

34. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at 200 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

35. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at 250 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

36. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 0.3 µg / kg for a cynomolgus monkey.

37. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 0.5 µg / kg for a cynomolgus monkey.

38. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 1 µg / kg for a cynomolgus monkey.

39. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 2 µg / kg for a cynomolgus monkey.

40. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 4 µg / kg for a cynomolgus monkey.

41. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 5 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg for a cynomolgus monkey.

42. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 10 µg / kg for a cynomolgus monkey.

43. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 15 µg / kg for a cynomolgus monkey.

44. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 20 µg / kg for a cynomolgus monkey.

45. The use of any one of claims 1-11, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 30 µg / kg for a cynomolgus monkey.

46. The use of any one of claims 1-45, wherein the use of the multispecific Fab fusion protein does not induce a cytokine storm.

47. The use of any one of claims 1-46, wherein the multispecific Fab fusion protein is for use consecutively at a first dose for a first period of time, and at a second dose for a second period of time, and wherein the second dose exceeds the first dose.

48. The use of claim 47, wherein the second period of time exceeds the first period of time.

49. The use of claim 47 or 48, wherein the multispecific Fab fusion protein is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at the second dose on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time.

50. The use of any one of claims 47-49, wherein the second dose is <com:Math><semantics>0.6μg / kg<annotation encoding="application / x-tex">0.6 \mu g / kg< / annotation>< / semantics>< / com:Math>.

51. The use of any one of claims 47-49, wherein the second dose is <com:Math><semantics>1.2μg / kg<annotation encoding="application / x-tex">1.2 \mu g / kg< / annotation>< / semantics>< / com:Math>.

52. The use of any one of claims 47-49, wherein the second dose is <com:Math><semantics>2.4μg / kg<annotation encoding="application / x-tex">2.4 \mu g / kg< / annotation>< / semantics>< / com:Math>.

53. The use of any one of claims 47-49, wherein the second dose is <com:Math><semantics>3.6μg / kg<annotation encoding="application / x-tex">3.6 \mu g / kg< / annotation>< / semantics>< / com:Math>.

54. The use of any one of claims 1-53, wherein the treatment further comprises use of an effective amount of a glucocorticoid.

55. The use of claim 54, wherein the glucocorticoid is dexamethasone, or a pharmaceutically acceptable ester, salt, or complex thereof.

56. The use of claim 54 or 55, wherein the glucocorticoid is for use simultaneously or prior to the first dose of the multispecific Fab fusion protein.

57. The use of claim 56, wherein the glucocorticoid is for further use after the first dose of the multispecific Fab fusion protein, and simultaneously or prior to the second dose of the multispecific Fab fusion protein.

58. The use of claim 57, wherein the multispecific Fab fusion protein is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at a second dose of 0.6 μg / kg on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time; and wherein the glucocorticoid is for use 1 hour prior to the first dose of the multispecific Fab fusion protein on day 1 of the 7-day period of time, and 1 hour prior to the second dose of the multispecific Fab fusion protein on day 1 of the 21-day period of time.

59. The use of claim 57, wherein the multispecific Fab fusion protein is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at a second dose of 1.2 μg / kg on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time; and wherein the glucocorticoid is for use 1 hour prior to the first dose of the multispecific Fab fusion protein on day 1 of the 7-day period of time, and 1 hour prior to the second dose of the multispecific Fab fusion protein on day 1 of the 21-day period of time.

60. The use of claim 57, wherein the multispecific Fab fusion protein is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at a second dose of 2.4 µg / kg on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time; and wherein the glucocorticoid is for use 1 hour prior to the first dose of the multispecific Fab fusion protein on day 1 of the 7-day period of time, and 1 hour prior to the second dose of the multispecific Fab fusion protein on day 1 of the 21-day period of time.

61. The use of claim 57, wherein the multispecific Fab fusion protein is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at a second dose of 3.6 µg / kg on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time; and wherein the glucocorticoid is for use 1 hour prior to the first dose of the multispecific Fab fusion protein on day 1 of the 7-day period of time, and 1 hour prior to the second dose of the multispecific Fab fusion protein on day 1 of the 21-day period of time.

62. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 0.1 mg / kg.

63. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 0.5 mg / kg.

64. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 1 mg / kg.

65. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 2 mg / kg.

66. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 3 mg / kg.

67. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 4 mg / kg.

68. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 5 mg / kg.

69. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 1 mg.

70. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 2 mg.

71. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 5 mg.

72. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 10 mg.

73. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 15 mg.

74. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 20 mg.

75. The use of any one of claims 54-61, wherein the glucocorticoid is for use at 25 mg.

76. The use of any one of claims 1-75, wherein the individual is a human.

77. The use of any one of claims 1-76, wherein the anti-CD3 Fab fragment is for specifically binding to the N-terminus of CD3 epsilon.

78. The use of claim 77, wherein the anti-CD3 Fab fragment is for specifically binding to an epitope within amino acids 1-27 of CD3 epsilon.

79. The use of claim 77 or 78, wherein the anti-CD3 Fab fragment comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:

6.

80. The use of claim 79, wherein the VH of the anti-CD3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 39-43, and / or wherein the VL of the anti-CD3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 44-47.

81. The use of claim 80, wherein the VH of the anti-CD3 Fab fragment comprises an amino acid sequence of SEQ ID NO: 7, and wherein the VL of the anti-CD3 Fab fragment comprises an amino acid sequence of SEQ ID NO:

8.

82. The use of any one of claims 79-81, wherein the anti-CD3 Fab fragment comprises a human immunoglobulin heavy chain constant region 1 (CH1) comprising the amino acid sequence of SEQ ID NO:

9.

83. The use of any one of claims 79-82, wherein the anti-CD3 Fab fragment comprises a human lambda light chain constant region comprising the amino acid sequence of SEQ ID NO:

10.

84. The use of any one of claims 79-83, wherein the CH1 and the CL of the anti-CD3 Fab fragment are connected by one or more disulfide bonds.

85. The use of any one of claims 79-84, wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and / or a second polypeptide comprising the amino acid sequence of SEQ ID NO:

12.

86. The use of any one of claims 79-85, wherein the multispecific Fab fusion protein comprises: (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 12; (ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 23; or (iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:

23.

87. The use of any one of claims 1-86, wherein the cancer is an EpCAM-positive solid cancer.

88. The use of claim 87, wherein the EpCAM-positive solid cancer is a carcinoma or an adenocarcinoma.

89. The use of any one of claims 1-88, wherein the cancer is selected from the group consisting of small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer, renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer.

90. The use of any one of claims 1-89, wherein the cancer is colorectal adenocarcinoma.

91. The use of any one of claims 1-89, wherein the cancer is lung adenocarcinoma.

92. The use of any one of claims 54-91, wherein the glucocorticoid is for reducing, inhibiting, or preventing: i) cytokine release syndrome (CRS), and / or ii) a side effect in liver in the individual.

93. The use of any one of claims 54-92, wherein the glucocorticoid is for reducing, inhibiting, or preventing: i) release of one or more cytokines selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, and IFN-γ; and / or ii) elevated serum level of one or more liver enzymes selected from the group consisting of alanine aminotransferase (ALT), total bilirubin (TBil), and alkaline phosphatase (ALP).

94. An anti-EpCAM antibody or antigen-binding fragment thereof, comprising an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 16, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:

18.

95. The anti-EpCAM antibody or antigen-binding fragment thereof according to claim 94, comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and / or a VL comprising the amino acid sequence of SEQ ID NO:

20.

96. The anti-EpCAM antibody or antigen-binding fragment thereof according to claim 94 or 95, wherein the anti-EpCAM antibody or antigen-binding fragment thereof is conjugated to another moiety, and wherein the another moiety is selected from the group consisting of a therapeutic agent, a cytotoxic agent, a toxin, a radioactive isotope, a vaccine, liposomes, DNA, and a label for detecting EpCAM.

97. The anti-EpCAM antibody or antigen-binding fragment thereof according to claim 96, wherein the another moiety is selected from the group consisting of a therapeutic agent, a cytotoxic agent, and a toxin.

98. The anti-EpCAM antibody or antigen-binding fragment thereof of any one of claims 94- 97, wherein the anti-EpCAM antibody or antigen-binding fragment thereof comprises an Fc fragment.

99. The anti-EpCAM antibody or antigen-binding fragment thereof of any one of claims 94- 98, which is a multispecific antibody.

100. The anti-EpCAM antigen-binding fragment of any one of claims 94-97 and 99, wherein the anti-EpCAM antigen-binding fragment is or comprises an anti-EpCAM scFv.

