HK1263052B - Multispecific fab fusion proteins and use thereof - Google Patents

Description

Multi-specificity Fab fusion protein and application thereof

Cross Reference to Related Applications

This application claims priority to chinese patent application No. 201610147227.8 filed on 2016, 3, 15, the entire contents of which are incorporated herein by reference in their entirety.

Sequence Listing text File

The contents of the following filed ASCII text files are incorporated herein by reference in their entirety: sequence Listing in Computer Readable Form (CRF) (filename: 720622000841SEQLIST. txt; recording date: 2017, 3 months, 13 days, size 48 KB).

Technical Field

The present invention relates to a multi-specific Fab fusion protein (MSFP) that specifically binds CD3 and EpCAM, and also provides pharmaceutical compositions comprising the Fab fusion protein, methods of using the MSFP to treat cancer, and kits comprising the MSFP.

Background

Certain antigens are overexpressed, mutated, or selectively mutated in tumor tissue. Thus, antibodies targeting cancer cell surface specific antigens are useful as cancer therapeutics. Epithelial cell adhesion factor (EpCAM, CD326) is a transmembrane glycoprotein with a molecular weight of 40kD, also known as 17-1A, ESA, AUA1, EGP40, etc., and has 314 amino acids. EpCAM is specifically expressed in a variety of epithelial cells and major types of human malignancies. For example, EpCAM is highly expressed in colon, lung, prostate, liver, pancreatic, breast and ovarian cancers. Thus, EpCAM has become a hot target in cancer therapy, including vaccines, murine or human monoclonal antibodies, as well as antibodies conjugated to bacterial toxins or chemotherapeutic drugs, such as EpCAM-specific antibody ING-1, alemtuzumab (Adecatumumab), edrecolomab (edrecolomab), and the like.

CD3, which comprises 3 different polypeptide chains (epsilon, delta, and gamma chains), is an antigen expressed by T cells. The 3 CD3 polypeptide chains associate with the T Cell Receptor (TCR) and zeta chain to form a TCR complex, which functions to activate a signal transduction cascade in T cells. Currently, various therapeutic strategies target TCR signaling to treat disease using anti-human CD3 monoclonal antibodies. The CD 3-specific antibody OKT3 was the first monoclonal antibody approved for human therapeutic use and was used clinically as an immunomodulator in the treatment of allograft rejection.

Over the last two decades, efforts in the field of bispecific antibodies have been increasingly successful clinically. In 2009, Cantomaxomab (anti-CD 3, an anti-EpCAM trifunctional antibody) was approved in the european union for the treatment of symptomatic malignant ascites. However, while bispecific antibodies have been shown to have potential in effectively killing cancer cells, serious side effects, including systemic immune activation, immunogenicity (anti-drug antibody effect), and the generally poor producibility of these molecules, have greatly limited the widespread use of such drugs. Another disadvantage of the CD19xCD3 bispecific scFv-scFv (single chain variable fragment) fusion protein (Blinatumomab) is that this drug requires daily intravenous (i.v.) administration due to its short half-life and incompatibility with subcutaneous administration; however, neurological reactions such as disorientation, disorganization, speech impairment, tremors or tics still occur during clinical trials (Science321: 974-. To better control these unwanted side effects, bispecific single chain antibodies (BiTE) are administered intravenously as continuous infusions over a longer period of time. US20120244161 discloses a phase I clinical trial of EpCAM x CD3 bispecific scFv-scFv fusion protein (MT110) wherein a low dose (1-12 μ g/kg/24 h) intravenous continuous infusion is applied for a long period of time and a glucocorticoid is administered prior to MT110 infusion or dose escalation. Furthermore, scFv-scFv fusion proteins have a tendency to aggregate.

The disadvantages of the current bispecific antibody formats remain a great challenge for the wide application of these drugs in the treatment of cancer patients with good efficacy and safety. Therefore, there is an urgent need in the art to develop new bispecific antibodies or therapeutic regimens with improved efficacy, stability, safety and producibility.

Brief description of the invention

The present invention provides a multi-specific Fab fusion protein, such as a bispecific Fab protein (BSFP), that specifically binds CD3 and EpCAM, and methods of treating cancer using the same.

In one aspect of the invention, there is provided a method of treating cancer in a subject (e.g., a human subject), the method comprising administering to the subject an effective amount of a multi-specific (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain that specifically binds EpCAM, wherein the binding domain is linked to the Fab fragment N-terminus, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (such as about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the binding domain is a scFv. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds EpCAM, wherein the first scFv is fused to the N-terminus of the Fab fragment VH and the second scFv is fused to the N-terminus of the Fab fragment VL. In some embodiments, the first scFv and the second scFv have the same sequence.

In some embodiments, the multi-specific Fab fusion protein is administered intravenously according to any one of the methods described above. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency (e.g., less than any of about 1, 2, 3, 4, 6, 9, or 12 months once, such as a single administration). In some embodiments, the multi-specific Fab fusion protein is administered at a dose equivalent to about 0.1 μ g/kg to about 100 μ g/kg (such as about 0.3 μ g/kg to about 5 μ g/kg, or about 5 μ g/kg to about 20 μ g/kg) to a cynomolgus monkey. In some embodiments, the multi-specific Fab fusion protein is administered at a dose that does not cause a cytokine storm. In some embodiments, the multi-specific Fab fusion protein is administered at a dose equivalent to no more than about 30 μ g/kg (e.g., no more than about 20 μ g/kg, 10 μ g/kg, or 1 μ g/kg) for a cynomolgus monkey. In some embodiments, the subject is a human subject.

In accordance with any one of the foregoing methods, in some embodiments, the multi-specific Fab fusion protein is administered to the individual at a first dose for a first period of time and subsequently, the multi-specific Fab fusion protein is administered to the individual at a second dose for a second period of time, and wherein the second dose exceeds the first dose. In some embodiments, the second time period exceeds the first time period. 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 μ g/kg. In some embodiments, the second dose is about 0.1 μ g/kg to about 10 μ g/kg.

In accordance with any one of the methods described above, in some embodiments, the method further comprises administering to the individual a glucocorticoid. In some embodiments, the glucocorticoid is dexamethasone. In some embodiments, the glucocorticoid is administered prior to the first dose of the multi-specific Fab fusion protein. In some embodiments, the glucocorticoid is administered at a dose of about 0.1mg/kg to about 5 mg/kg.

According to any one of the above methods, in some embodiments, the Fab fragment specifically binds to the N-terminus of CD3 e, such as an epitope within amino acids 1-27 of CD3 e. In some embodiments, the VH of the Fab fragment comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3. In some embodiments, the VL of the Fab fragment comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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% (e.g., 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% (e.g., 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 the heavy chain constant region 1(CH1) of a human immunoglobulin, wherein the heavy chain constant region 1 comprises the amino acid sequence of SEQ ID NO. 9. In some embodiments, the Fab fragment comprises a human λ light chain constant region comprising the amino acid sequence of SEQ ID No. 10. In some embodiments, CH1 and CL of the Fab fragment are linked 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% (e.g., 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% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID No. 12.

According to any one of the above methods, in some embodiments, the cancer is EpCAM positive solid cancer. In some embodiments, the EpCAM-positive cancer is a carcinoma (carcinosoma) or an adenocarcinoma (adenocarinoma). In some embodiments, the EpCAM positive cancer is selected from the group consisting of: small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer. In some embodiments, the cancer is colorectal adenocarcinoma. In some embodiments, the cancer is lung adenocarcinoma.

According to any one of the above methods, in some embodiments, the binding domain (e.g., scFv) comprises an N-VH-VL-C fusion polypeptide. In some embodiments, the VH of the scFv comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15. In some embodiments, the VL of the scFv comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and 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% (e.g., 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% (e.g., 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% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 21.

In some embodiments, the multi-specific Fab fusion protein comprises a first polypeptide comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the multi-specific Fab fusion protein comprises a second polypeptide comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID No. 23.

The invention also provides an anti-EpCAM antibody. In some embodiments, an anti-EpCAM antibody or antigen-binding fragment thereof is provided, comprising a heavy chain variable region comprising: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and (3) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region comprises: (1) HVR-L1 comprising the amino acid sequence of SEQ ID NO 16; (2) HVR-L2 comprising the amino acid sequence of SEQ ID NO 17; and (3) 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% (e.g., 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% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 20.

According to any one of the above anti-EpCAM antibodies, in some embodiments, 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.

According to any one of the above anti-EpCAM antigen-binding fragments, in some embodiments, 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% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 21.

In some embodiments, there is provided a multi-specific (e.g. bispecific) Fab fusion protein comprising any one of the anti-EpCAM antigen-binding fragments described hereinbefore. In some embodiments, the multi-specific (e.g., bispecific) Fab fusion protein comprises a Fab fragment that specifically binds CD3, a first copy of an anti-EpCAM antigen-binding fragment, and a second copy of an anti-EpCAM antigen-binding fragment; wherein a first copy of the anti-EpCAM antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment; and wherein a 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: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3. In some embodiments, the VL of the Fab fragment comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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% (e.g., 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% (e.g., 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 the 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 λ light chain constant region comprising the amino acid sequence of SEQ ID No. 10. In some embodiments, CH1 and CL of the Fab fragment are linked 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% (e.g., 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% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID No. 12. In some embodiments, the multi-specific (e.g., bispecific) Fab fusion protein comprises a first polypeptide comprising an amino acid sequence having at least about 85% (e.g., 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% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 23.

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

The invention still further provides an isolated nucleic acid molecule encoding a multi-specific Fab fusion protein (MSFP) as described above, an anti-EpCAM antibody or antigen-binding fragment thereof, an expression vector comprising the isolated nucleic acid molecule, an isolated host cell comprising the expression vector, and a method of producing the MSFP, anti-EpCAM antibody or antigen-binding fragment thereof, comprising culturing the isolated host cell and recovering the MSFP, anti-EpCAM antibody or antigen-binding fragment thereof from the cell culture.

The invention also provides uses, compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture comprising any of the aforementioned multi-specific Fab fusion proteins or anti-EpCAM antibodies or antigen-binding fragments thereof.

The invention also provides the use of any one of the above-described multi-specific Fab fusion proteins or any one of the above-described anti-EpCAM antibodies or antigen-binding fragments thereof in the manufacture of a medicament for the treatment of cancer.

These and other aspects and advantages of the present invention will be apparent from the detailed description and appended claims that follow. It is to be understood that one, some or all of the features of the various embodiments described herein may be combined with other embodiments of the invention.

The disclosures of all publications, patents, patent applications and published patent applications mentioned herein are hereby incorporated by reference in their entirety.

Drawings

Figure 1 depicts the structure of an exemplary EpCAM x CD3Fab fusion protein.

Figure 2A depicts an SDS-PAGE gel of a purified exemplary EpCAM x CD3Fab fusion protein (hereinafter ITAB1002) under non-reducing conditions. Non-reducing SDS-PAGE showed that the molecular weight of the purified protein was about 100kD, similar to the theoretical molecular weight of the EpCAM × CD3Fab fusion protein.

Figure 2B depicts an SDS-PAGE gel of purified ITAB1002 under reducing conditions. Reduced SDS-PAGE showed that the apparent molecular weight of the purified protein was between about 45kD and about 66 kD.

FIG. 3A depicts the results of a CE-SDS analysis of purified ITAB1002 under non-reducing conditions. The non-reducing CE-SDS analysis showed a single protein peak at a migration time of about 21.59 minutes.

FIG. 3B depicts the results of a CE-SDS analysis of purified ITAB1002 under reducing conditions. The reduced CE-SDS analysis showed two single protein peaks at migration times of approximately 18.37 minutes, 18.84 minutes, corresponding to the light and heavy chains of the Fab fusion protein, respectively.

Figure 4A depicts the binding affinity of ITAB1002 to human PBMCs expressing the cell surface antigen CD 3.

Figure 4B depicts the binding affinity of ITAB1002 to cynomolgus monkey PBMCs expressing the cell surface antigen CD 3.

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

FIG. 6A depicts ITAB1002 and OKT3 activating CD4 under different conditions⁺The ability of human PBMCs.

FIG. 6B depicts ITAB1002 and OKT3 activating CD8 under different conditions⁺The ability of human PBMCs.

FIG. 6C depicts ITAB1002 stimulating CD4 under various conditions⁺The ability of human PBMCs to proliferate.

FIG. 6D depicts ITAB1002 stimulating CD8 under various conditions⁺The ability of human PBMCs to proliferate.

Figure 7 depicts ITAB 1002-mediated cytotoxicity of human or cynomolgus monkey PBMCs against SW480 tumor cells.

Figure 8 depicts ITAB 1002-mediated cytotoxicity of human PBMCs against several representative cancer cell lines.

Figure 9A depicts the growth inhibitory effect of varying doses of ITAB1002 on subcutaneous SW480 xenografts co-inoculated with human PBMCs in mice.

Figure 9B shows photographs of tumors at the end of the experiment from mice in different treatment groups.

Figure 10A depicts the growth inhibitory effect of varying doses of ITAB1002 on SW480 xenografts in immunodeficient mice reconstituted with the human PBMC immune system.

Figure 10B shows photographs of tumors at the end of the experiment from mice in different treatment groups.

FIG. 10C shows human CD3 in a leukocyte sample from mice at the end of the experiment⁺Percentage of cells.

FIG. 11 depicts the growth inhibitory effect of ITAB1002 at different doses on NCI-H1975 xenografts in immunodeficient mice reconstituted with the human PBMC immune system.

Figure 12A depicts CD4 in cynomolgus monkey blood following intravenous administration of ITAB1002 at different doses⁺The number of T cells varied with time (X axis: H ═ H; D ═ day).

Figure 12B depicts CD8 in cynomolgus monkey blood following intravenous administration of ITAB1002 at different doses⁺The number of T cells varied with time (X axis: H ═ H; D ═ day).

Figure 13A depicts IL-2 concentration in cynomolgus monkey serum over time after intravenous administration of ITAB1002 at different doses.

Figure 13B depicts IL-4 concentration in cynomolgus monkey serum over time after intravenous administration of ITAB1002 at different doses.

Figure 13C depicts IL-5 concentration in cynomolgus monkey serum over time after intravenous administration of ITAB1002 at different doses.

Figure 13D depicts IL-6 concentration in cynomolgus monkey serum over time after intravenous administration of ITAB1002 at different doses.

Figure 13E depicts TNF concentrations in cynomolgus monkey serum over time following intravenous administration of ITAB1002 at different doses.

Figure 13F depicts IFN- γ concentration in cynomolgus monkey serum over time after intravenous administration of ITAB1002 at different doses.

Figure 14 depicts the change in ITAB1002 concentration in cynomolgus monkey serum over time following a single intravenous administration of ITAB1002 at different doses. .

Figure 15 compares ITAB1002 and ITAB 1012-mediated cytotoxicity of human PBMC on SW480 tumor cells.

Figure 16 depicts MSFP-mediated killing activity against SW480 cells in the presence or absence of Dexamethasone (DXM).

FIG. 17 depicts MSFP-mediated IL-6 release from human T cells in the presence or absence of Dexamethasone (DXM).

Figure 18A shows alanine Aminotransferase (ALT) serum levels in monkeys treated with ITAB1002 alone.

Figure 18B shows alanine Aminotransferase (ALT) serum levels in monkeys treated with ITAB1002 and Dexamethasone (DXM) pretreatment.

Figure 19A shows total bilirubin (TBil) serum levels in monkeys treated with ITAB1002 alone.

Figure 19B shows total bilirubin (TBil) serum levels in monkeys treated with ITAB1002 and Dexamethasone (DXM) pretreatment.

Figure 20A shows alkaline phosphatase (ALP) serum levels in monkeys treated with ITAB1002 alone.

Fig. 20B shows alkaline phosphatase (ALP) serum levels in monkeys treated with ITAB1002 and Dexamethasone (DXM) pretreatment.

Figure 21A shows IL-6 levels in monkeys treated with ITAB1002 alone.

Figure 21B shows IL-6 levels in monkeys treated with ITAB1002 and Dexamethasone (DXM) pretreatment.

Detailed Description

The present invention provides methods for treating cancer by administering a multi-specific Fab fusion protein (MSFP) comprising a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM. In some embodiments, the MSFP comprises an anti-CD 3Fab fragment whose N-termini of the heavy and light chain polypeptides are each fused to an scFv of anti-EpCAM. In contrast to the current state of the art anti-cancer bispecific antibodies which suffer from poor producibility, aggregation, short half-lives, severe side effects, long infusion times, the multi-specific Fab fusion proteins described herein have an improved stability and safety profile, which enables therapeutic approaches with lower doses and reduced administration frequency, avoiding undesired side effects such as the induction of cytokine storms. The reduced frequency of administration and shortened infusion time facilitates treatment of the patient, and is beneficial to improving the quality of life of the patient. For example, it has been surprisingly found that the MSFP described herein can be administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg, such as about 0.01 μ g/kg to 5 μ g/kg, about 0.1 μ g/kg to 30 μ g/kg, or about 2.5 μ g/kg to 100 μ g/kg. Novel anti-EpCAM antibodies or antigen-binding fragments thereof are also provided.