101. The anti-EpCAM antigen-binding fragment of claim 100, wherein the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO:

21.

102. A multispecific Fab fusion protein comprising the anti-EpCAM antigen-binding fragment as defined in any one of claims 94, 95, 100, and 101.

103. The multispecific Fab fusion protein of claim 102, wherein the multispecific Fab fusion protein comprises an anti-CD3 Fab fragment, wherein: (i) the anti-EpCAM antigen-binding fragment is fused to the N-terminus of the VH or the VL of the anti-CD3 Fab fragment; or (ii) the multispecific Fab fusion protein comprises a first copy of the anti-EpCAM antigen-binding fragment and a second copy of the anti-EpCAM antigen binding fragment, and wherein the first copy of the anti-EpCAM antigen-binding fragment is fused to the N-terminus of the VH of the anti-CD3 Fab fragment, and the second copy of the anti-EpCAM antigen-binding fragment is fused to the N-terminus of the VL of the anti-CD3 Fab fragment.

104. The multispecific Fab fusion protein of claim 103, wherein the anti-CD3 Fab fragment is for specifically binding to the N-terminus of CD3 epsilon.

105. The multispecific Fab fusion protein of claim 104, wherein the anti-CD3 Fab fragment is for specifically binding to an epitope within amino acids 1-27 of CD3 epsilon.

106. The multispecific Fab fusion protein of claim 104 or 105, wherein the anti-CD3 Fab fragment comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:

6.

107. The multispecific Fab fusion protein of claim 106, wherein the VH of the anti-CD3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 39-43; and / or wherein the VL of the anti-CD3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 44-47.

108. The multispecific Fab fusion protein of claim 107, wherein the VH of the anti-CD3 Fab fragment comprises an amino acid sequence of SEQ ID NO: 7, and wherein the VL of the anti- CD3 Fab fragment comprises an amino acid sequence of SEQ ID NO:

8.

109. The multispecific Fab fusion protein of any one of claims 106-108, wherein the anti-CD3 Fab fragment comprises a human CH1 comprising the amino acid sequence of SEQ ID NO:

9.

110. The multispecific Fab fusion protein of any one of claims 106-109, wherein the anti-CD3 Fab fragment comprises a human lambda light chain constant region comprising the amino acid sequence of SEQ ID NO:

10.

111. The multispecific Fab fusion protein of claim 110, wherein the CH1 and the CL of the anti-CD3 Fab fragment are connected by one or more disulfide bonds.

112. The multispecific Fab fusion protein of any one of claims 106-111, wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and / or a second polypeptide comprising the amino acid sequence of SEQ ID NO:

12.

113. The multispecific Fab fusion protein of any one of claims 103-112, wherein: (i) the anti- EpCAM antigen-binding fragment is an anti-EpCAM scFv; or (ii) the first copy of the anti- EpCAM antigen-binding fragment and the second copy of the anti-EpCAM antigen-binding fragment are both anti-EpCAM scFvs.

114. The multispecific Fab fusion protein of any one of claims 103-113, wherein the first copy of the anti-EpCAM antigen-binding fragment and the second copy of the anti-EpCAM antigen- binding fragment have different sequences.

115. The multispecific Fab fusion protein of any one of claims 103-113, wherein the first copy of the anti-EpCAM antigen-binding fragment and the second copy of the anti-EpCAM antigen- binding fragment have the same sequence.

116. The multispecific Fab fusion protein of claim 113, wherein: (i) the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 21; or (ii) the first copy of the anti-EpCAM antigen-binding fragment and the second copy of the anti-EpCAM antigen-binding fragment are both anti-EpCAM scFvs each comprising the amino acid sequence of SEQ ID NO:

21.

117. The multispecific Fab fusion protein of claim 116, wherein the multispecific Fab fusion protein comprises: (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 12; (ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 23; or (iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:

23.

118. An isolated nucleic acid encoding the polypeptide portion of the anti-EpCAM antibody or antigen-binding fragment thereof as defined in any one of claims 94-101, or the multispecific Fab fusion protein as defined in any one of claims 102-117.