Compared with other multi-specific Fab fusion proteins known in the field, the MSFP provided by the invention has the following advantages: the MSFP has higher stability and enhanced efficacy of killing cancer cells. The extended half-life of the MSFP of the invention enables lower frequency of administration and shorter infusion times, providing more convenience to the patient. The cross-reactivity of the MSFP of the invention with primates such as cynomolgus monkeys facilitates toxicology studies. The MSFP of the invention has fewer side effects, including reduced neurological effects, and good safety and tolerability in cynomolgus monkeys.

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

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

Kits and articles of manufacture for use in the methods of the invention are also provided.

I. Term(s)

The practice of the present invention will employ, unless specifically indicated to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA technology within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained in detail in the literature below. 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 rd edition, Wiley & Sons, 1995; sambrook and Russell, Molecular Cloning: A Laboratory Manual (3 rd edition, 2001); molecular Cloning, A Laboratory Manual (1982); DNA Cloning: A Practical Approach, volumes I and II (compiled by D.Glover); oligonucleotide Synthesis (n. gait eds., 1984); nucleic Acid Hybridization (B.Hames & S.Higgins eds, 1985); transcription and transformation (B.Hames & S.Higgins eds, 1984); animal Cell Culture (ed. r. freshney, 1986); perbal, A Practical Guide to Molecular Cloning (1984) and other similar references.

As used herein, the term "treatment" refers to clinical interventions designed to alter the natural course of the treated individual or cell in the course of clinical pathology. Desirable therapeutic effects include reducing the chance of disease progression, improving or alleviating the disease state, and ameliorating or improving prognosis. For example, an individual is considered to be successfully "treated" if one or more symptoms associated with the tumor are reduced or eliminated, including but not limited to reducing the proliferation of or destroying cancer cells, reducing symptoms resulting from the disease, increasing the quality of life of those (patients) afflicted with the disease, reducing the dose of other drugs required to treat the disease, and/or prolonging survival of the individual.

As used herein, the term "effective amount" refers to an amount of an agent or drug effective for a disease or condition in a subject. In the case of cancer, an effective amount of the agent can reduce the number of cancer cells; reducing the volume of the tumor; inhibit (i.e., slow to some extent and preferably prevent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more symptoms associated with cancer to some extent. As understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" can be considered in the context of administering one or more therapeutic agents, and it is contemplated that a single agent can be administered in an effective amount if (in combination with one or more other agents) a desired result is achieved or achieved.

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

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. The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody".

The terms "natural antibody," "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably to refer to an antibody in substantially intact form, rather than an antibody fragment as defined below. The term specifically refers to an antibody having a heavy chain comprising an Fc region. Natural antibodies are typically heterotetrameric glycoproteins of about 150kD in size, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin subtypes. Each weightThe chains and light chains also have regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain (V) at one end _H) Then a plurality of constant domains. Each light chain has a variable domain (V) at one end_L) And a constant domain at its other end; the light chain constant domain 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. It is believed that particular amino acid residues form the interface between the light and heavy chain variable domains.

The term "constant region" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to the rest of the immunoglobulin molecule (the variable region that comprises the antigen binding site). Constant region comprising heavy chain C_H1、C_H2 and C_H3 domain (commonly known as CH) and CHL (or CL) of the light chain.

The "variable region" or "variable domain" refers to the amino-terminal domain of an antibody heavy or light chain. 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 typically the most variable parts of an antibody and contain an antigen binding site.

The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of the antibody. It is concentrated in three segments called hypervariable regions (HVRs, also called CDRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called the Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, which mostly adopt a β -sheet configuration, connected by three HVRs that form a loop junction (and in some cases form part of the β -sheet structure). The HVRs in each chain are held together in close proximity by the FR region and, together with HVRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, 5 th edition, National Institute of Health, Bethesda, Md. (1991)). Constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as participation of antibodies in antibody-dependent cellular cytotoxicity.

The "light chains" of antibodies (immunoglobulins) from any mammalian species can be divided into two distinct classes, called "kappa" and "lambda", based on the amino acid sequences of their constant domains.

The term "isotype" or "subclass" as used herein means any subclass of immunoglobulins defined by the chemical or antigenic characteristics of their constant regions.

Depending on the amino acid sequence of the constant domains of the heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM. A number of subclasses (isotypes) can be further divided, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to different types of immunoglobulins may be referred to as: α, γ, ε, γ, and μ. The subunit structures and three-dimensional configurations of different types of immunoglobulins are known and are generally described, for example, in Abbas et al, Cellular and mol. An antibody may be part of a larger fusion molecule formed by covalent or non-covalent attachment of the antibody to one or more other proteins or peptides.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. In some embodiments, the antibody fragment is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab ', F (ab') ₂And Fv fragments; a diabody; a linear antibody; single chain antibody molecules and multispecific antibodies formed from antibody fragments.

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, the name of which reflects its ability to crystallize readily. Pepsin treatment of antibodies to F (ab')₂A fragment which has two antigen binding sites and is still capable of cross-linking antigens.

"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In some embodiments, a two-chain Fv consists of a dimer of one heavy chain variable region and one light chain variable region in tight, non-covalent association. In single chain Fv (scfv), one heavy chain variable domain and one light chain variable domain may be covalently linked by a flexible peptide linker, so that the heavy and light chains can associate in a "dimeric" structure similar to a two-chain Fv. In this configuration, 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, albeit with a lower affinity than the entire binding site.

The Fab fragment has two polypeptide chains, comprising a heavy chain and a light chain variable domain, and further comprising the constant region of the light chain and the first constant domain of the heavy chain (CH 1). 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 residues of the constant domains carry a free thiol group. F (ab ') 2 antibody fragments were originally produced as paired Fab ' fragments with hinge cysteines between the Fab ' fragments. Other chemical conjugates of antibody fragments are also known.

"Single chain Fv", or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present as a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For reviews on scFv see, for example, Pluckth ü n, The Pharmacology of Monoclonal Antibodies, Springer Berlin Heidelberg, 1994.269-315.

The Fc fragment contains the carboxy terminal portions of two H chains held together by disulfide bonds. The effector function of an antibody is determined by sequences in the Fc region, which is also the portion recognized by Fc receptors (fcrs) found on certain types of cells.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprised in the population are identical except for possible mutations that may be present in minor amounts (e.g., naturally occurring mutations). Thus, the modifier "monoclonal" indicates that the antibody is characterized as not being a mixture of discrete antibodies. In some embodiments, such monoclonal antibodies generally include an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be to select a unique clone from a plurality of clones (e.g., hybridoma clones, phage clones, or pools of recombinant DNA clones). It will be appreciated that the target binding sequence selected may be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its yield 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 the invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins.

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, Monoclonal Antibodies to be used in accordance with the present invention can 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, 2 nd edition, 1988); Hammerling et al, Monoclonal Antibodies and T-Cell Hybridoma 563-681(Elsevier, N.Y., 1981)); recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567), phage display techniques (see, for example, 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-, Bio/Technology 10:779-783 (1992); lonberg et al, Nature 368:856-859 (1994); morrison, Nature 368: 812-; fishwild et al, Nature Biotechnol.14: 845-; neuberger, Nature Biotechnol.14:826 (1996); and Lonberg and Huszar, Intern.Rev.Immunol.13:65-93 (1995)).

Such monoclonal antibodies specifically include "chimeric" antibodies wherein a portion of the heavy and/or light chain is identical to 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 is identical to 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; Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies herein include "primatizedAn "antibody, wherein the antigen-binding region of the antibody is derived from an antibody produced by, for example, immunizing a cynomolgus monkey (macaque monkey) with an antigen of interest.

"humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced with residues from an HVR of a non-human species (donor antibody), such as a mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and/or capacity. In some embodiments, FR residues of a human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications can be made to further improve antibody performance. In general, a humanized antibody will comprise substantially all of the variable domains in at least one, and typically two, 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 more details see, e.g., Jones et al, Nature 321:522-525 (1986); riechmann et al, Nature 332: 323-E329 (1988); and Presta, curr, Op, Structure, 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-; hurle and Gross, curr. Op. Biotech.5: 428-; and U.S. patent nos. 6,982,321 and 7,087,409.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibody specifically excludes humanized antibodies that comprise non-human antigen binding residues. Human antibodies can be generated using a variety of 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 useful for preparing human Monoclonal Antibodies are the methods described in the literature (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 made by administering an antigen to a transgenic animal, such as an immunized XENOMOUSE (xenomic), that has been modified to produce human antibodies in response to antigenic stimuli, but whose endogenous genome has been disabled (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for Xenomose^TMA technique). See also, e.g., Li et al, Proc. Natl. Acad. Sci. USA,103:3557-3562(2006), Guanyu Human antibodies generated via human B-cell hybridoma technology.

As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region of an antibody variable domain which is highly variable in sequence and/or forms structurally defined loops. Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Among natural antibodies, H3 and L3 showed the greatest diversity among the six HVRs, and H3 was specifically thought to play a unique role in conferring precise 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 eds., Human Press, Totowa, N.J., 2003). In fact, naturally occurring camelid (camelid) antibodies, which consist of only heavy chains, are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al, Nature 363: 446-; sheriff et al, Nature struct.biol.3:733-736 (1996). HVRs are also referred to as "CDRs" or "complementarity determining regions".

The structure and position of immunoglobulin variable regions can be determined by reference to Kabat, e.a. et al, Sequences of Proteins of Immunological interest, 4 th edition, US Department of Health and Human services, 1987 and its updates, currently available on the internet (immunol.

"framework" or "FR" residues refer to those variable domain residues in the variable domain other than the HVR residues defined herein.

As used herein, the terms "bind," "specific binding," or "specific for …" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that binds or specifically binds a target (which may be an epitope) is an antibody that binds this target with greater affinity (affinity), avidity (avidity), more readily, and/or for a greater duration than it binds other targets. In one embodiment, the extent to which the antibody binds to an unrelated target is less than about 10% of the extent to which the antibody binds to the target, as measured, for example, by Radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds a target has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, or less than or equal to 0.1 nM. In some embodiments, the antibody specifically binds to an epitope on the protein that is conserved among proteins from different species. In another embodiment, specific binding may include, but need not be exclusive binding.

As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide or antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular peptide chain or polypeptide sequence, by 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 determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill in the art, e.g., using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN^TM(DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared.

Amino acid substitutions can include, but are not limited to, substitution of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in table a. Amino acid substitutions can be introduced into an antibody of interest and the product screened for a desired activity, e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

TABLE A

Amino acids can be grouped according to common side chain properties as: (1) hydrophobic amino acid: norleucine (Norleucine), Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic amino acids: cys, Ser, Thr, Asn, Gln; (3) acidic amino acids: asp, Glu; (4) basic amino acids: his, Lys, Arg; (5) residues that influence chain orientation: gly, Pro; (6) aromatic amino acids: trp, Tyr, Phe. Non-conservative substitutions will require the replacement of a member of one of these classes with an amino acid of another class.

As used herein, a "Fab fusion protein" refers to a protein having a Fab fragment covalently linked to one or more binding domains, wherein the binding domains have different properties relative to the Fab fragment. The property may be a biological property, such as an in vitro or in vivo activity. The property may also be a simple chemical or physical property, such as binding to a target molecule, a catalytic reaction, etc. The Fab fragment and the one or more binding domains may be linked by a peptide bond directly or via a peptide linker, but in frame with each other.

The term "multispecific" when used in conjunction with an antibody (e.g., a Fab fusion protein) means that the antibody (e.g., Fab fusion protein) has polyepitopic specificity (e.g., is capable of specifically binding two, three, or more different epitopes on one biomolecule, or is capable of specifically binding two, three, or more different epitopes on different biomolecules).

The term "bispecific" when used in conjunction with an antibody (e.g., a Fab fusion protein) means that the antibody (e.g., a Fab fusion protein) is capable of specifically binding to two different epitopes on one biomolecule, or is capable of specifically binding to epitopes on two different biomolecules. The order of the antigens bound by the bispecific antibody listed in the bispecific antibody or Fab fusion protein name is arbitrary, i.e., the terms "anti-CD 3/EpCAM", "anti-EpCAM/CD 3", "EpCAM × CD 3" and "CD 3 × EpCAM" are used interchangeably to refer to bispecific antibodies (e.g., bispecific Fab fusion proteins) that specifically bind CD3 and EpCAM, among other things.

The terms "multi-specific Fab fusion protein" and "MSFP" are used interchangeably herein to refer to a Fab fusion protein with multi-epitope specificity.

As used herein, the "C-terminus" of a polypeptide refers to the last amino acid residue of the polypeptide which contributes its amino group to form a peptide bond with the carboxyl group of its adjacent amino acid residue. As used herein, the "N-terminus" of a polypeptide refers to the first amino acid of the polypeptide, which contributes its carboxyl group to form a peptide bond with the amino group of its adjacent amino acid residue.

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 vectors which are self-replicating nucleic acid structures, as well as vectors which are integrated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked, and such vectors are referred to herein as "expression vectors".

The term "cell" encompasses a primary subject cell and its progeny.

The term "cytokine storm," also known as "cytokine cascade" or "hypercytokinemia," is a potentially lethal immune response generally consisting of a positive feedback loop between cytokines and immune cells, with highly elevated levels of various cytokines (e.g., INF- γ, IL-10, IL-6, CCL2, etc.).

It is to be understood that the embodiments described herein include embodiments "consisting of …" and/or "consisting essentially of …".

Reference to "about" a value or parameter includes (and describes) variations that point to the value or parameter itself. For example, a description of "about X" includes a description of "X".

As used herein, reference to a "non/non" value or parameter is generally intended and described as "in addition to" a value or parameter ". For example, the method is not used to treat type X cancer means that the method is used to treat cancer other than type X.

The term "about X-Y" has the same meaning as "about X to about Y".

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

Methods of treating cancer

In one aspect of the present invention, there is provided a method of treating cancer in an individual (e.g. human) comprising administering to the individual an effective amount of a multi-specific (e.g. bi-specific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain (e.g., scFv) is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method does not cause a cytokine storm.

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

In some embodiments, there is provided a method of inhibiting the proliferation of a (e.g., human) cancer cell in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain (e.g., scFv) is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method does not cause a cytokine storm. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone).

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

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

In some embodiments, the MSFP specifically binds more than two (e.g., 3 or more) epitopes. In some embodiments, the MSFP is a trispecific Fab fusion protein further comprising a binding domain (e.g., scFv) that specifically binds to a cell surface protein (e.g., not EpCAM). In some embodiments, the MSFP comprises a multispecific (e.g., bispecific) binding domain.

In some embodiments, the MSFP comprises a single binding domain that specifically binds EpCAM. In some embodiments, the single binding domain comprises a single polypeptide chain. In some embodiments, the binding domain is a 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.

In some embodiments, the MSFP comprises two binding domains that specifically bind 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, the two binding domains each comprise a single polypeptide chain. In some embodiments, the two binding domains are each 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.

Thus, in some embodiments, there is provided a method of treating cancer in an individual (e.g., human) comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds CD3, (2) a first binding domain (e.g., scFv) that specifically binds EpCAM, (3) a second binding domain (e.g., scFv) that specifically binds 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 multi-specific (e.g., bispecific) Fab fusion protein is administered at a dose of about 0.01 to about 250 μ g/kg (e.g., about 0.01 to about 5 μ g/kg, about 0.1 to about 30 μ g/kg, about 2.5 to about 100 μ g/kg). In some embodiments, the first binding domain (e.g., scFv) and the second binding domain (e.g., scFv) have the same sequence. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone).

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

In some embodiments, there is provided a method of inhibiting cancer cell proliferation in an individual (e.g., human) comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds CD3, (2) a first binding domain (e.g., scFv) that specifically binds EpCAM, (3) a second binding domain (e.g., scFv) that specifically binds 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 multi-specific (e.g., bispecific) Fab fusion protein is administered at a dose of about 0.01 to about 250 μ g/kg (e.g., about 0.01 to about 5 μ g/kg, about 0.1 to about 30 μ g/kg, about 2.5 to about 100 μ g/kg). In some embodiments, the first binding domain (e.g., scFv) and the second binding domain (e.g., scFv) have the same sequence. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone).

In some embodiments, there is provided a method of inducing peripheral T cell redistribution in an individual (e.g., human) comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds CD3, (2) a first binding domain (e.g., scFv) that specifically binds EpCAM, (3) a second binding domain (e.g., scFv) that specifically binds 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 multi-specific (e.g., bispecific) Fab fusion protein is administered at a dose of about 0.01 to about 250 μ g/kg (e.g., about 0.01 to about 5 μ g/kg, about 0.1 to about 30 μ g/kg, about 2.5 to about 100 μ g/kg). In some embodiments, the first binding domain (e.g., scFv) and the second binding domain (e.g., scFv) have the same sequence. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone).

The Fab fragment may be derived from any suitable anti-CD 3 antibody known in the art. In some embodiments, the Fab fragment specifically binds to the N-terminus of CD 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 SP 34. In some embodiments, the Fab fragment comprises any one, two, or three HVRs (or CDRs) of the SP34 heavy chain variable region, such as the HVRs comprising the amino acid sequence 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 the HVRs comprising the amino acid sequence 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 the group consisting of SEQ ID NO: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 the heavy chain constant region 1(CH1) of a human immunoglobulin, such as CH1 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, CH1 and CL of the Fab fragment are linked 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.

Thus, in some embodiments, there is provided a method of treating cancer in an individual (e.g., human) comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) Fab fusion protein, wherein the Fab fusion protein comprises: a Fab fragment that specifically binds CD3, wherein the binding domain is fused to the N-terminus of the Fab fragment, and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the Fab fragment comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the heavy chain variable region comprises: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and (3) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; the light chain variable region comprises: (1) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, (2) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and (3) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 6; wherein the multi-specific (e.g., bispecific) Fab fusion protein is administered at a dose of about 0.01 to about 250 μ g/kg (e.g., about 0.01 to about 5 μ g/kg, about 0.1 to about 30 μ g/kg, about 2.5 to about 100 μ g/kg). In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 7 and 39-43 and/or a light chain variable region (VL) comprising 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 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) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone).

In some embodiments, there is provided a method of treating cancer in an individual (e.g., human) comprising administering to the individual an effective amount of a multi-specific (e.g., 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) and/or a light chain variable region (VL), wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO. 6; (2) a first binding domain (e.g., scFv) that specifically binds EpCAM; and (3) a second binding domain (e.g., scFv) that specifically binds EpCAM; wherein the first binding domain (e.g., scFv) is fused to the N-terminus of the VH of the Fab fragment, wherein the second binding domain (e.g., scFv) is fused to the N-terminus of the VL of the Fab fragment, and wherein the multispecific (e.g., bispecific) Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the first binding domain (e.g., scFv) and the second binding domain (e.g., 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 the group consisting of SEQ ID NOS: 7 and 39-43 and/or a light chain variable region (VL) comprising 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 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) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone).

In some embodiments, the binding domain that specifically binds EpCAM (also referred to 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 an scFv. In some embodiments, the scFv comprises an N-VH-VL-C fusion polypeptide. In some embodiments, the EpCAM binding domain is derived from an EpCAM antibody of the invention comprising any one, two, or three HVRs of the heavy chain variable region, which HVRs comprise the amino acid sequence of SEQ ID NOs 13-15. In some embodiments, the EpCAM binding domain comprises any one, two, or three HVRs of a light chain variable region, wherein the HVRs comprise 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 to the subject a glucocorticoid (e.g., dexamethasone).

Thus, in some embodiments, there is provided a method of treating cancer in an individual (e.g., human) comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) Fab fusion protein comprising: (1) fab fragments that specifically bind CD3 (e.g., amino acids 1-27 of the N-terminus of CD 3. epsilon.); (2) a first scFv that specifically binds EpCAM; and (3) a second scFv that specifically binds EpCAM; wherein a first scFv is fused to the N-terminus of the VH of the Fab fragment, wherein a second scFv is fused to the N-terminus of the VL of the Fab fragment, wherein the first and/or second scFv comprises a heavy chain variable region (VH) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18; wherein the multi-specific (e.g., bispecific) Fab fusion protein is administered at a dose of about 0.01 to about 250 μ g/kg (e.g., about 0.01 to about 5 μ g/kg, about 0.1 to about 30 μ g/kg, about 2.5 to about 100 μ 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) inhibiting cancer cell proliferation; and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone).

In some embodiments, there is provided a method of treating cancer in an individual (e.g., human) comprising administering to the individual an effective amount of a multi-specific (e.g., 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) and/or a light chain variable region (VL), wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO. 6; (2) a first scFv that specifically binds EpCAM; and (3) a second scFv that specifically binds EpCAM; wherein a first scFv is fused to the N-terminus of the VH of the Fab fragment, wherein a second scFv is fused to the N-terminus of the VL of the Fab fragment, wherein the first and/or second scFv comprises a heavy chain variable region (VH) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18; wherein the multi-specific (e.g., bispecific) Fab fusion protein is administered at a dose of about 0.01 to about 250 μ g/kg (e.g., about 0.01 to about 5 μ g/kg, about 0.1 to about 30 μ g/kg, about 2.5 to about 100 μ g/kg). In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 7 and 39-43 and/or a light chain variable region (VL) comprising 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 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) inhibiting cancer cell proliferation; and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone).

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 by a linker, such as a flexible peptide linker, e.g., a peptide linker comprising glycine and serine. In some embodiments, the multi-specific (e.g., bispecific) Fab fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the multi-specific (e.g., bispecific) Fab fusion protein comprises a second polypeptide comprising the amino acid sequence of SEQ ID NO: 23.

Thus, in some embodiments, there is provided a method of treating cancer in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., 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 multi-specific Fab fusion protein is administered at a dose of about 0.01 to about 250 μ g/kg (e.g., about 0.01 to about 5 μ g/kg, about 0.1 to about 30 μ g/kg, about 2.5 to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting cancer cell proliferation; and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone).

The method provided by the invention can be implemented by auxiliary setting. In some embodiments, the method is a new adjuvant setting, i.e., the method can be performed prior to primary/definitive treatment. In some embodiments, the method is used to treat an individual who has been previously treated. Any of the methods of treatment provided by the present invention can be used to treat a previously untreated individual. 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 to the subject a glucocorticoid (e.g., dexamethasone).

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

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

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

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

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

In some embodiments, there is provided a method of increasing the time to disease progression of cancer in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method extends the time to disease progression by at least any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone).

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

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

The methods of the invention are suitable for treating a variety of cancers, including solid and liquid tumors. The methods are applicable to all stages of cancer, including early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, or cancer in remission. The method can be used as an adjuvant or neoadjuvant setting in a first therapy, a second therapy, a third therapy, or in combination with other cancer treatment regimens 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, radiofrequency ablation, and the like. In some embodiments, the cancer is refractory to existing therapies.

The types of cancers that can be treated by the methods of the invention include, but are not limited to, adrenocortical cancer, AIDS-related cancers (e.g., AIDS-related lymphomas), anal cancer, appendiceal cancer, astrocytomas (e.g., cerebellar and cerebral), basal cell carcinoma, cholangiocarcinoma (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumors (e.g., gliomas, brain stem gliomas, cerebellar or cerebral astrocytomas (e.g., hairy cell astrocytomas, diffuse astrocytomas, anaplastic (malignant) astrocytomas), malignant gliomas, ependymomas, oligodendrogliomas, meningiomas, craniopharyngiomas, angioblastomas, medulloblastomas, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas, and glioblastoma), breast cancer, bronchial adenocarcinoma/carcinoid-carcinoma, cancer, melanoma, and melanoma Carcinoid tumors (e.g., gastrointestinal carcinoid tumors), unknown primary cancers, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative diseases, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, ewing's family tumors, ocular cancers (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (gastric) cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumors (GIST), germ cell tumors, (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumors, head and neck cancer, hepatocellular (liver) cancer (e.g., liver cancer (hepatoma) and hepatoma (heptoma)), hypopharynx cancer, islet cell cancer (endocrine pancreas), laryngeal cancer, leukemia, lip and oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, cervical cancer, pancreatic cancer, and pancreatic cancer, and pancreatic cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung); lymphoid tumors (e.g., lymphoma), medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer, oral cancer, multiple endocrine tumor syndrome, myelodysplastic/myelo-and extramyeloproliferative diseases, cancers of the nasal cavity and sinuses, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer (e.g., epithelial ovarian cancer, ovarian germ cell tumor, tumor of low malignant potential of the ovary), pancreatic cancer, parathyroid cancer, penile cancer, cancer of the peritoneum, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, pleural blastoblastoma, lymphoma, primary central nervous system lymphoma (microglioma), rectal cancer, renal pelvis and ureter cancer (transitional cell carcinoma), rhabdomyosarcoma, salivary gland carcinoma, neuroblastoma, bladder carcinoma of the head and throat, Skin cancers (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma, merkel cell carcinoma), small intestine cancer, squamous cell carcinoma, testicular cancer, throat cancer, thymoma and carcinoma of the thymus, thyroid cancer, urinary tract cancer, vaginal cancer, vulvar cancer, wilms' tumor, and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with nevus maculatus, edema (e.g., associated with brain tumors), and megger syndrome.

In some embodiments, the methods are suitable for treating a cancer that overexpresses EpCAM on the surface of a cancer cell, such as an EpCAM-positive solid cancer. In some embodiments, the cancer cell expresses EpCAM in the individual at any one of 2, 5, 10, 20, 50, 100, 200, 500, 1000 or more fold greater compared to a normal cell. 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, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

Thus, in some embodiments, there is provided a method of treating an EpCAM positive solid cancer (e.g., carcinoma or adenocarcinoma) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). 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, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

In some embodiments, there is provided a method of treating an EpCAM positive solid cancer (e.g., carcinoma or adenocarcinoma) in an individual comprising administering to the individual an effective amount of a multispecific (e.g., bispecific) Fab fusion protein comprising: (1) a Fab fragment that specifically binds CD3, (2) a first binding domain (e.g., scFv) that specifically binds EpCAM, (3) a second binding domain (e.g., scFv) that specifically binds 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 multi-specific (e.g., bispecific) Fab fusion protein is administered at a dose of about 0.01 to about 250 μ g/kg (e.g., about 0.01 to about 5 μ g/kg, about 0.1 to about 30 μ g/kg, about 2.5 to about 100 μ g/kg). In some embodiments, the first binding domain (e.g., scFv) and the second binding domain (e.g., scFv) have the same sequence. In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). 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, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

In some embodiments, there is provided a method of treating an EpCAM positive solid cancer (e.g., carcinoma or adenocarcinoma) in an individual comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) Fab fusion protein, wherein the Fab fusion protein comprises: a Fab fragment that specifically binds CD3, wherein the binding domain is fused to the N-terminus of the Fab fragment, and a binding domain (e.g., scFv) that specifically binds EpCAM, the Fab fragment comprising a heavy chain variable region (VL) comprising: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and (3) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; the light chain variable region comprises: (1) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, (2) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and (3) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 6; wherein the multi-specific Fab fusion protein is administered at a dose of about 0.01 to about 250 μ g/kg (such as about 0.01 to about 5 μ g/kg, about 0.1 to about 30 μ g/kg, about 2.5 to about 100 μ g/kg). In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 7 and 39-43 and/or a light chain variable region (VL) comprising 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 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) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). 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, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

In some embodiments, there is provided a method of treating an EpCAM positive solid cancer (e.g., carcinoma or adenocarcinoma) in an individual comprising administering to the individual an effective amount of a multispecific (e.g., bispecific) Fab fusion protein comprising: (1) fab fragments that specifically bind CD3 (e.g., amino acids 1-27 of the N-terminus of CD 3. epsilon.); (2) a first scFv that specifically binds EpCAM; and (3) a second scFv that specifically binds EpCAM; wherein a first scFv is fused to the N-terminus of the VH of the Fab fragment, wherein a second scFv is fused to the N-terminus of the VL of the Fab fragment, wherein the first and/or second scFv comprises a heavy chain variable region (VH) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18; wherein the multi-specific (e.g., bispecific) Fab fusion protein is administered at a dose of about 0.01 to about 250 μ g/kg (e.g., about 0.01 to about 5 μ g/kg, about 0.1 to about 30 μ g/kg, about 2.5 to about 100 μ 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) inhibiting cancer cell proliferation; and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). 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, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

In some embodiments, there is provided a method of treating an EpCAM positive solid cancer (e.g., carcinoma or adenocarcinoma) in an individual comprising administering to the individual an effective amount of a multispecific (e.g., 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) and/or a light chain variable region (VL), wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and HVR-L3 comprising the amino acid sequence of SEQ ID NO. 6; (2) a first scFv that specifically binds EpCAM; and (3) a second scFv that specifically binds EpCAM; wherein a first scFv is fused to the N-terminus of the VH of the Fab fragment, wherein a second scFv is fused to the N-terminus of the VL of the Fab fragment, wherein the first and/or second scFv comprises a heavy chain variable region (VH) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18; wherein the multi-specific (e.g., bispecific) Fab fusion protein is administered at a dose of about 0.01 to about 250 μ g/kg (e.g., about 0.01 to about 5 μ g/kg, about 0.1 to about 30 μ g/kg, about 2.5 to about 100 μ g/kg). In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 7 and 39-43 and/or a light chain variable region (VL) comprising 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 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) inhibiting cancer cell proliferation; and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). 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, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

In some embodiments, a method of treating an EpCAM positive solid cancer (e.g., carcinoma or adenocarcinoma) in an individual is provided comprising administering to the individual an effective amount of a multi-specific (e.g., 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 multi-specific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells; (2) inhibiting cancer cell proliferation; and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). 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, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, breast cancer, bile duct cancer, and head and neck cancer.

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

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

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 multi-specific (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). Examples of non-small cell lung cancer include, but are not limited to, large cell carcinoma, adenocarcinoma, neuroendocrine lung tumor, and squamous cell carcinoma.

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 multi-specific (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone).

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 (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). In some embodiments, the liver cancer is hepatocellular carcinoma, a fibrolamellar variant of hepatocellular carcinoma, or mixed hepatocellular cholangiocarcinoma.

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

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 multi-specific (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; and/or a light chain variable region, wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). In some embodiments, the pancreatic cancer is a serous cystic tumor, a mucinous cystic tumor, intraductal papillary myxoma, pancreatic cancer, adenosquamous carcinoma, squamous cell carcinoma, signet ring cell carcinoma (significant ring cell carcinoma), undifferentiated carcinoma with giant cells, solid pseudopapillary tumor, ampulla, or pancreatic neuroendocrine tumor. In some embodiments, the pancreatic cancer is pancreatic adenocarcinoma.

In some embodiments, there is provided a method of treating a skin cancer in an individual, comprising administering to the individual an effective amount of a multi-specific (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; and/or a light chain variable region, wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). In some embodiments, the skin cancer is melanoma. In some embodiments, the melanoma is superficial invasive melanoma, malignant lentigo melanoma, nodular melanoma, mucosal melanoma, polypoid melanoma, desmoplastic melanoma, melanophore-free melanoma, soft tissue melanoma, or acromatic lentigo melanoma.

In some embodiments, there is provided a method of treating a renal cancer in an individual, comprising administering to the individual an effective amount of a multispecific (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; and/or a light chain variable region, wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the renal cell carcinoma is adenocarcinoma. In some embodiments, the renal cell carcinoma is clear cell renal cell carcinoma, papillary renal cell carcinoma (also known as eosinophilic renal cell carcinoma), chromophobe renal cell carcinoma, collecting duct renal cell carcinoma, granular renal cell carcinoma, mixed granular renal cell carcinoma, renal vascular smooth muscle lipoma, or spindle renal cell carcinoma.

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 (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; and/or a light chain variable region, wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). In some embodiments, the bladder cancer is 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, T1Cis, Cis with Ta and/or T1). In some embodiments, the bladder cancer is transitional cell carcinoma or urothelial cancer (such as metastatic urothelial cancer), including, but not limited to, papillomas and squamous cell carcinoma. In some embodiments, the cystoma is a squamous cell carcinoma, a non-squamous cell carcinoma, an adenocarcinoma, or a small cell carcinoma.

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 (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; and/or a light chain variable region, wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). In some embodiments, the thyroid cancer is papillary carcinoma, follicular carcinoma, herchle's (Hurthle) cell carcinoma, medullary thyroid carcinoma, undifferentiated carcinoma, thyroid lymphoma, thyroid sarcoma, or parathyroid carcinoma.

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 multi-specific (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; and/or a light chain variable region, wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). In some embodiments, the prostate cancer is adenocarcinoma. In some embodiments, the prostate cancer is a sarcoma, neuroendocrine tumor, small cell carcinoma, ductal carcinoma, or lymphoma.

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 (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; and/or a light chain variable region, wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method does not cause a cytokine storm. In some embodiments, the ovarian cancer is an ovarian epithelial cancer.

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 (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; and/or a light chain variable region, wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). In some embodiments, the endometrial cancer is an adenocarcinoma, a carcinosarcoma, a squamous cell carcinoma, an undifferentiated carcinoma, a small cell carcinoma, or a transitional cell carcinoma.