119. A vector comprising the isolated nucleic acid as defined in claim 118.

120. A host cell comprising the isolated nucleic acid as defined in claim 118, or the vector as defined in claim 119.

121. A method of producing an anti-EpCAM antibody or antigen-binding fragment thereof, or a multispecific Fab fusion protein, comprising: i) culturing an isolated host cell comprising the isolated nucleic acid as defined in claim 118 or the vector as defined in claim 119, or culturing the host cell as defined in claim 120, and ii) recovering the expressed anti-EpCAM antibody or antigen-binding fragment thereof, or the multispecific Fab fusion protein, from the cell culture.

122. A composition comprising: (i) the anti-EpCAM antibody or antigen-binding fragment thereof as defined in any one of claims 94-101, or the multispecific Fab fusion protein as defined in any one of claims 102-117, and (ii) a pharmaceutically acceptable carrier.

123. A use, for treating a cancer in an individual, of an effective amount of the anti-EpCAM antibody or antigen-binding fragment thereof as defined in claim 97, the multispecific Fab fusion protein as defined in any one of claims 102-117, or the composition as defined in claim 122.

124. The use of claim 123, wherein the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is formulated for intravenous use.

125. The use of any one of claims 123 or 124, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at a low frequency.

126. The use of any one of claims 123-125, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use twice weekly.

127. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math>.

128. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>0.05μg / kg<annotation encoding="application / x-tex">0.05 \mu g / kg< / annotation>< / semantics>< / com:Math>.

129. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>0.1μg / kg<annotation encoding="application / x-tex">0.1 \mu g / kg< / annotation>< / semantics>< / com:Math>.

130. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>0.3μg / kg<annotation encoding="application / x-tex">0.3 \mu g / kg< / annotation>< / semantics>< / com:Math>.

131. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>0.5μg / kg<annotation encoding="application / x-tex">0.5 \mu g / kg< / annotation>< / semantics>< / com:Math>.

132. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>0.6μg / kg<annotation encoding="application / x-tex">0.6 \mu g / kg< / annotation>< / semantics>< / com:Math>.

133. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at 1 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

134. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at 1.2 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

135. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>2μg / kg<annotation encoding="application / x-tex">2 \mu g / kg< / annotation>< / semantics>< / com:Math>.

136. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>2.4μg / kg<annotation encoding="application / x-tex">2.4 \mu g / kg< / annotation>< / semantics>< / com:Math>.

137. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>2.5μg / kg<annotation encoding="application / x-tex">2.5 \mu g / kg< / annotation>< / semantics>< / com:Math>.

138. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>3.6μg / kg<annotation encoding="application / x-tex">3.6 \mu g / kg< / annotation>< / semantics>< / com:Math>.

139. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at 4 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

140. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at 5 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

141. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>10μg / kg<annotation encoding="application / x-tex">10 \mu g / kg< / annotation>< / semantics>< / com:Math>.

142. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at 15 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

143. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>20μg / kg<annotation encoding="application / x-tex">20 \mu g / kg< / annotation>< / semantics>< / com:Math>.

144. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at 25 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

145. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>30μg / kg<annotation encoding="application / x-tex">30 \mu g / kg< / annotation>< / semantics>< / com:Math>.

146. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>50μg / kg<annotation encoding="application / x-tex">50 \mu g / kg< / annotation>< / semantics>< / com:Math>.

147. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at <com:Math><semantics>100μg / kg<annotation encoding="application / x-tex">100 \mu g / kg< / annotation>< / semantics>< / com:Math>.

148. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at 150 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

149. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at 200 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

150. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at 250 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

151. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at a dose equivalent to 0.3 µg / kg for a cynomolgus monkey.

152. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at a dose equivalent to <com:Math><semantics>0.5μg / kg<annotation encoding="application / x-tex">0.5 \mu g / kg< / annotation>< / semantics>< / com:Math> for a cynomolgus monkey.

153. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at a dose equivalent to 1 µg / kg for a cynomolgus monkey.

154. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at a dose equivalent to 2 µg / kg for a cynomolgus monkey.

155. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at a dose equivalent to 4 µg / kg for a cynomolgus monkey.

156. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at a dose equivalent to 5 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg for a cynomolgus monkey.

157. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at a dose equivalent to 10 µg / kg for a cynomolgus monkey.

158. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at a dose equivalent to 15 µg / kg for a cynomolgus monkey.

159. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at a dose equivalent to 20 µg / kg for a cynomolgus monkey.

160. The use of any one of claims 123-126, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use at a dose equivalent to 30 µg / kg for a cynomolgus monkey.

161. The use of any one of claims 123-160, wherein the use of the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof does not induce a cytokine storm.

162. The use of any one of claims 123-161, wherein the anti-EpCAM antibody or antigen- binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use consecutively at a first dose for a first period of time, and at a second dose for a second period of time, and wherein the second dose exceeds the first dose.

163. The use of claim 162, wherein the second period of time exceeds the first period of time.

164. The use of claim 162 or 163, wherein the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at the second dose on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time.

165. The use of any one of claims 162-164, wherein the second dose is <com:Math><semantics>0.6μg / kg<annotation encoding="application / x-tex">0.6 \mu g / kg< / annotation>< / semantics>< / com:Math>.

166. The use of any one of claims 162-164, wherein the second dose is <com:Math><semantics>1.2μg / kg<annotation encoding="application / x-tex">1.2 \mu g / kg< / annotation>< / semantics>< / com:Math>.

167. The use of any one of claims 162-164, wherein the second dose is <com:Math><semantics>2.4μg / kg<annotation encoding="application / x-tex">2.4 \mu g / kg< / annotation>< / semantics>< / com:Math>.

168. The use of any one of claims 162-164, wherein the second dose is <com:Math><semantics>3.6μg / kg<annotation encoding="application / x-tex">3.6 \mu g / kg< / annotation>< / semantics>< / com:Math>.

169. The use of any one of claims 123-168, wherein the treatment further comprises use of an effective amount of a glucocorticoid.

170. The use of claim 169, wherein the glucocorticoid is dexamethasone, or a pharmaceutically acceptable ester, salt, or complex thereof.

171. The use of claim 169 or 170, wherein the glucocorticoid is for use simultaneously or prior to the first dose of the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof.

172. The use of claim 171, wherein the glucocorticoid is for further use after the first dose of the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof, and simultaneously or prior to the second dose of the anti- EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof.

173. The use of claim 172, wherein the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at a second dose of 0.6 μg / kg on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time; and wherein the glucocorticoid is for use 1 hour prior to the first dose of the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof on day 1 of the 7-day period of time, and 1 hour prior to the second dose of the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof on day 1 of the 21-day period of time.

174. The use of claim 172, wherein the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at a second dose of 1.2 μg / kg on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time; and wherein the glucocorticoid is for use 1 hour prior to the first dose of the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof on day 1 of the 7-day period of time, and 1 hour prior to the second dose of the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof on day 1 of the 21-day period of time.

175. The use of claim 172, wherein the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at a second dose of 2.4 μg / kg on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time; and wherein the glucocorticoid is for use 1 hour prior to the first dose of the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof on day 1 of the 7-day period of time, and 1 hour prior to the second dose of the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof on day 1 of the 21-day period of time.

176. The use of any one of claims 169-172, wherein the the anti-EpCAM antibody or antigen- binding fragment thereof, multispecific Fab fusion protein, or the composition thereof is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at a second dose of 3.6 µg / kg on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time; and wherein the glucocorticoid is for use 1 hour prior to the first dose of the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof on day 1 of the 7-day period of time, and 1 hour prior to the second dose of the anti-EpCAM antibody or antigen-binding fragment thereof, the multispecific Fab fusion protein, or the composition thereof on day 1 of the 21-day period of time.

177. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 0.1 mg / kg.

178. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 0.5 mg / kg.

179. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 1 mg / kg.

180. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 2 mg / kg.

181. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 3 mg / kg.

182. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 4 mg / kg.

183. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 5 mg / kg.

184. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 1 mg.

185. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 2 mg.

186. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 5 mg.

187. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 10 mg.

188. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 15 mg.

189. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 20 mg.

190. The use of any one of claims 169-176, wherein the glucocorticoid is for use at 25 mg.

191. The use of any one of claims 169-190, wherein the glucocorticoid is for reducing, inhibiting, or preventing: i) CRS, and / or ii) a side effect in liver in the individual.

192. The use of any one of claims 169-191, wherein the glucocorticoid is for reducing, inhibiting, or preventing: i) release of one or more cytokines selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, and IFN-γ; and / or ii) elevated serum level of one or more liver enzymes selected from the group consisting of ALT, TBil, and ALP.