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 multi-specific (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; and/or a light chain variable region, wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). In some embodiments, the breast cancer is early 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 assisted setting, or breast cancer in a neo-assisted setting. In some embodiments, the breast cancer is fibroadenocarcinoma, or intraductal papillomas. In some embodiments, the breast cancer is HER2 positive or HER2 negative. In some embodiments, the breast cancer is a triple negative breast cancer.

In some embodiments, there is provided a method of treating a biliary tract cancer in an individual, comprising administering to the individual an effective amount of a multispecific (e.g., bispecific) Fab fusion protein comprising: a Fab fragment that specifically binds CD3 and a binding domain (e.g., scFv) that specifically binds EpCAM, wherein the binding domain is fused to the N-terminus of the Fab fragment, wherein the multispecific Fab fusion protein is administered at a dose of about 0.01 μ g/kg to about 250 μ g/kg (e.g., about 0.01 μ g/kg to about 5 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 2.5 μ g/kg to about 100 μ g/kg). In some embodiments, the method has one or more of the following biological activities: (1) killing cancer cells, (2) inhibiting cancer cell proliferation, and (3) inducing peripheral T cell redistribution. In some embodiments, the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM and a second scFv that specifically binds 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. 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 (e.g., amino acids 1-27 of the N-terminus) of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; and/or a light chain variable region, wherein the light chain variable region comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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) and/or a light chain variable region (VL); wherein the heavy chain variable region (VH) comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific Fab fusion protein is administered intravenously. In some embodiments, the multi-specific Fab fusion protein is administered at a low frequency. In some embodiments, the method further comprises administering to the subject a glucocorticoid (e.g., dexamethasone). In some embodiments, the cholangiocarcinoma is intrahepatic cholangiocarcinoma, peripulmonary cholangiocarcinoma, distal cholangiocarcinoma. In some embodiments, the cholangiocarcinoma is cholangiocarcinoma (cholangiocarcinoma), sarcoma, lymphoma, or small cell carcinoma.

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

Exemplary routes of administration of the multi-specific Fab fusion protein (MSFP) include, but are not limited to, oral, intravenous, intracavity, intratumoral, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, ocular, topical, intraperitoneal, intracranial, intrapleural, and transepidermal routes, or delivery 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.

In some embodiments, the MSFP is administered by intravenous infusion. In some embodiments, the MSFP is infused into a subject for a period of no more than about 24 hours, 20 hours, 15 hours, 10 hours, 8 hours, 6 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or less. In some embodiments, the MSFP is infused into the subject for any one of a time period from about 30 minutes to about 1 hour, from about 1 hour to about 2 hours, from about 2 hours to about 4 hours, from about 4 hours to about 6 hours, from about 6 hours to about 8 hours, from about 8 hours to about 10 hours, from about 10 hours to about 12 hours, from about 12 hours to about 18 hours, from about 18 hours to about 24 hours, from about 30 minutes to about 2 hours, from about 2 hours to about 5 hours, from about 5 hours to about 10 hours, from about 10 hours to about 20 hours, from about 30 minutes to about 10 hours, or from about 30 minutes to about 20 hours. The MSFP may be infused into an individual at any suitable rate. In some embodiments, the MSFP may be greater than 0.01 μ g/kg/hr, 0.02 μ g/kg/hr, 0.05 μ g/kg/hr, 0.1 μ g/kg/hr, 0.2 μ g/kg/hr, 0.5 μ g/kg/hr, 0.6 μ g/kg/hr, 0.7 μ g/kg/hr, 0.8 μ g/kg/hr, 0.9 μ g/kg/hr, 1 μ g/kg/hr, 1.5 μ g/kg/hr, 2 μ g/kg/hr, 3 μ g/kg/hr, 4 μ g/kg/hr, 5 μ g/kg/hr, 10 μ g/kg/hr, 15 μ g/kg/hr, 20 μ g/kg/hr, 25 μ g/kg/hr, 50 μ g/hr, At any rate of 75 μ g/kg/hr, 100 μ g/kg/hr or more.

The administration regimen of the MSFP administered to an individual will vary with the particular MSFP composition, method of administration, and the particular type and stage of cancer being treated. In some embodiments, the effective amount of the MSFP is below a level that causes a toxicological effect (e.g., an effect that exceeds a clinically acceptable toxicity level), or a level at which potential side effects can be controlled or tolerated when the composition is administered to an individual.

In some embodiments, the effective amount of the MSFP is below a level that causes central nervous system side effects, e.g., side effects observed in antibody therapy are infusion-related side effects such as cytokine release syndrome ("CRS"), a severe state known as "cytokine storm. When a "cytokine storm" is induced, the immune system of healthy individuals is activated and releases large amounts of proinflammatory cytokines such as IFN-. gamma., CCL2, IL-10, IL-6, etc. It is a potentially lethal immune response, usually consisting of a positive feedback loop between cytokines and immune cells, with highly elevated levels of various cytokines. Other adverse side effects associated with CRS are fatigue, vomiting, tachycardia, hypertension, back pain, and also central nervous system reactions (CNS reactions) such as epilepsy, encephalopathies, cerebral edema, aseptic meningitis, and headache. In some embodiments, the MSFP is administered at a dose that does not induce a cytokine release syndrome, such as a 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-gamma. In some embodiments, the significant release of the cytokine is a sustained release of the cytokine that progresses over a period of at least any one of about 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 the cytokine is a serum or blood concentration level of the cytokine of at least any one of about 1, 5, 10, 20, 50, 100, 200, 500, 1000pg/mL or more. Without being bound by any theory, the MSFP described herein need to bind EpCAM on target cancer cells to recruit and activate T cells. This requirement can greatly reduce undesirable cytokine storms and undesirable T cell activation in the absence of the desired target cancer cells.

In some embodiments, the MSFP is administered at a dose of no more than any one of about 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1 μ g/kg. In some embodiments, the dose of the MSFP is administered in any one of the following ranges, with an upper limit of any one of 0.05, 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1 μ g/kg, and an independently selected lower limit of any one of 0.01, 0.05, or 1 μ g/kg, 0.1. mu.g/kg, 0.5. mu.g/kg, 1. mu.g/kg, 2. mu.g/kg, 5. mu.g/kg, 10. mu.g/kg, 15. mu.g/kg, 20. mu.g/kg, 25. mu.g/kg, 30. mu.g/kg, 50. mu.g/kg, 100. mu.g/kg, 150. mu.g/kg, 200. mu.g/kg, 250. mu.g/kg, 300. mu.g/kg, 400. mu.g/kg, 500. mu.g/kg, 600. mu.g/kg, 700. mu.g/kg, 800. mu.g/kg, or 900. mu.g/kg, and the lower limit is smaller than the upper limit. In some embodiments, the MSFP is administered at a rate of about 0.01 to about 0.05 μ g/kg, about 0.05 to about 0.1 μ g/kg, about 0.1 to about 0.5 μ g/kg, about 0.5 to about 1 μ g/kg, about 0.01 to about 0.1 μ g/kg, about 0.1 to about 1 μ g/kg, about 1 to about 5 μ g/kg, about 5 to about 10 μ g/kg, about 10 to about 15 μ g/kg, about 15 to about 20 μ g/kg, about 20 to about 25 μ g/kg, about 25 to about 30 μ g/kg, about 5 to about 15 μ g/kg, about 10 to about 30 μ g/kg, About 30 μ g/kg to about 50 μ g/kg, about 50 μ g/kg to about 100 μ g/kg, about 0.01 μ g/kg to about 1 μ g/kg, about 0.01 μ g/kg to about 5 μ g/kg, about 0.01 μ g/kg to about 30 μ g/kg, about 0.01 μ g/kg to about 250 μ g/kg, about 0.1 μ g/kg to about 10 μ g/kg, about 0.1 μ g/kg to about 30 μ g/kg, about 0.1 μ g/kg to about 250 μ g/kg, about 1 μ g/kg to about 10 μ g/kg, about 1 μ g/kg to about 20 μ g/kg, about 1 μ g/kg to about 30 μ g/kg, about 1 μ g/kg to about 250 μ g/kg, about 100 μ g/kg to about 250 μ g/kg, about 5 μ g/kg to about 250 μ g/kg, A dose of any of about 250 μ g/kg to about 500 μ g/kg, about 500 μ g/kg to about 1000 μ g/kg, or about 0.01 μ g/kg to about 1000 μ g/kg. The dosage described herein may refer to dosages suitable for cynomolgus monkeys as well as their human equivalent or equivalent dosages for a particular species of individual. In some embodiments, the multi-specific Fab fusion protein is administered at a dose equivalent to about 0.1 μ g/kg to about 100 μ g/kg (such as about 0.3 μ g/kg to about 5 μ g/kg, or about 5 μ g/kg to about 20 μ g/kg) to a cynomolgus monkey. In some embodiments, the multi-specific Fab fusion protein is administered at a dose equivalent to no more than about 30 μ g/kg (e.g., no more than about 20, 15, or 10 μ g/kg) for a cynomolgus monkey.

In some embodiments, the MSFP is administered at a dose of about 0.01 μ g/kg to about 10 μ g/kg, such as any of about 0.3, 0.5, 0.6, 1, 1.2, 2, 2.4, 3.6, or 4 μ g/kg.

The effective amount of the MSFP may be administered in a single dose or in multiple doses. For treatment methods comprising administering the MSFP in multiple doses, exemplary frequencies of administration include, but are not limited to, daily uninterrupted, weekly uninterrupted, two of three weeks weekly, three of four weeks weekly, every three weeks, every two weeks, monthly, every 6 months, or yearly, etc. In some embodiments, the MSFP is administered at a frequency of 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 a frequency of at least about 1, 2, 3, 4, 5, 6, or 7 times per week (i.e., daily). In some embodiments, the interval between each administration is less than any one of about 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, each administration is separated by greater than about any one 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 administration period.

In some embodiments, the MSFP is administered to a subject at a first dose for a first period of time and continuously the MSFP is administered to the subject at a second dose for a second period of time, wherein the second dose exceeds the first dose. The first time period and the second time period may be any suitable length, including, for example, any of about 1, 2, 3, 4, 5, 6 weeks or more. 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 any one of about 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or more. The first dose and the second dose may be any suitable dose described above. In some embodiments, the first dose is no more than about any of 2, 1.5, 1, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 μ g/kg or less. In some embodiments, the second dose is about 0.1 μ g/kg to about 10 μ g/kg, such as about 0.3 μ g/kg to about 5 μ g/kg. In some embodiments, the second dose is about any one of 0.3, 0.6, 1.2, 2.4, or 3.6 μ g/kg.

In some embodiments, the MSFP is administered at a low frequency, e.g., at a frequency of no more than any one of 1 every month, 1 every 2 months, 1 every 3 months, 1 every 4 months, 1 every 5 months, 1 every 6 months, 1 every 7 months, 1 every 8 months, 1 every 9 months, 1 every 10 months, 1 every 11 months, 1 every year, 1 every 18 months, 1 every 2 years, 1 every 3 years, or less. In some embodiments, the MSFP is administered in a single administration. In some embodiments, the MSFP is administered twice weekly.

The administration of the MSFP may be extended for a period of time, for example, from 1 day to about one week, from about one week to about one month, from about one month to about one year, from about one year to about several years. In some embodiments, the MSFP is administered for a period of at least any one 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.

In some embodiments, the method further comprises administering to the subject one or more glucocorticoids. Glucocorticoids (GCs) are a class of steroid hormones that bind to the Glucocorticoid Receptor (GR), which is present in almost all vertebrate cells, including humans. Regardless of the cause of inflammation, such compounds are potent anti-inflammatory agents. In some embodiments, the glucocorticoid inhibits the 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-gamma. Suitable glucocorticoids include, but are not limited to, cortisone, hydrocortisone, cortisol, chloroprolol, prednisone, prednisolone, methylprednisolone, deflazacort, fludrocortisone, triamcinolone, dexamethasone, and betamethasone. In some embodiments, the glucocorticoid is selected from the group consisting of: cortisone, hydrocortisone, cortisol, chloroprolol, prednisone, prednisolone, methylprednisolone, deflazacort, fludrocortisone, triamcinolone, dexamethasone, betamethasone and pharmaceutically acceptable esters, salts or complexes thereof. In some embodiments, the glucocorticoid is dexamethasone. In some embodiments, the glucocorticoid is a pharmaceutically acceptable ester, salt, or complex of dexamethasone.

The glucocorticoid may be administered to the subject simultaneously with the MSFP or prior to administration of the MSFP. The glucocorticoid may be administered no more than about any of 3 hours, 2 hours, 1 hour, 30 minutes, or less prior to administration of the MSFP. In some embodiments, the glucocorticoid is administered at the same time as or prior to each dose of the MSFP. In some embodiments, the glucocorticoid is administered at the same time 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 continuously at a second dose for a second period of time, the glucocorticoid is administered prior to (e.g., about 1 hour prior to) the first administration of the MSFP and the glucocorticoid is administered prior to (e.g., 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 administration of the MSFP when the individual has elevated liver enzyme levels (e.g., ALT, TBil, and/or ALP) and/or elevated cytokine levels (e.g., IL-6). The glucocorticoid can be administered in any suitable dose, including, for example, at least about any one of 0.1, 0.5, 1, 2, 3, 4, 5mg/kg or more. In some embodiments, the glucocorticoid is administered in a dose of at least about any one of 1, 2, 5, 10, 15, 20, 25mg, or more.

In some embodiments, the glucocorticoid is dexamethasone. In some embodiments, the method comprises administering dexamethasone to the subject prior to administering the first dose of the MSFP. In some embodiments, the glucocorticoid is administered at a dose of about 0.1mg/kg to about 5 mg/kg.

Multispecific Fab fusion proteins

The multi-specific Fab fusion protein (MSFP) used in the methods of the invention comprises an anti-CD 3Fab fragment, a first EpCAM binding domain fused to the N-terminus of the VH of the Fab fragment, and/or a second EpCAM binding domain fused to the N-terminus 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 linked to the VH or VL of the anti-CD 3Fab fragment by a linker. An exemplary bispecific Fab fusion protein for use in the methods of the invention is shown in figure 1.

Fab fragments

Fab fragments in the MSFP of the invention are capable of specifically binding to CD3, such as human CD 3. "CD 3" is known in the art as a six-chain multi-protein complex (see Abbas and Lichtman, 2003; Janeway et al, p172 and 178,1999). In mammals, the complex comprises a homodimer of the CD3 γ chain, the CD3 δ chain, the two CD3 ε chains, and the CD3 ζ chain. The CD3 γ chain, CD3 δ chain, and CD3 ε chain are highly related cell surface proteins of the immunoglobulin superfamily that comprise a single immunoglobulin domain. The transmembrane regions of the CD3 γ, CD3 δ and CD3 ε chains are negatively charged, a feature that allows these chains to bind to positively charged T cell receptor chains. The intracellular tails of the CD3 γ, CD3 δ and CD3 ε chains each contain a conserved motif called the immunoreceptor tyrosine-based activation motif or ITAM, while each CD3 ζ chain has three. Without wishing to be bound by theory, it is believed that ITAMs are important for the signaling capacity of the TCR complex. CD3 as used herein can be from different animal species including human, primate, mouse, rat, or other mammal.

In some embodiments, the Fab fragment of MSFP specifically binds to a CD3 chain, such as a CD3 gamma chain, a CD3 delta chain, or a CD3 epsilon chain, in an individual. In some embodiments, the Fab fragment specifically binds to a complex formed by two or more individual CD3 chains (e.g., a complex of more than one CD3 epsilon chain, a complex of a CD3 gamma chain and a CD3 epsilon chain, a complex of a CD3 delta chain and a CD3 epsilon chain). In some embodiments, the Fab fragment specifically binds to the CD3 epsilon chain. In some embodiments, the Fab fragment specifically binds to the N-terminus of the CD3 epsilon chain. In some embodiments, the Fab fragment specifically binds to amino acids 1-27 of the epsilon chain of CD 3.

Fab fragments of the disclosure can be produced as described herein or by a variety of methods known in the art (see, e.g., U.S. Pat. No. 6,291,161; No. 6,291,158). The source of Fab includes monoclonal antibodies or antigen binding fragments thereof from different species, including human, camelid antibodies (from camels, dromedary 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.Natl.Acad.Sci. (USA)95:11804), fish (Nguyen et al (2002) Immunogenetics,54:39), rodents, birds, or sheep. In some embodiments, the Fab fragment is derived from a human or humanized antibody.