193. The use of any one of claims 123-192, wherein the cancer is an EpCAM-positive solid cancer.

194. The use of claim 193, wherein the EpCAM-positive solid cancer is a carcinoma or an adenocarcinoma.

195. The use of any one of claims 123-194, wherein the cancer is selected from the group consisting of small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer, renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer.

196. The use of any one of claims 123-195, wherein the cancer is colorectal adenocarcinoma.

197. The use of any one of claims 123-195, wherein the cancer is lung adenocarcinoma.

198. The use of any one of claims 123-197, wherein the individual is a human.

199. A use, for treating a cancer in an individual, of an effective amount of a glucocorticoid and an effective amount of a multispecific Fab fusion protein, wherein the multispecific Fab fusion protein comprises an anti-CD3 Fab fragment and an anti-EpCAM binding domain, wherein the anti-EpCAM binding domain is fused to an N-terminus of the anti-CD3 Fab fragment, wherein the anti-EpCAM binding domain comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 13, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 14, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 15, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 16, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO: 18; wherein the glucocorticoid is for use simultaneously or prior to the multispecific Fab fusion protein, and wherein the use of the glucocorticoid is for reducing, inhibiting, or preventing: i) CRS, and / or ii) a side effect in liver in the individual.

200. The use of claim 199, wherein the use of glucocorticoid is for reducing, inhibiting, or preventing: i) release of one or more cytokines selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, and IFN-γ; and / or ii) elevated serum level of one or more liver enzymes selected from the group consisting of ALT, TBil, and ALP.

201. The use of claim 199 or 200, wherein the glucocorticoid is dexamethasone, or a pharmaceutically acceptable ester, salt, or complex thereof.

202. The use of any one of claims 199-201, wherein the anti-EpCAM binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 19, and / or a VL comprising the amino acid sequence of SEQ ID NO:

20.

203. The use of any one of claims 199-202, wherein the anti-EpCAM binding domain is an anti-EpCAM scFv.

204. The use of claim 203, wherein: i) the C-terminus of the anti-EpCAM scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment; or ii) the C-terminus of the anti-EpCAM scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment.

205. The use of claim 203, wherein the multispecific Fab fusion protein comprises a first anti- EpCAM scFv and a second anti-EpCAM scFv, wherein the C-terminus of the first anti-EpCAM scFv is fused to the N-terminus of the VH of the anti-CD3 Fab fragment, and wherein the C- terminus of the second anti-EpCAM scFv is fused to the N-terminus of the VL of the anti-CD3 Fab fragment.

206. The use of claim 205, wherein the first anti-EpCAM scFv and the second anti-EpCAM scFv have the same sequence.

207. The use of any one of claims 203-206, wherein: (i) the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO: 21, or (ii) the first anti-EpCAM scFv and the second anti- EpCAM scFv each comprises the amino acid sequence of SEQ ID NO:

21.

208. The use of claim 205, wherein the first anti-EpCAM scFv and the second anti-EpCAM scFv have different sequences.

209. The use of any one of claims 199-208, wherein the anti-CD3 Fab fragment is for specifically binding to the N-terminus of CD3 epsilon.

210. The use of claim 209, wherein the anti-CD3 Fab fragment is for specifically binding to an epitope within amino acids 1-27 of CD3 epsilon.

211. The use of claim 209 or 210, wherein the anti-CD3 Fab fragment comprises an HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, an HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, an HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3, an HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and an HVR-L3 comprising the amino acid sequence of SEQ ID NO:

6.

212. The use of claim 211, wherein the VH of the anti-CD3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 39-43; and / or wherein the VL of the anti-CD3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 44-47.

213. The use of claim 212, wherein the VH of the anti-CD3 Fab fragment comprises an amino acid sequence of SEQ ID NO: 7, and wherein the VL of the anti-CD3 Fab fragment comprises an amino acid sequence of SEQ ID NO:

8.

214. The use of any one of claims 211-213, wherein the anti-CD3 Fab fragment comprises a human CH1 comprising the amino acid sequence of SEQ ID NO:

9.

215. The use of any one of claims 211-214, wherein the anti-CD3 Fab fragment comprises a human lambda light chain constant region comprising the amino acid sequence of SEQ ID NO:

10.

216. The use of any one of claims 211-215, wherein the CH1 and the CL of the anti-CD3 Fab fragment are connected by one or more disulfide bonds.