In some embodiments, the Fab fragment specifically binds human or non-human primate (e.g., cynomolgus monkey) CD3, and exemplary anti-human CD3 antibodies that cross-react with monkey CD3 include, but are not limited to, SP34 mouse monoclonal antibodies (see, e.g., 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. anim49:97-110,2000; Conrad M. L. et al, cytometric 71A:925-33, 2007; and Yang et al, J.munol.137: 1097-1100: 1986). MSFP with anti-CD 3Fab fragments, cross-reactive with monkey CD3, facilitates toxicity studies in non-human primates, which may provide more relevant safety assessments for human clinical trial candidates without the need for toxicity studies in chimpanzees or using alternative molecules.

In some embodiments, the Fab fragments are from an anti-CD 3 antibody that is not cross-reactive with non-human primates, exemplary anti-CD 3 antibodies include the Cris-7 monoclonal antibody (Reinherz, E.L. et al (eds.), Leucocyte type II, Springer Verlag, New York, (1986)), the BC3 monoclonal antibody (Anaceti et al (1990) J.Exp.Med.172:1691), OKT3(Ortho multicenter graft Study Group (1985) N.Engl.J.Med.313:337), and derivatives thereof such as OKT3 a-ala (Herold et al (2003) J.Clin.invest.11:409), Visilimab (Visili Immab) (Carpenter et al (2002) Blood 99:2712), and 145-2C11 (Hirsch.26. J.1988: 37140). Other CD3 binding molecules contemplated herein include UCHT-1(Beverley, P C and Callad, R.E (1981) Eur.J.Immunol.11: 329-334) and WO 2004/106380; WO 2010/037838; WO 2008/119567; WO 2007/042261; CD3 binding molecules as described in WO 2010/0150918.

In some embodiments, the Fab fragment comprises one constant and one variable region of an immunoglobulin heavy chain and one constant and variable region of an immunoglobulin light chain. In some embodiments, the heavy chain constant and variable regions are heterodimerized with the light chain constant and variable regions and passed through the heavy chainCovalently linked to disulfide bonds between light chain constant regions. In some embodiments, the Fab fragment has NH₂-VL-CL-S-S-CH1-VH-NH₂The basic structure of (2). In some embodiments, the CH1 of the Fab fragment is linked to the CL by one or more disulfide bonds. In some embodiments, the number of disulfide bonds between the heavy chain first constant region (CH1) and the light chain constant region (CL) of the Fab fragment is at least 1, e.g., 2, 3, 4, or more. In some embodiments, cysteine residues in the Fab fragment (e.g., in the CH1 or CL region) are engineered to introduce a disulfide bond.

In some embodiments, the Fab fragment of the MSFP does not comprise a disulfide bond, e.g., the heavy and light chains can be engineered in a way so as to stably interact without the need for disulfide bonds. In some embodiments, the heavy and light chains can be engineered to remove cysteine residues, wherein the heavy and light chains remain stably interacting and functional as a Fab. In some embodiments, mutagenesis to allow stable interaction between the heavy and light chains, e.g., an Engineering strategy of "knob-into-hole" may be used for dimerization between the heavy and light chains of Fab (see, e.g., 1996Protein Engineering,9: 617-621). Also contemplated for use in the present invention are variant Fab fragments designed for a particular purpose, e.g., amino acid changes in the CH1 and/or CL constant regions, and removal of disulfide bonds or addition of purification tags, among others.

In some embodiments, the configuration of the variable and constant regions within the Fab fragment may be different from that found in a native Fab. In some embodiments, the variable and constant regions may be oriented VH-CL in one chain, and VL-CH1 in the other chain (see, e.g., Schaefer et al (2011), PNAS,108: 11187-92).

In some embodiments, the Fab fragment of MSFP is derived from a monoclonal antibody. Suitable monoclonal antibodies may be of any type, including IgA, IgM, IgD, IgG, IgE and subtypes thereof (e.g., IgG1, IgG2, IgG3, and IgG 4). In some embodiments, the light chain domain may be derived from a kappa chain or a lambda chain. In some embodiments, the Fab fragment is recombinantly designed.

In some embodiments, the Fab fragment comprises human immunoglobulin CH 1. In some embodiments, the human immunoglobulin CH1 comprises the amino acid sequence of SEQ ID No. 9. In some embodiments, the Fab fragment comprises a human lambda light chain constant region. In one embodiment, the human λ light chain constant region comprises the amino acid sequence of SEQ ID NO 10.

SEQ ID NO 9 (human CH1)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS

SEQ ID NO 10 (human lambda CL)

GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTE

The Fab fragment of MSFP specifically binds to CD3 via the antigen binding site formed between the heavy chain variable region (VH) and the light chain variable region (VL). The antigen binding site comprises at least one (e.g., 1, 2, or 3) HVR of an immunoglobulin heavy chain and/or at least one (e.g., 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 the VH and VL sequences of a full length antibody that specifically binds CD 3.

In some embodiments, the Fab fragment is derived from SP 34. In some embodiments, the Fab fragment is a CD3Fab fragment described in U.S. patent No.8,846,042. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising 1, 2, or 3 HVRs (or CDRs) from SEQ ID NO:7 and/or a light chain variable region (VL); the light chain variable region (VL) comprises 1, 2 or 3 HVRs (or CDRs) from SEQ ID NO: 8. In some embodiments, a Fab fragment comprises a heavy chain variable region (VH) comprising 3 HVRs from SEQ ID NO:7 and/or a light chain variable region (VL); the light chain variable region (VL) comprises 3 HVRs from SEQ ID NO: 8. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising 1, 2, or 3 HVRs selected from SED ID Nos. 1-3; the light chain variable region (VL) comprises 1, 2 or 3 HVRs selected from SED ID Nos 4-6. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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 having 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%, at least about 99% identity to a 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 having 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%, at least about 99% identity to a sequence selected from SEQ ID NOs 8 and 44-47. In some embodiments, a VH 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 a reference sequence, but the Fab fragment comprising that sequence retains the ability to bind CD 3. In some embodiments, one or two amino acids are substituted, inserted, and/or deleted in any one or more HVRs. In some embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising an amino acid 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 selected from the group consisting of 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); the light chain variable region (VL) comprises 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; the light chain polypeptide comprises the amino acid sequence of SEQ ID NO 12.

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)

EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTMVTVSS

SEQ ID NO:8(CD3 VL)

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

SEQ ID NO. 11(CD3 heavy chain)

EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS

SEQ ID NO. 12(CD3 light chain)

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTE

SEQ ID NO:39(CD3 VH)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTMVTVSS

SEQ ID NO:40(CD3 VH)

EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTMVTVSS

SEQ ID NO:41(CD3 VH)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARHGNFGNSYVSWFAYWGQGTMVTVSS

SEQ ID NO:42(CD3 VH)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTMVTVSS

SEQ ID NO:43(CD3 VH)

EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS

SEQ ID NO:44(CD3 VL)

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

SEQ ID NO:45(CD3 VL)

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

SEQ ID NO:46(CD3 VL)

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

SEQ ID NO:47(CD3 VL)

QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWYQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVL

In some embodiments, specific VH and/or VL of an anti-CD 3Fab fragment may be used to screen a library of complementary variable regions to identify VH/VL with desired properties (e.g., increased affinity for CD 3). Such methods are described, for example, in Portolano et al, J.Immunol. (1993)150: 880-887; clarkson et al, Nature (1991)352: 624-; 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-.

EpCAM binding domain

The MSFP of the invention comprises one or two binding domains that specifically bind EpCAM. Epithelial cell adhesion molecule (EpCAM, CD326) is a transmembrane glycoprotein with a molecular weight of 40kD, composed of 314 amino acids, and is also known as 17-1A, ESA, AUA1, EGP40, and the like. EpCAM is involved in cell signaling, migration, proliferation and differentiation. EpCAM is specifically expressed in a variety 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, and thus, can be used as a diagnostic marker for various cancers. EpCAM is also a potential target for immunotherapeutic strategies including vaccines, murine or humanized monoclonal antibodies, and antibodies conjugated to bacterial toxins or chemotherapeutic drugs, such as EpCAM-specific antibody ING-1, alemtuzumab (adelimumab), Edrecolomab (Edrecolomab), and the like.

The binding domains in MSFP, including the EpCAM binding domain, not only provide additional binding specificity and enhanced properties (e.g., increased serum half-life, or activation of other immune activation cascades), but also provide 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 combined with other Fab fusion proteins (e.g., TRIBODIES)^TMWhich fuses an additional binding domain to the C-terminus of the Fab fragment) in direct comparison (see, e.g., Journal of Immunology,2000,165: 7050-. Unlike other known fusion proteins (e.g., those described in WO2008/024188 and WO 2009/149185), the binding domains are not intended to dimerize. The MSFP is further characterized in that when the MSFP is not knottedIn binding to a cell surface target (e.g., EpCAM) on a cancer cell, the binding domain reduces the binding affinity of the Fab fragment to CD 3.

In some embodiments, the MSFP has an extended half-life in vivo as compared to the anti-CD 3Fab alone. In some embodiments, the half-life of the MSFP is at least any one of about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times the half-life of the anti-CD 3Fab fragment alone.

The binding domain, including EpCAM, may be selected from an antigen binding domain, such as scFv or scTCR, an extracellular domain of a receptor, a ligand of a cell surface molecule/receptor, or a receptor binding domain thereof, and a tumor binding protein. In some embodiments, the antigen binding domain is selected from the group consisting of scFv, VH, VL, domain antibody variants (dabs), camelid antibodies (VHHs), fibronectin 3 domain variants, ankyrin repeat variants, and other antigen-specific binding domains derived from other protein scaffolds.

In some embodiments, the EpCAM binding domain is an scFv that specifically binds EpCAM (also referred to herein as an anti-EpCAM scFv). In some embodiments, the VH and VL of the anti-EpCAM scFv are interconnected by a peptide linker (e.g., a flexible linker comprising glycine and/or serine). In some embodiments, the VH and VL of the anti-EpCAM scFv are directly linked to each other. 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.

The EpCAM binding domain (e.g., scFv) can 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 EpCAM in both human and non-human primates (e.g., cynomolgus monkeys). In some embodiments, the EpCAM binding domain specifically recognizes human EpCAM, but is not cross-reactive with a non-human primate. Exemplary anti-EpCAM antibodies are known in the art, see, e.g., U.S. patent 8,884,602. The anti-EpCAM binding domain can comprise at least 1 (e.g., 1, 2, or 3) HVRs of an immunoglobulin heavy chain and/or at least 1 (e.g., 1, 2, or 3) HVRs of an immunoglobulin light chain. In some embodiments, the anti-EpCAM binding domain comprises 1, 2, 3, 4, 5, or all 6 HVRs of the VH and VL sequences of a full length antibody that specifically binds EpCAM.

In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising 1, 2, or 3 HVRs (or CDRs) from SEQ ID NO:19 and/or a light chain variable region (VL); the light chain variable region (VL) comprises 1, 2 or 3 HVRs (or CDRs) from SEQ ID NO: 20. In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising 3 HVRs from SEQ ID NO:19 and/or a light chain variable region (VL); the light chain variable region (VL) comprises 3 HVRs from SEQ ID NO: 20. In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising 1, 2, or 3 HVRs selected from SED ID Nos:13-15 and or a light chain variable region (VL); the light chain variable region (VL) comprises 1, 2 or 3 HVRs selected from SED ID Nos: 16-18. In some embodiments, the anti-EpCAM scFv comprises a heavy chain variable region (VH) comprising: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and 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 that is 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; the light chain variable region comprises an amino acid sequence that is 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); the light chain variable region (VL) comprises 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(EpCAMHVR-H2)

NIKQDGSEKFYADSVKG

SEQ ID NO:15(EpCAMHVR-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)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQDGSEKFYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGPSWEQDYWGQGTLVTVSA

SEQ ID NO:20(EpCAM VL)

GAQSVLTQPPSVSGAPGQRVTISCTGSSSNIGSYYGVHWYQQLPGTAPKLLIYSDTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDKGFGHRVFGGGTKLTVL

SEQ ID NO:21(EpCAM scFv)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQDGSEKFYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGPSWEQDYWGQGTLVTVSAGGGGSGGGGSGGGGSGAQSVLTQPPSVSGAPGQRVTISCTGSSSNIGSYYGVHWYQQLPGTAPKLLIYSDTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDKGFGHRVFGGGTKLTVL

Joint

The MSFP of the invention may comprise a linker (e.g. a peptide linker) between the VH or VL of the Fab fragment and the binding domain. In some embodiments, the linker between the VH of the Fab fragment and the first binding domain and the linker between the VL of the Fab fragment and the first/second binding domain are the same. In some embodiments, the linker between the VH of the Fab fragment and the first binding domain and the linker between the VL of the Fab fragment and the first/second binding domain are different. In some embodiments, the anti-EpCAM scFv comprises a linker (e.g., a peptide linker) between the VH and VL of the scFv, which may be the same or different from any linker between the VH or VL and the binding domain of the Fab fragment.

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

In some embodiments, the joint is a flexible joint. Exemplary flexible linkers include glycine polymers (G)_nGlycine-serine polymers (including, for example, (GS)_n、(GSGGS)_nAnd (GGGS)_nWhere n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and may therefore be able to act as neutral linkers between components. Glycine is significantly more accessible to the phi-psi space than alanine and is much less restricted than residues with longer side chains (see Scheraga, rev. comparative chem.11173-142 (1992)). An exemplary flexible joint comprisesBut are not limited to, 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-Ser-Gly (SEQ ID NO:29), and the like. In some embodiments, the linker between the VH and the EpCAM binding domain (e.g., scFv) of the anti-CD 3Fab fragment is Gly-Gly. In some embodiments, the linker between the VL of the anti-CD 3Fab fragment and the EpCAM binding domain (e.g., scFv) is Gly-Gly. One of ordinary skill in the art will recognize that the design of a MSFP may include joints that are fully or partially flexible, such that the joints may include flexible joint portions and one or more portions that impart less flexible structure to provide the desired MSFP structure.

In some embodiments, the linker between the Fab and the binding domain is a stable linker (not cleaved by proteases, particularly MMPs).

In some embodiments, the linker is a cleavable linker. In some embodiments, the linker between the VH or VL of the Fab and the binding domain comprises a protease substrate cleavage sequence, e.g., an MMP substrate cleavage sequence. The known peptide sequence of PLGLAG (SEQ ID NO:30) in the substrate is cleaved by most MMPs. The sequence of substrates that can be cleaved by MMPs has 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.

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. The invention also provides MSFP and compositions (e.g., 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

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQDGSEKFYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGPSWEQDYWGQGTLVTVSAGGGGSGGGGSGGGGSGAQSVLTQPPSVSGAPGQRVTISCTGSSSNIGSYYGVHWYQQLPGTAPKLLIYSDTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDKGFGHRVFGGGTKLTVLGGEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIR SKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCPPCS

SEQ ID NO:23

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQDGSEKFYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGPSWEQDYWGQGTLVTVSAGGGGSGGGGSGGGGSGAQSVLTQPPSVSGAPGQRVTISCTGSSSNIGSYYGVHWYQQLPGTAPKLLIYSDTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDKGFGHRVFGGGTKLTVLGGQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGG TNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECPPCS

EpCAM antibodies

The present application also provides novel anti-EpCAM antibodies or antigen-binding fragments thereof, including multi-specific (e.g., bispecific) Fab fusion proteins, comprising an antigen-binding fragment derived from the anti-EpCAM antibody. The anti-EpCAM antibodies and antigen-binding fragments thereof described herein have enhanced stability and ability to be expressed compared to anti-EpCAM antibodies and fragments known in the art. Furthermore, the anti-EpCAM antibodies and antigen-binding fragments thereof described in the present invention have a cross-reactivity to EpCAM from both human and non-human primates (e.g., cynomolgus monkeys), which is beneficial for extrapolation from toxicity and efficacy studies in monkeys to human clinical studies for evaluation of EpCAM antibodies or derivatives thereof (e.g., MSFP).

The antibodies of the invention bind with an equilibrium of ≦ 1 μ M, e.g. ≦ 100nM, preferably ≦ 10nM, more preferably ≦ 1nMConstant (K)_d) Binds to an EpCAM epitope. For example, anti-EpCAM antibodies provided herein exhibit a K in the range of about ≦ 1nM to about 1pM_d。

The anti-EpCAM antibodies of the invention are useful for modulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with the functional activity of a broad distribution of EpCAM, in whole or in part. An EpCAM antibody is considered to fully modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with EpCAM functional activity when the level of EpCAM functional activity in the presence of said anti-EpCAM antibody is reduced by at least 95%, such as by 96%, 97%, 98%, 99%, or 100% as compared to the level of EpCAM functional activity in the absence of binding to an anti-EpCAM antibody described herein. An anti-EpCAM antibody is considered to significantly block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with EpCAM functional activity when the level of EpCAM functional activity in the presence of the anti-EpCAM antibody is reduced by at least 50%, such as by 55%, 60%, 75%, 80%, 85%, or 90% as compared to the level of EpCAM functional activity in the absence of binding to an anti-EpCAM antibody described herein. An anti-EpCAM antibody is considered to partially modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with EpCAM functional activity when the level of EpCAM functional activity in the presence of said anti-EpCAM antibody is reduced by less than 95%, e.g., by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90% as compared to the level of EpCAM functional activity in the absence of binding to an anti-EpCAM antibody described herein.

In some embodiments, the anti-EpCAM antibody moiety specifically binds EpCAM present on the surface of a cell. In some embodiments, the surface of the cell exhibits an abnormally high level 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.

In some embodiments, the anti-EpCAM antibody portion comprises a specific sequence or certain variants of these sequences. In some embodiments, the amino acid substitution in the variant sequence does not substantially reduce the ability of the anti-EpCAM antibody portion to bind EpCAM. For example, changes that do not substantially reduce EpCAM binding affinity can be made. Alterations that substantially improve the binding affinity of EpCAM or affect other properties such as specificity, immunogenicity, ADCC or CDC, and/or cross-reactivity with EpCAM-related variants are also contemplated.

In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof (e.g., scFv) comprises a heavy chain variable region (VH) comprising 1, 2, or 3 hvrs (cdrs) from SEQ ID NO:19 and/or a light chain variable region (VL); the light chain variable region (VL) comprises 1, 2 or 3 HVRs (CDRs) from SEQ ID NO: 20. In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof (e.g., scFv) comprises a heavy chain variable region (VH) comprising 3 hvrs (cdrs) from SEQ ID NO:19 and/or a light chain variable region (VL); the light chain variable region (VL) comprises 3 HVRs (CDRs) from SEQ ID NO: 20.

In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof (e.g., scFv) comprises a heavy chain variable region (VH) comprising HVR-H3 comprising the amino acid sequence of SED ID No. 15 and or a light chain variable region (VL); the light chain variable region (VL) comprises HVR-L3 comprising the amino acid sequence of SED ID NO: 18.

In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising: (1) 13, (2) 14, and/or (3) 15, with one or two amino acid substitutions, HVR-H1 of SEQ ID NO, (2) 14, and/or 3; the light chain variable region (VL) comprises: (1) HVR-L1 of SEQ ID NO 16 with one or two amino acid substitutions, (2) HVR-L2 of SEQ ID NO 17 with one or two amino acid substitutions, and/or (3) HVR-L3 of SEQ ID NO 18 with one or two amino acid substitutions.

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

In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof (e.g., scFv) comprises a heavy chain variable region (VH) comprising an amino acid sequence having 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% identity to the sequence of SEQ ID No. 19; the light chain variable region (VL) comprises an amino acid sequence having 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% identity to the sequence of SEQ ID NO. 20. In some embodiments, a VH 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 a reference sequence, but the Fab fragment comprising that sequence retains the ability to bind to EpCAM. In some embodiments, one or both amino acids are substituted, inserted, and/or deleted in any one or more HVRs. In some embodiments, the substitution, insertion, or deletion occurs in a region outside of the HVR (i.e., in the FR). In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof (e.g., 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); the light chain variable region (VL) comprises the amino acid sequence of SEQ ID NO: 20.

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 (e.g., any one of IgA, IgD, IgE, IgG, and IgM). In some embodiments, the full-length anti-EpCAM antibody comprises an Fc sequence of an IgG (e.g., any one of IgG1, IgG2, IgG3, or IgG 4). 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 is altered or otherwise altered such that it has the function of enhancing antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) effector function.

The invention also provides an isolated antibody or antigen-binding fragment thereof that competes with any of the anti-EpCAM antibodies described herein for binding to EpCAM. In some embodiments, an isolated antibody or antigen-binding fragment thereof that binds to the same epitope of any of the anti-EpCAM antibodies described herein is also provided. Methods of screening for antibodies with the desired specificity include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and other immune-mediated techniques known in the art.

Those skilled in the art will recognize that: it is possible to determine without undue experimentation whether a monoclonal antibody has the same specificity as a monoclonal antibody of the invention (e.g., an anti-EpCAM antibody having the heavy chain variable region of SEQ ID NO:19 and the light chain variable region of SEQ ID NO: 20) by confirming whether the former prevents the latter from binding to EpCAM. If the monoclonal antibodies tested compete with the monoclonal antibodies of the invention, as indicated by the reduced binding of the monoclonal antibodies of the invention, then the two monoclonal antibodies are capable of binding the same or closely related epitopes.

An alternative method of determining whether a monoclonal antibody has the specificity of a monoclonal antibody of the invention is to pre-incubate an antibody of the invention with a soluble EpCAM protein (with which it has normal reactivity) and then add the monoclonal antibody tested to determine whether the tested monoclonal antibody is inhibited in its ability to bind EpCAM. If the monoclonal antibody tested is inhibited, it is likely that it has the same or functionally equivalent epitope specificity as the monoclonal antibody of the invention.

In some embodiments, the anti-EpCAM antibody is a monoclonal antibody, e.g., a monovalent antibody. In some embodiments, the anti-EpCAM antigen-binding fragment is in the form of: fab, Fab ', F (ab)'2, single chain Fv (scFv), Fv fragment, diabody (diabody), or linear antibody.

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

In some embodiments, the anti-EpCAM antibody is a multispecific antibody that is capable of not only binding EpCAM, but also binding to and optionally inhibiting the function of one or more other targets. Multispecific antibodies are monoclonal, preferably human or humanized antibodies that have binding specificities for two or more different antigens (e.g., bispecific antibodies having binding specificities for at least two antigens). For example, one of the binding specificities may be for the EpCAM protein and the other may be for any other antigen. In some embodiments, the additional antigen is a cell surface protein or receptor subunit, e.g., the cell surface protein can be CD3, such as CD3 epsilon. Thus, according to one embodiment, a bispecific antibody of the invention can bind both EpCAM and, for example, a second cell surface receptor.

In some embodiments, the multispecific anti-antibodyEpCAM molecules are, for example, diabodies (Db), single chain diabodies (scDb), tandem scDb (tandab), linear dimeric scDb (LD-scDb), cyclic dimeric scDb (CD-scDb), diabodies, tandem scFv, tandem bis scFv (e.g., bispecific T cell engager), tandem trisscFv, triabodies, bispecific Fab₂A bimini antibody, a tetravalent antibody, a scFv-Fc-scFv fusion, a Dual Affinity Retargeting (DART) antibody, a Dual Variable Domain (DVD) antibody, an IgG-scFab, scFab-ds-scFv, Fv2-Fc, an IgG-scFv fusion, a base-and-lock (DNL) antibody, a well-in-socket (KiH) antibody (bispecific IgG prepared by KiH technology), a DuoBody (bispecific IgG prepared by Duobody technology), a heteromultimeric antibody, or a heteroconjugate antibody. In some embodiments, the multispecific anti-EpCAM molecule is a tandem scFv (e.g., a tandem bis scFv, such as a bispecific T cell engager).

The invention still further provides a fusion protein, conjugate, or isolated cell comprising any of the anti-EpCAM antibodies or antigen-binding fragments thereof described herein.

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

In some embodiments, the multi-specific 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 multi-specific Fab fusion protein comprises two anti-EpCAM antigen binding fragments or two copies of an anti-EpCAM antigen binding fragment.

In some embodiments, there is provided a multi-specific (e.g., 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, wherein the anti-EpCAM antigen-binding fragment comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), the heavy chain variable region (VH) comprising: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18. In some embodiments, the multi-specific 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 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 and the second anti-EpCAM antigen-binding fragment have the same sequence. In some embodiments, the anti-EpCAM antigen-binding fragment comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, an anti-EpCAM antigen-binding fragment comprises a light chain variable region (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.

A multi-specific Fab fusion protein comprising one or more anti-EpCAM antigen-binding fragments described herein can further comprise one or more of the features of the multi-specific Fab fusion protein described in the "multi-specific Fab fusion protein" section "method of treating cancer" section II, above.

In some embodiments, the multi-specific 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 the CD3 epsilon chain. In some embodiments, the Fab fragment binds to a cell surface target selected from the group consisting of Fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIIIa, NKG2D, CD25, CD28, CD137, CTLA-4, FAS, FGFR1, FGFR2, FGFR3, FGFR4, GITR, LT β R, TLR, TRAIL receptor 1, TRAIL receptor 2, EGFR, Her2/neu, and ErbB 3.

In some embodiments, the Fab fragment specifically binds to CD3, e.g., the N-terminus of CD3 epsilon, e.g., the N-terminal amino acids 1-27 of CD3 epsilon. In some embodiments, the Fab fragment comprises a heavy chain variable region (VH) comprising: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3; the light chain variable region (VL) comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and 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 selected from the group consisting of SEQ ID NOS: 7 and 39-43 and/or a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 8 and 44-47. In some embodiments, the multi-specific 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 EQ ID NO. 23.

In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent (e.g., cytotoxic drugs, radioisotopes, and chemotherapeutic drugs) or a label for detecting EpCAM in a patient sample or in vivo by imaging (e.g., radioisotopes, fluorescent dyes, and enzymes). In some embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof is conjugated to a toxin.

In some embodiments, there is provided an anti-EpCAM Chimeric Antigen Receptor (CAR) comprising: a) an extracellular domain comprising any one of the anti-EpCAM antibodies described herein or an antigen-binding fragment thereof; and b) an intracellular signaling domain. A transmembrane domain may be present between the extracellular domain and the intracellular domain. There may be a spacer domain, such as a peptide linker (e.g., a flexible peptide linker), 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. Examples of intracellular signaling domains used by the anti-EpCAM CARs of the present invention include cytoplasmic sequences of T Cell Receptors (TCRs) and co-receptors that act synergistically to trigger signaling upon antigen receptor engagement, as well as derivatives or variants of any of these sequences, and any synthetic sequence with the same functional capacity. Any method for producing a CAR can be used in the present invention. See, e.g., 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 investment 118: 1294-.

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

The invention also provides isolated cells (e.g., CAR-T or TCR-T cells) expressing the anti-EpCAM CARs or TCRs, and methods of treating diseases (e.g., cancer) using the anti-EpCAM CARs or TCRs, or cells expressing the anti-EpCAM CARs or TCRs thereof.

The anti-EpCAM antibodies or antigen-binding fragments of the present invention can be used in a variety of therapeutic and diagnostic methods. The invention also provides a method of treating cancer in an individual comprising administering to the individual an effective amount of an anti-EpCAM antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof. For example, an anti-EpCAM antibody (or antigen-binding fragment thereof) can be used alone or in combination with other agents to treat diseases characterized by aberrant EpCAM expression, including, but not limited to, head and neck cancer, pancreatic cancer, colorectal cancer, and lung cancer. The antibodies provided by the invention can also be used to detect EpCAM protein in a patient or patient sample.

Monoclonal antibodies

The method of screening for monoclonal antibodies of the invention can be performed, for example, by measuring EpCAM-mediated signaling and determining whether the tested monoclonal antibody is capable of modulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with EpCAM-mediated signaling. These assays may include competitive binding assays. In addition, these assays can measure biological readings.

A variety of methods known in the art can be used to generate monoclonal Antibodies against EpCAM or against derivatives, fragments, analogs, homologs or orthologs thereof (see, e.g., Antibodies: A Laboratory Manual, Harlow E and Lane D, 1998, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., incorporated herein by reference). Fully human antibodies refer to antibody molecules in which all of the sequences of the light and heavy chains (including the CDRs) are derived from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies". For example, human monoclonal antibodies can be prepared using the methods described in the examples below. Human monoclonal antibodies can also be prepared using hybridoma technology, human B cell hybridoma technology (see Kozbor et al, 1983 immunological Today 4: 72); and the use of EBV hybridoma technology to produce human MONOCLONAL ANTIBODIES (see Cole et al, 1985, MONOCLONAL ANTIBODIES AND CANCER THERAPY (MONOCLONAL ANTIBODIES and cancer therapies), ARL Inc. (Alan R.Liss, Inc.), pages 77-96). Human MONOCLONAL ANTIBODIES can be used and can be generated by using human hybridomas (see Cote et al, 1983.Proc Natl Acad Sci USA 80: 2026-.

Antibodies can be purified by well-known techniques, such as affinity chromatography using protein a or protein G, which provides primarily the IgG fraction of the immune serum. Subsequently or alternatively, a specific antigen or epitope thereof targeted by the immunoglobulin may be immobilized on a column to purify the immunospecific antibody by immunoaffinity chromatography. Wilkinson, for example, discusses purification of immunoglobulins (published by The Scientist, Scientist press, Inc., philadelphia, pennsylvania, volume 14, phase 8 (4/17/2000), pages 25-28).

The anti-EpCAM antibodies of the invention are monoclonal antibodies. Monoclonal antibodies capable of modulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with EpCAM-mediated cell signaling can be produced, for example, by immunizing an animal with membrane-bound and/or soluble EpCAM (e.g., human EpCAM or an immunogenic fragment, derivative, or variant thereof); alternatively, immunizing an animal with a cell transfected with a vector comprising a nucleic acid molecule encoding EpCAM such that EpCAM is expressed and conjugated to the surface of the transfected cell; alternatively, the antibody is obtained by screening a library comprising sequences for binding to an EpCAM antibody or antigen binding domain. The library is prepared, for example, in a phage, the proteins or peptides are fused to phage coat proteins expressed on the surface of the assembled phage particle, and the encoded DNA sequences are contained within the phage particle (i.e., a "phage display library"). Hybridomas derived from myeloma/B cell fusions were then screened for reactivity to EpCAM.

For example, monoclonal antibodies can be prepared using a hybridoma method, such as those described by Kohler and Milstein, Nature 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable 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, lymphocytes may be immunized in vitro.

The immunizing agent typically includes a protein antigen, fragment thereof, or fusion protein thereof. Typically, peripheral blood lymphocytes are used if cells of human origin are desired, and spleen cells or lymph node cells are used if non-human mammalian sources are desired. Lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice), Academic Press (1986) pp 59-103). Immortalized cell lines are typically transfected mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Myeloma cells from rat or mouse are often used. The hybridoma cells may be cultured in a suitable medium preferably containing one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parental cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the hybridoma culture medium typically comprises hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of the antibody by the selected antibody-producing cell, and are sensitive to a culture medium (e.g., HAT medium). More preferred immortalized Cell lines are murine myeloma Cell lines available, for example, from the Solker Cell Distribution Center (Salk Institute Cell Distribution Center) of san Diego, Calif. and the American Type Culture Collection (American Type Culture Collection) of Marnsas, Vaginia. Human myeloma and murine-human heteromyeloma cell lines are also described for the production of monoclonal antibodies. (see Kozbor, J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications (Monoclonal Antibody Production Techniques and Applications), Marcel Dekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies to the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined using immunoprecipitation or an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined by Scatchard (Scatchard) analysis, e.g., of Munson and Pollard, anal. biochem.,107:220 (1980). Furthermore, in the therapeutic application of monoclonal antibodies, it is important to identify antibodies with high specificity and high affinity for the target antigen.

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

Monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification methods, such as protein a-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

Monoclonal antibodies can also be prepared by recombinant DNA methods, as 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 methods (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention can be used as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into a host cell (e.g., Chinese Hamster Ovary (CHO), human embryonic kidney (HEK293) cells, simian COS cells, per. c6, NS0 cells, SP2/0, YB2/0, or myeloma cells that do not produce immunoglobulins) to obtain synthesis of monoclonal antibodies in the recombinant host cell. The DNA may also be modified, for example, by replacing homologous murine sequences with the coding sequences for the constant domains of the human heavy and light chains (see U.S. Pat. No. 4,816,567; Morrison, Nature 368,812-13(1994)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides may be substituted with a constant region of an antibody of the invention, or with a variable domain of an antigen-binding site of an antibody of the invention to produce a chimeric bivalent antibody.

Human antibodies and antibody humanization

The monoclonal antibodies of the invention include fully human antibodies or humanized antibodies. These antibodies are suitable for administration to humans without eliciting an immune response by the human being against the administered immunoglobulin.

anti-EpCAM antibodies can be obtained using any method known in the art. For example, EpCAM antibodies of the invention can be identified in mice and subsequently in hybridomas using a modified RIMMS (multiple site repeat immunization) immunization strategy. In other alternative methods, anti-EpCAM antibodies are developed, for example, using phage display methods that use antibodies that contain only human sequences. Such methods are known in the art, for example in WO92/01047 and U.S. Pat. No. 6,521,404, which are incorporated herein by reference. In this method, a combinatorial library of combinatorial phages carrying random light and heavy chain pairs is screened using EpCAM of natural or recombinant origin or a fragment thereof. In another method, an anti-EpCAM antibody can be produced by a process, wherein at least one step of the process comprises immunizing a transgenic non-human animal with a human EpCAM protein. In this method, some of the endogenous heavy and/or kappa light chain loci of the xenogeneic non-human animal are disabled and unable to undergo the rearrangement required to produce genes encoding immunoglobulins that respond to antigens. In addition, at least one human heavy chain locus and at least one human light chain locus have been stably transfected into an animal. Thus, in response to an administered antigen, the human site rearrangement has provided genes encoding human variable regions that are immunospecific for the antigen. Thus, after immunization, transgenic mice produce B cells that secrete fully human immunoglobulins.