217. The use of any one of claims 211-216, wherein the anti-CD3 Fab fragment comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and / or a second polypeptide comprising the amino acid sequence of SEQ ID NO:

12.

218. The use of any one of claims 211-217, wherein the multispecific Fab fusion protein comprises: (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 12; (ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 11, and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 23; or (iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22, and a second polypeptide comprising the amino acid sequence of SEQ ID NO:

23.

219. The use of any one of claims 199-218, wherein the multispecific Fab fusion protein is formulated for intravenous use.

220. The use of any one of claims 199-219, wherein the multispecific Fab fusion protein is for use at a low frequency.

221. The use of any one of claims 199-220, wherein the multispecific Fab fusion protein is for use twice weekly.

222. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>0.01μg / kg<annotation encoding="application / x-tex">0.01 \mu g / kg< / annotation>< / semantics>< / com:Math>.

223. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>0.05μg / kg<annotation encoding="application / x-tex">0.05 \mu g / kg< / annotation>< / semantics>< / com:Math>.

224. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>0.1μg / kg<annotation encoding="application / x-tex">0.1 \mu g / kg< / annotation>< / semantics>< / com:Math>.

225. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>0.3μg / kg<annotation encoding="application / x-tex">0.3 \mu g / kg< / annotation>< / semantics>< / com:Math>.

226. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>0.5μg / kg<annotation encoding="application / x-tex">0.5 \mu g / kg< / annotation>< / semantics>< / com:Math>.

227. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>0.6μg / kg<annotation encoding="application / x-tex">0.6 \mu g / kg< / annotation>< / semantics>< / com:Math>.

228. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at 1 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

229. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>1.2μg / kg<annotation encoding="application / x-tex">1.2 \mu g / kg< / annotation>< / semantics>< / com:Math>.

230. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>2μg / kg<annotation encoding="application / x-tex">2 \mu g / kg< / annotation>< / semantics>< / com:Math>.

231. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>2.4μg / kg<annotation encoding="application / x-tex">2.4 \mu g / kg< / annotation>< / semantics>< / com:Math>.

232. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>2.5μg / kg<annotation encoding="application / x-tex">2.5 \mu g / kg< / annotation>< / semantics>< / com:Math>.

233. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>3.6μg / kg<annotation encoding="application / x-tex">3.6 \mu g / kg< / annotation>< / semantics>< / com:Math>.

234. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at 4 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

235. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>5μg / kg<annotation encoding="application / x-tex">5 \mu g / kg< / annotation>< / semantics>< / com:Math>.

236. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>10μg / kg<annotation encoding="application / x-tex">10 \mu g / kg< / annotation>< / semantics>< / com:Math>.

237. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at 15 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

238. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>20μg / kg<annotation encoding="application / x-tex">20 \mu g / kg< / annotation>< / semantics>< / com:Math>.

239. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at 25 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

240. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>30μg / kg<annotation encoding="application / x-tex">30 \mu g / kg< / annotation>< / semantics>< / com:Math>.

241. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>50μg / kg<annotation encoding="application / x-tex">50 \mu g / kg< / annotation>< / semantics>< / com:Math>.

242. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at <com:Math><semantics>100μg / kg<annotation encoding="application / x-tex">100 \mu g / kg< / annotation>< / semantics>< / com:Math>.

243. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at 150 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

244. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at 200 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

245. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at 250 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg.

246. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 0.3 µg / kg for a cynomolgus monkey.

247. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 0.5 μg / kg for a cynomolgus monkey.

248. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 1 µg / kg for a cynomolgus monkey.

249. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 2 µg / kg for a cynomolgus monkey.

250. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 4 µg / kg for a cynomolgus monkey.

251. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 5 <com:Math><semantics>μ<annotation encoding="application / x-tex">\mu< / annotation>< / semantics>< / com:Math>g / kg for a cynomolgus monkey.

252. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 10 µg / kg for a cynomolgus monkey.

253. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 15 µg / kg for a cynomolgus monkey.

254. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 20 µg / kg for a cynomolgus monkey.

255. The use of any one of claims 199-221, wherein the multispecific Fab fusion protein is for use at a dose equivalent to 30 µg / kg for a cynomolgus monkey.

256. The use of any one of claims 199-255, wherein the use of the multispecific Fab fusion protein does not induce a cytokine storm.

257. The use of any one of claims 199-256, wherein the multispecific Fab fusion protein is for use consecutively at a first dose for a first period of time, and at a second dose for a second period of time, and wherein the second dose exceeds the first dose.

258. The use of claim 257, wherein the second period of time exceeds the first period of time.

259. The use of claim 257 or 258, wherein the multispecific Fab fusion protein is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at the second dose on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time.

260. The use of any one of claims 257-259, wherein the second dose is <com:Math><semantics>0.6μg / kg<annotation encoding="application / x-tex">0.6 \mu g / kg< / annotation>< / semantics>< / com:Math>.

261. The use of any one of claims 257-259, wherein the second dose is <com:Math><semantics>1.2μg / kg<annotation encoding="application / x-tex">1.2 \mu g / kg< / annotation>< / semantics>< / com:Math>.

262. The use of any one of claims 257-259, wherein the second dose is <com:Math><semantics>2.4μg / kg<annotation encoding="application / x-tex">2.4 \mu g / kg< / annotation>< / semantics>< / com:Math>.

263. The use of any one of claims 257-259, wherein the second dose is <com:Math><semantics>3.6μg / kg<annotation encoding="application / x-tex">3.6 \mu g / kg< / annotation>< / semantics>< / com:Math>.

264. The use of any one of claims 257-263, wherein the glucocorticoid is for use simultaneously or prior to the first dose of the multispecific Fab fusion protein.

265. The use of claim 264, wherein the glucocorticoid is for further use after the first dose of the multispecific Fab fusion protein, and simultaneously or prior to the second dose of the multispecific Fab fusion protein.

266. The use of claim 265, wherein the multispecific Fab fusion protein is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at a second dose of 0.6 µg / kg on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time; and wherein the glucocorticoid is for use 1 hour prior to the first dose of the multispecific Fab fusion protein on day 1 of the 7-day period of time, and 1 hour prior to the second dose of the multispecific Fab fusion protein on day 1 of the 21-day period of time.

267. The use of claim 265, wherein the multispecific Fab fusion protein is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at a second dose of 1.2 µg / kg on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time; and wherein the glucocorticoid is for use 1 hour prior to the first dose of the multispecific Fab fusion protein on day 1 of the 7-day period of time, and 1 hour prior to the second dose of the multispecific Fab fusion protein on day 1 of the 21-day period of time.

268. The use of claim 265, wherein the multispecific Fab fusion protein is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at a second dose of 2.4 µg / kg on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time; and wherein the glucocorticoid is for use 1 hour prior to the first dose of the multispecific Fab fusion protein on day 1 of the 7-day period of time, and 1 hour prior to the second dose of the multispecific Fab fusion protein on day 1 of the 21-day period of time.

269. The use of claim 265, wherein the multispecific Fab fusion protein is for use consecutively at a first dose of 0.3 µg / kg on day 1 and day 4 of a 7-day period of time, and at a second dose of 3.6 µg / kg on day 1, day 4, day 8, day 11, day 15, and day 18 of a 21-day period of time; and wherein the glucocorticoid is for use 1 hour prior to the first dose of the multispecific Fab fusion protein on day 1 of the 7-day period of time, and 1 hour prior to the second dose of the multispecific Fab fusion protein on day 1 of the 21-day period of time.

270. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 0.1 mg / kg.

271. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 0.5 mg / kg.

272. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 1 mg / kg.

273. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 2 mg / kg.

274. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 3 mg / kg.

275. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 4 mg / kg.

276. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 5 mg / kg.

277. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 1 mg.

278. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 2 mg.

279. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 5 mg.

280. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 10 mg.

281. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 15 mg.

282. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 20 mg.

283. The use of any one of claims 199-269, wherein the glucocorticoid is for use at 25 mg.

284. The use of any one of claims 199-283, wherein the cancer is an EpCAM-positive solid cancer.

285. The use of claim 284, wherein the EpCAM-positive solid cancer is a carcinoma or an adenocarcinoma.

286. The use of any one of claims 199-285, wherein the cancer is selected from the group consisting of small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, gastric cancer, pancreatic cancer, skin cancer, renal cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer.

287. The use of any one of claims 199-286, wherein the cancer is colorectal adenocarcinoma.

288. The use of any one of claims 199-286, wherein the cancer is lung adenocarcinoma.

289. The use of any one of claims 199-288, wherein the individual is a human.