A variety of techniques for producing xenogenic non-human animals are well known in the art. See, for example, U.S. patent nos. 6,075,181 and 6,150,584, which are incorporated herein by reference in their entirety. As published in 1994, the general strategy is demonstrated along with the first XenoMouse^TMGeneration of germline lines. See Green et al, Nature Genetics 7:13-21(1994), which is incorporated herein by reference in its entirety. See also U.S. patent nos. 6,162,963; 6,150,584; 6,114,598, respectively; 6,075,181; and 5,939,598 and Japanese patent Nos. 3068180B 2, 3068506B 2 and 3068507B 2 and European patent No. EP 0463151B1 and International patent application Nos. WO 94/02602, WO96/34096, WO98/24893, WO 00/76310 and related family members.

In an alternative approach, others have utilized a "minilocus" approach, in which the foreign Ig locus is mimicked by the inclusion of fragments (individual genes) from the Ig locus. Thus, one or more VH genes, one or more D_HGenes, one or more J_HThe gene, mu constant region and second constant region (preferably gamma constant region) form a construct for insertion into an animal. See, for example, U.S. Pat. nos. 5,545,806; 5,545,807, respectively; 5,591,669, respectively; 5,612,205; 5,625,825, respectively; 5,625,126, respectively; 5,633,425, respectively; 5,643,763, respectively; 5,661,016, respectively; 5,721,367, respectively; 5,770,429, respectively; 5,789,215; 5,789,650, respectively; 5,814, 318; 5,877,397, respectively; 5,874,299, respectively; 6,023,010 and 6,255,458; and european patent No. 0546073B 1; and international patent application numbers WO92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852 and WO 98/24884 and related family members.

The generation of human antibodies from mice, in which large fragment chromosomes or whole chromosomes are introduced by minicell (minicell) fusion, has also been demonstrated. See european patent application nos. 773288 and 843961.

Human anti-mouse antibody (HAMA) responses have led to the industry for the production of chimeric or other humanized antibodies. Although chimeric antibodies have human constant regions and murine variable regions, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, particularly in chronic or multi-dose uses of the antibodies. It would therefore be desirable to provide fully human antibodies against EpCAM to attenuate or mitigate concerns and/or effects on HAMA or HACA responses.

The production of low immunogenic antibodies can also be achieved by humanization, chimerization and display techniques using appropriate libraries. It is understood that murine antibodies or antibodies from other species may be humanized or primatized using techniques well known in the art. References are, for example, Winter and Harris, Immunol Today 14:4346(1993) and Wright et al, Crit, reviews in Immunol.12125-168 (1992). Antibodies of interest can be engineered by recombinant DNA techniques to replace CH1, CH2, CH3, the hinge domain and/or the framework domain with the corresponding human sequences (see WO 92102190 and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,792; 5,714,350 and 5,777,085). Furthermore, the use of Ig cDNAs to construct 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 hybridomas or other cells that produce antibodies and are used to produce cDNA. The cDNA of interest can be amplified by polymerase chain reaction using specific primers (U.S. Pat. nos. 4,683,195 and 4,683,202). Alternatively, libraries are prepared and screened to isolate sequences of interest. The DNA sequence encoding the variable region of the antibody is then fused to the human constant region sequence. The sequence of the human constant region genes can be found in Kabat et al (1991) Sequences of Proteins of immunological Interest (sequence of the immune protein of Interest), N.I.H. Pub. Nos. 91-3242. The human C region gene can be readily obtained from known clones. The choice of isotype can be guided by the desired effector function (e.g., complement fixation) or activity in antibody-dependent cellular cytotoxicity. Preferred isotypes are IgG1, IgG2, IgG3, and IgG 4. Any of the human light chain constant region, κ or λ may be used. The chimeric, humanized antibody is then expressed using conventional methods.

Antibody fragments can be prepared by cleavage of intact proteins, e.g., by protease or chemical cleavage (e.g., Fv, F (ab')₂And Fab). Alternatively, truncated genes are designed. For example, a part of F (ab')₂The chimeric gene of the fragment comprises a DNA sequence encoding the CH1 domain and the H-strand hinge region, followed by a translation stop codon to produce a truncated molecule.

The consensus sequences of regions H and L J can be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent ligation of V region fragments with human C region fragments. Site-directed mutagenesis can be used to modify the C region cDNA to place restriction sites at similar positions in the human sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV-derived episomes, and the like. A convenient vector is one which encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction sites, and which is adapted to allow for easy insertion and expression of any VH or VL sequence. In such vectors, splicing typically occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, as well as on the splice region in the human CH exon. Polyadenylation and transcription termination occur at natural chromosomal sites downstream of the coding region. The chimeric antibodies produced may be linked to any strong promoter, including retroviral LTRs, such as the 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)). It is also understood that native Ig promoters and the like may be used.

In addition, human antibodies or antibodies from other species can be prepared by display-type techniques, including but not limited to phage display, retroviral display, ribosome display, and other techniques, using techniques well known in the art and in which the resulting molecule can be additionally matured (e.g., affinity matured), such techniques being well known in the art. Wright et al, Crit, Reviews in immunol.12125-168 (1992), Hanes and Pl ü ckthun, PNAS USA 94: 4937-; 10:80-8A (1992) and U.S. Pat. No. 5,733,743. If non-human antibodies are generated using display technology, such antibodies can be humanized as described above.

Antibodies to EpCAM-expressing cells, soluble forms of EpCAM, epitopes or peptides thereof, and expression libraries thereof can be generated using these techniques (see, e.g., U.S. patent No. 5,703,057), and these antibodies can then be screened for activity as described herein above.

The anti-EpCAM antibody of the present invention can be expressed by a vector comprising a DNA fragment encoding the single chain antibody described above.

These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, and the like. Vectors include chemical conjugates, such as chemical conjugates having a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine), as described in WO 93/64701, viral vectors (e.g., DNA or RNA viral vectors), fusion proteins (e.g., as described in PCT/US95/02140(WO 95/22618), which are fusion proteins comprising a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., protamine)), plasmids, phage, and the like. The vector may be chromosomal, non-chromosomal or synthetic.

Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These include poxvirus vectors (e.g., orthopoxvirus or fowlpox virus vectors), herpesvirus vectors (e.g., herpes simplex virus type I (HSV) vectors) (see, Geller, A.I. et al, J.Neurochem,64:487 (1995); Lim, F. et al, in DNA Cloning: Mammali Systems (DNA Cloning: Mammalian Systems, D.Glover, eds. (Oxford Univ. Press), Oxylan Oxford et al) (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 (2004), adenovirus vectors (see, LeGal LaSalle et al, (Sci., 988: 259: Danviron et al; Yat et al, Natt. Natt.S.S.Sci., USA 1990, 1994, J. 148: 78, J. 1993); et al (Gene clone M. J. 8, 1995) (see, Gene J. 1993)).

Poxvirus vectors introduce genes into the cytoplasm. Fowlpox viral vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and Herpes Simplex Virus (HSV) vectors are preferred for introducing nucleic acids into neural cells. The short term expression (about 2 months) caused by adenoviral vectors is shorter than that of adeno-associated virus (about 4 months), which is shorter than HSV vectors. The particular vector chosen will depend on the target cell and the condition being treated. Introduction can be accomplished by standard techniques, such as infection, transfection, transduction, or transformation. Examples of gene transfer patterns include, for example, naked DNA, CaPO₄Precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cellular microinjection, and viral vectors.

These vectors can be used to target any desired target cell. For example, stereotactic injection can be used to localize a vector (e.g., adenovirus, HSV) to a desired location. In addition, particles may be delivered by intraventricular (icv) infusion using a micro-pump infusion system, such as the cisco loume (syncromed) infusion system. A bulk flow (termed convection) based approach has also been shown to be effective in delivering macromolecules to extended regions of the brain and can be used to deliver vectors to target cells. (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 may be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injections, as well as oral or other known routes of administration.

These vectors can be used to express large quantities of antibodies that can be used in a variety of ways. For example, the presence or absence of EpCAM in the sample is detected. The antibodies may also be used to attempt to bind and disrupt EpCAM-mediated signaling.

Techniques can be adapted to the production of single chain antibodies specific for the antigenic proteins of the invention (see, e.g., U.S. Pat. No. 4,946,778). Furthermore, the methods can be adapted to the construction of Fab expression libraries (see, e.g., Huse et al, 1989Science 246: 1275-. Antibody fragments comprising an isotype to a protein antigen can be generated using techniques known in the art, including but not limited to: (i) f (ab') produced by pepsin digestion of antibody molecules₂A fragment; (ii) by reduction of F (ab')₂A Fab fragment generated by the disulfide bridge of (1); (iii) fab fragments generated by treating antibody molecules with papain and a reducing agent; and (iv) F_vAnd (3) fragment.

The invention also includes F_vFab, Fab 'and F (ab')₂An EpCAM fragment, a single chain EpCAM antibody, a single domain antibody (e.g., a nanobody or a VHH), a multispecific (e.g., bispecific) anti-EpCAM antibody, and a heteroconjugated anti-EpCAM antibody.

Methods for making bispecific antibodies are known in the art. Typically, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy/light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature,305:537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (quadromas) may produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The correct molecule must usually be purified using an affinity chromatography step. A similar approach is disclosed in WO93/08829 at 13.5.1993 and in Traunecker et al, EMBO J.,10:3655-3659 (1991).

Antibody variable domains with the desired binding specificity (antibody-antigen binding site) can be fused to immunoglobulin constant region sequences. Preferably to an immunoglobulin heavy chain constant region comprising at least part of the hinge region, CH2 and CH3 regions. Preferably, the first heavy chain constant region (CH1) containing the site necessary for binding to the light chain is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain, is inserted into separate expression vectors and co-transfected into a suitable host organism. For further details on the generation of bispecific antibodies see, e.g., Suresh et al, Methods in Enzymology,121:210 (1986).

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 recovered from recombinant cell culture. Preferably the interface comprises at least part of the CH3 region of the antibody constant domain. In this approach, one or more smaller amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By substituting a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine), a compensatory "hole" of the same or similar size to the larger side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers relative to undesired end products like homodimers.

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments are described in the literature. Example (b)For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science 229:81(1985) describe a method in which intact antibodies are proteolytically cleaved to yield F (ab')₂And (3) fragment. Reduction of these fragments in the presence of the dithiol complexing agent sodium arsenite stabilizes the adjacent dithiols and prevents intermolecular disulfide bond formation. The resulting Fab' fragments are then converted to mercaptonitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then converted back to the Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as selective immunological reagents for enzymes.

In addition, Fab' fragments can be recovered directly from E.coli and chemically conjugated to form bispecific antibodies. Shalaby et al, J.Exp.Med.175:217-225(1992) describe a fully humanized bispecific antibody F (ab')₂And (4) generation of molecules. Each Fab' fragment was secreted separately from e.coli (e.coli) and chemically coupled in vitro to form a bispecific antibody.

Various techniques for the direct preparation and isolation of bispecific antibody fragments from recombinant cell cultures have also been described. For example, bispecific antibodies are generated using leucine zippers. Kostelny et al, J.Immunol.148(5):1547-1553 (1992). The leucine zipper peptides of the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The homodimeric antibody is reduced in the hinge region to form monomers, which are then oxidized to form heterodimeric antibodies. Antibody homodimers can also be produced using this method. The "bispecific antibody" technology described by Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-. These fragments comprise a linker to the variable region of the light chain (V)_L) Linked heavy chain variable region (V)_H) The linker is short enough not to allow pairing between two domains on the same strand. Thus, V of a segment _HAnd V_LThe domains are forced to complement V of another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another use sheet is also reportedChain fv (sFv) dimers strategy for the preparation of bispecific antibody fragments. See Gruber et al, J.Immunol.152:5368 (1994).

Antibodies more than bivalent are contemplated. For example, trispecific antibodies can be prepared. Tutt et al, J.Immunol.147:60 (1991).

Exemplary bispecific antibodies can bind to two different epitopes, at least one of which is derived from a protein antigen of the invention. Alternatively, the anti-antigenic arms of an immunoglobulin molecule can be combined with arms of a priming molecule (e.g., a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7) or an Fc receptor (FcyR) of IgG, such as FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16) that bind to leukocytes that express a particular antigen.

Heterologous conjugate antibodies are also within the scope of the invention. The heteroconjugate antibody consists of two covalently linked antibodies. For example, such antibodies have been proposed to target immune system cells to unwanted cells (see U.S. Pat. No. 4,676,980), and are useful in the treatment of HIV infection (see WO 91/00360, WO 92/200373, and EP 03089). It is contemplated that the antibodies can be prepared by known methods of synthetic protein chemistry, including those using cross-linking agents. For example, immunotoxins may 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 (butylimidate) and those disclosed, for example, in U.S. Pat. No. 4,676,980.

It may be desirable to modify the antibodies of the invention in terms of effector function to enhance, for example, the effectiveness of the antibodies in treating diseases and disorders associated with aberrant EpCAM signaling. For example, cysteine residues may be introduced into the Fc region, thereby forming interchain disulfide bonds in this region. The homodimeric antibody so produced may have improved internalization capacity and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (see Caron et al, J.ExpM, eds. 176:1191-1195(1992) and shop, J.Immunol.,148:2918-2922 (1992)). Alternatively, antibodies with dual Fc regions can be engineered to have improved complement lysis and ADCC capabilities. (see Stevenson et al, Anti-Cancer Drug Design,3:219-230 (1989)).

The invention also relates 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 a fragment thereof) or a radioisotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that may be used include diphtheria a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin a chain, abrin a chain, modeccin a chain, alphabroom, Aleurites fordii protein, dianthin protein, phytolacca americana protein (PAPI, PAPII and PAP-S), Momordica charantia (momordia charrantia) inhibitors, curcin, crotin, saponaria officinalis (Sapaonaria officinalis) inhibitors, gelonin, mitotoxin (mitogellin), restrictocin, phenomycin, enomycin and trichothecene toxins (tricothecenes). A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include²¹²Bi、¹³¹I、¹³¹In、⁹⁰Y and¹⁸⁶Re。

conjugation of the antibody to the cytotoxic agent may be accomplished using a variety of bifunctional protein conjugating agents, such as N-succinimidyl-3- (2-pyridinedithiol) propionate (SPDP), Iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene). For example, ricin immunotoxins may be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methylenedioxytriaminepentaacetic acid (1-isothiocyanatobenzyl-3-methylidene triaminepentaacetic acid, MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. (see WO 94/11026).

One of ordinary skill in the art will recognize that a wide variety of possible moieties may be conjugated to the resulting antibodies of the invention. (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology "), J.M.Cruse and R.E.Lewis, eds., Kagill Press, New York, (1989), the entire contents of which are incorporated herein by reference in their entirety.

Conjugation may be accomplished by any chemical reaction that is capable of binding two molecules, provided that the antibody and the other moiety retain their respective activities. The linkage may include a number of chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, cooperative bonding, and complexation. But preferably the binding is covalent. Covalent bonding can be accomplished by direct condensation of existing side chains or by integration of external bridging molecules. Many bivalent or multivalent linking agents can be used to conjugate protein molecules (e.g., antibodies of the invention) to other molecules. For example, representative conjugating agents may include organic compounds such as thioesters, carbodiimides, succinimidyl esters, diisocyanates, glutaraldehyde, diazobenzenes, and 1, 6-hexanediamine. This list is not intended to be exhaustive of the various classes of conjugation agents known in the art, but rather is exemplary of the more commonly used conjugation agents. (see Killen and Lindstrom, journal.Immun.133: 1335-2549 (1984); Jansen et al, Immunological Reviews 62:185-216 (1982); and Vitetta et al, Science 238:1098 (1987)).

Preferred linkers are described in the literature. (see, e.g., Ramakrishan, S. et al, Cancer Res.44:201-208(1984), which describes MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester) usage). See also U.S. Pat. No. 5,030,719, which describes conjugation of halogenated acethydrazide derivatives to antibodies via oligopeptide linkers. Specifically, the preferred joint comprises: (i) EDC (1-ethyl-3- (3-dimethylamino-propyl) carbodiimide, (ii) SMPT (4-succinimidyloxycarbonyl- α -methyl- α - (2-pyridyldithio) -toluene (Pierce chem. Co., catalog No. 21558G), (iii) SPDP (ethyl succinimidyl-6 [3- (2-pyridyldithio) propionamido ] hexanoate, Pierce chem. Co., catalog No. 21651G), (iv) sulfo-LC-SPDP (ethyl sulfosuccinimidyl-6 [3- (2-pyridyldithio) propionamido ] hexanoate, Pierce chem. Co., catalog No. 2165-G), and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce. chem. Co., Ltd.) conjugated to EDC Pierce chem. co.), catalog No. 2451).

The linkers described above comprise components with different properties, resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. The NHS-ester containing linker is less soluble than the sulfo-NHS ester. In addition, the linker SMPT comprises a sterically hindered disulfide bond and can form conjugates of increased stability. Disulfide linkers are generally less stable than other linkers because disulfide linkers are cleaved in vitro, resulting in fewer conjugates being available. sulfo-NHS is particularly capable of enhancing the stability of carbodiimide conjugation. Carbodiimide conjugation (such as EDC) when used in conjunction with sulfo-NHS forms esters that are more resistant to hydrolysis than the carbodiimide conjugation reaction alone.

The antibodies disclosed herein can also be formulated as immunoliposomes. Antibody-containing liposomes can be prepared by methods known in the art, e.g., 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. patent nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be produced by reverse phase evaporation methods using lipid compositions comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a pore-sized filter to obtain liposomes having a desired diameter. Fab' fragments of the antibodies of the invention can be conjugated to liposomes by a disulfide interchange reaction as described in Martin et al, J.biol.chem.,257:286-288 (1982).

Use of anti-EpCAM antibodies

It is to be understood that the therapeutic entities according to the present invention may be administered with suitable carriers, excipients and other agents incorporated into the formulation, thereby providing improved transfer, delivery, tolerance, etc. Many suitable formulations can be found among those known to all medicinal chemists: remington's Pharmaceutical Sciences (Remington Pharmaceutical Sciences) (15 th edition, Mark Publishing Company (Mack Publishing Company), Iston, Pennsylvania, (1975)), particularly chapter 87, authored by Blaug, Seymour, among others. These include, for example, powders, pastes, salves, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (e.g., lipofectins) ^TM) DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of different molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures are suitable for use in the treatments and therapies according to the present invention, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible and tolerated by the route of administration. See also Baldrick p. "Pharmaceutical excipient course: the need for the new for clinical guidance "Regul. Toxicol Pharmacol.32(2):210-8(2000), Wang W. "Lyophilization and development of solid protein drugs" int.J.pharm.203(1-2):1-60(2000), Charman WN "Lipids, lipographic drugs, and oral drug delivery-some emerging concepts" J Pharm sci.89(8):967-78(2000), Powell et al, "Complex of excipients for parenteral formulations" PDA J Pharm Sci Technol.52:238-, and references therein to other information relating to formulations, excipients and carriers known to medicinal chemists.

In one embodiment, an antibody of the invention, including a monoclonal antibody of the invention, is useful as a therapeutic agent. Such agents are typically used to diagnose, prognose, monitor, treat, ameliorate, and/or prevent a disease or disorder associated with aberrant EpCAM expression, activity, and/or signaling in a subject. A treatment regimen may be implemented by using standard methods to identify a subject (e.g., a human patient) having or at risk of developing a disease or disorder associated with aberrant EpCAM expression, activity, and/or signaling (e.g., cancer or other neoplastic disorder). The subject is administered an antibody preparation, preferably one with high specificity and high affinity for its target antigen, which is generally effective due to its binding to the target. Administration of the antibody may eliminate or inhibit or interfere with expression, activity, and/or signaling function of the target (e.g., EpCAM). Administration of the antibody can abolish or inhibit or interfere with binding of the target (e.g., EpCAM) to its endogenous ligand bound in its native state. For example, the antibody binds to the target and modulates, blocks, inhibits, decreases, antagonizes, neutralizes, or otherwise interferes with the expression, activity, and/or signaling of EpCAM.

As a non-limiting example, diseases or disorders involving aberrant EpCAM expression, activity and/or signaling include hematologic cancers and/or solid tumors. Hematologic cancers include, for example, leukemia, lymphoma, and myeloma. By way of non-limiting example, certain forms of leukemia include Acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), myeloproliferative disease/tumor (MPDS), and myelodysplastic syndrome. As non-limiting examples, certain forms of lymphoma include hodgkin's lymphoma, indolent and aggressive non-hodgkin's lymphoma, burkitt's lymphoma, and follicular lymphoma (both small and large). By way of non-limiting example, some forms of myeloma include Multiple Myeloma (MM), giant cell myeloma, heavy chain myeloma, and light chain or bense-jones myeloma. Solid tumors include, for example, breast, ovarian, lung, pancreatic, prostate, melanoma, colorectal, lung, head and neck, bladder, esophageal, liver, and kidney cancers.

Symptoms associated with cancer and other neoplastic conditions include, for example, inflammation, fever, general malaise, fever, pain, frequent localization to the inflamed area (soft localized to the inflamed area), decreased appetite, decreased body weight, edema, headache, fatigue, rash, anemia, muscle weakness, muscle fatigue, and abdominal symptoms (such as abdominal pain, diarrhea, or constipation).

A therapeutically effective amount of an antibody of the invention generally relates to the amount required to achieve a therapeutic target. As described above, in some cases, the interaction between an antibody and its target antigen can interfere with the function of the target. The amount required for administration further depends on the binding affinity of the antibody to its specific antigen and also on the rate at which the administered antibody is depleted from free volume in the subject receiving administration. By way of non-limiting example, a typical range of therapeutically effective doses of an antibody or antibody fragment of the invention is from about 0.1mg/kg body weight to about 100mg/kg body weight. Common dosage frequency ranges are, for example, from twice daily to once weekly.

The effectiveness of the treatment can be determined in conjunction with any known method for diagnosing or treating a particular inflammation-related disorder. Alleviation of the symptoms of one or more inflammation-related disorders indicates that the antibody has clinical benefit.

In another embodiment, antibodies directed to EpCAM can be used in methods known in the art that correlate with EpCAM localization and/or quantification (e.g., for measuring the level of EpCAM in a suitable physiological sample, for diagnostic methods, for protein imaging, etc.). In a particular embodiment, an antibody specific for EpCAM comprising an antigen binding domain derived from an antibody or a derivative, fragment, analogue or homologue thereof is used as the pharmacologically active compound (hereinafter referred to as "therapeutic agent").

In another embodiment, EpCAM polypeptides can be isolated using EpCAM-specific antibodies by standard methods (e.g., immunoaffinity, chromatography, or immunoprecipitation). Antibodies to EpCAM protein (or fragments thereof) can be used in diagnostics as part of a clinical testing procedure to monitor protein levels in tissues, for example, to determine the effectiveness of a particular therapeutic regimen. Conjugation (i.e., physical attachment) of the antibody to a detectable substance may facilitate detection. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, anda radioactive material. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include: umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include: luciferase, luciferin and photoprotein; examples of suitable radioactive materials include ¹²⁵I、¹³¹I、³⁵S or³H。

In yet another embodiment, the antibodies of the invention can be used as reagents to detect the presence of EpCAM (or a protein fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. The antibody is polyclonal, or more preferably, monoclonal. Use of intact antibodies or fragments thereof (e.g. Fab, scFv or F (ab')₂). The term "label" with respect to a probe or antibody is intended to encompass direct labeling of the probe or antibody by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and labeling of the ends of the DNA probes with biotin such that they can be detected using fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and biological fluids present in a subject. Thus, blood and blood fractions or components (including serum, plasma or lymph) are included in the use of the term "biological sample". In other words, the detection method of the present invention can be used to detect analyte mRNA, protein or genomic DNA in a biological sample in vitro as well as in vivo. For example, techniques for detecting analyte mRNA in vitro include Northern hybridization and in situ hybridization. Techniques for in vitro detection of analyte proteins include enzyme-linked immunosorbent assays (ELISA), Western blots, immunoprecipitations, and immunofluorescence. For in vitro detection of analyte-based Techniques for genomic DNA include Southern hybridization. Methods for performing immunoassays can be found in, for example, "ELISA: Theroy and Practice: Methods in Molecular Biology (ELISA: Methods in Theory and Practice: Methods in Molecular Biology)", volume 42, eds. J.R.Crowther, Human Press, Towa, N.J., 1995; "Immunoassay" (Immunoisay) ", E.Diamandis and T.Christopous, Academic Press Inc. (Academic Press Inc.), san Diego, Calif., 1996; and "Practice and Theory of Enzyme Immunoassays" (Practice and Theory of enzymatic Immunoassays), p.tijssen, Elsevier Science, amsterdam, 1985. In addition, techniques for in vivo detection of analyte proteins include introducing labeled anti-analyte protein antibodies into a subject. For example, the presence and location of a radiolabel in a subject may be detected by standard imaging techniques using a radiolabel-labeled antibody.

Preparation method

The MSFP or anti-EpCAM antibodies (or antigen-binding fragments thereof) of the invention can be prepared using protein expression and purification methods known in the art.

In some embodiments, the invention provides an isolated nucleic acid encoding one or more polypeptide chains of any of the MSFP or anti-EpCAM antibodies (or antigen-binding fragments thereof) described herein. In some embodiments, the isolated nucleic acid comprises the nucleic acid sequence of SEQ ID NO 31 or SEQ ID NO 32. The isolated nucleic acid may be DNA or RNA.

SEQ ID NO:31 (nucleic acid encoding SEQ ID NO: 22)

gaggtgcagctggtggagtcagggggaggcttggtccagcctgggggatccctgagactctcctgtgcagcctctggattcacctttagtaattattggatgagctgggtccgccaggctccagggaaggggctggagtgggtggccaacataaagcaagatggaagtgagaaattctatgcggactctgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaagacacggctgtctattactgtgcgagagtggggccgtcctgggagcaggactactggggccagggaaccctggtcactgtctcggccggtggcggtggcagcggcggtggtgggtccggtggcggcggatctggcgcgcagtctgtactgactcaaccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcgggtcttattatggtgtgcactggtaccagcagcttccaggaacagcccccaaactcctcatctattctgacactaatcgaccctcaggggtccctgaccgattctctggctccaagtctggcacctcggcctccctggccatcactgggctccaggctgaggatgaggctgattattactgccagtcgtatgacaagggcttcgggcaccgggtgttcggcggagggaccaagctgaccgtcctagggggcgaggtgcagctggtggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttaacacctacgccatgaactgggtccgccaggctccagggaaggggctggagtgggtcgcacgcataagaagtaaatataataattatgcaacatattatgccgattcagtgaaagaccggttcaccatctccagagacgattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgtgagacatgggaacttcggtaatagctacgtttcctggtttgcttactggggccaagggacaatggtcaccgtctcttcagctagcaccaagggcccatccgtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtccaccgtgctcatga

SEQ ID NO:32 (nucleic acid encoding SEQ ID NO: 23)

gaggtgcagctggtggagtcagggggaggcttggtccagcctgggggatccctgagactctcctgtgcagcctctggattcacctttagtaattattggatgagctgggtccgccaggctccagggaaggggctggagtgggtggccaacataaagcaagatggaagtgagaaattctatgcggactctgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaagacacggccgtctattactgtgcgagagtggggccgtcctgggagcaggactactggggccagggaaccctggtcactgtctcggccggtggcggtggcagcggcggtggtgggtccggtggcggcggatctggcgcgcagtctgtactgactcaaccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcgggtcttattatggtgtgcactggtaccagcagcttccaggaacagcccccaaactcctcatctattctgacactaatcgaccctcaggggtccctgaccgattctctggctccaagtctggcacctcggcctccctggccatcactgggctccaggctgaggatgaggctgattattactgccagtcgtatgacaagggcttcgggcaccgggtgttcggcggagggaccaagctgaccgtcctagggggccaggctgtggtgactcaggagccctcactgactgtgtccccaggagggacagtcactctcacctgtcgctcaagtactggggctgttacaactagtaactatgccaactgggtccagcagaaacctggacaagcacccaggggtctgattggtggtaccaacaagcgagctccaggtacccctgcccggttctcaggctccctccttgggggcaaagctgccctgacactgtcaggtgtgcagcctgaggacgaggctgagtattactgcgctctatggtacagcaacctctgggtgttcggcggagggaccaagctgaccgtcctaggccaaccgaaagcggcgccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgtccaccgtgctcatga

In some embodiments, the isolated nucleic acid is inserted into a vector, such as a viral vector or a cloning vector. To express the nucleic acid, the vector may be introduced into a host cell to express the polypeptide in the host cell. Expression vectors may contain a variety of elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selectable markers, and signal sequences. Those skilled in the art may select these elements as appropriate. For example, the promoter sequence may be selected to facilitate transcription of the polynucleic acid in the vector. Suitable promoter sequences include, but are not limited to, the T7 promoter, the T3 promoter, the SP6 promoter, the β -actin promoter, the EF1a promoter, the CMV promoter, and the SV40 promoter. Enhancer sequences can be selected to enhance transcription of the polynucleic acid. The selectable marker may be selected so as to allow selection of host cells into which the vector is inserted from host cells into which the vector is not inserted, e.g., the selectable marker may be a gene conferring resistance to an antibiotic. The signal sequence may be selected so as to allow the expressed polypeptide to be transported out of the host cell. In some embodiments, the isolated nucleic acid further comprises a nucleic acid sequence encoding a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO 33. In some embodiments, the nucleic acid sequence encoding a signal peptide comprises the nucleic acid sequence of SEQ ID NO 34.

SEQ ID NO 33 (Signal peptide)

MEWSWVFLFFLSVTTGVHS

SEQ ID NO. 34 (nucleic acid encoding a signal peptide)

atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgtccactcc

In some embodiments, an isolated host cell comprising a vector provided herein is provided. Host cells comprising the vector may be used for the expression or cloning of the polynucleic acid comprised in said vector. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Expression of antibodies and antigen-binding fragments in prokaryotic cells such as E.coli has been established in the art. For a review see, e.g., Pluckthun, A.Biotechnology 9:545-551 (1991). Culture expression in eukaryotic cells is also used by those skilled in the art as an option for the production of antibodies or antigen-binding fragments thereof, see recent reviews, e.g., Ref, M.E (1993) curr. opinion biotech.4: 573-576; trill J.J.et al (1995) curr.opinion Biotech 6: 553-560. Higher eukaryotic cells, particularly those derived from multicellular organisms, can be used for expression of the glycosylated polypeptides provided herein. Suitable higher eukaryotic cells include, but are not limited to, invertebrate cells and insect cells, and vertebrate cells.

The vector may be introduced into the host cell using any suitable method known in the art, including but not limited to: DEAE-dextran mediated delivery, calcium phosphate precipitation, cationic lipid mediated delivery, liposome mediated transfection, electroporation, microprojectile bombardment, receptor mediated gene delivery, delivery mediated by polylysine, histones, chitosan and peptides. Standard methods for transfection and transformation of cells for expression of the vector of interest are known in the art. In some embodiments, the host cell comprises a first vector encoding a first polypeptide and a second vector encoding a second polypeptide. In some embodiments, the host cell comprises a single vector comprising an isolated nucleic acid encoding a first polypeptide and a second polypeptide.

In some embodiments, the invention provides a method of expressing any one of the MSFP or anti-EpCAM antibodies (or antigen-binding fragments thereof) of the present disclosure, comprising culturing an isolated host cell comprising a vector, said isolated host cell being cultured under conditions that allow expression of a nucleic acid inserted in the vector, and recovering said MSFP or anti-EpCAM antibody (or antigen-binding fragment thereof) from the culture, suitable conditions for expressing a polynucleic acid may include, but are not limited to: suitable media, suitable density of host cells in the media, presence of necessary nutrients, presence of supplemental factors, suitable temperature and humidity, and absence of microbial contaminants. One of ordinary skill in the art can optionally select suitable conditions for expression purposes.

In some embodiments, the polypeptide expressed in the host cell may form a dimer and thereby produce a MSFP or anti-EpCAM antibody (or antigen-binding fragment thereof) of the invention. In some embodiments, the polypeptides expressed in the host cell may form a polypeptide complex of homodimers. In some embodiments, a first polypeptide and a second polypeptide are expressed in a host cell, which can form a polypeptide complex of a heterodimer.

In some embodiments, the polypeptide complex (e.g., MSFP or anti-EpCAM antibody or antigen-binding fragment thereof) can be formed within a host cell, e.g., a dimer can be formed within a host cell with the aid of an associated enzyme and/or cofactor. In some embodiments, the polypeptide complex may be secreted extracellularly. In some embodiments, the first polypeptide and the second polypeptide can be secreted extracellularly and form a dimer (e.g., MSFP or anti-EpCAM antibody or antigen-binding fragment thereof) outside the host cell.

In some embodiments, the first and second polypeptides are separately expressible and allowed to dimerize under suitable conditions to form a MSFP or anti-EpCAM antibody or antigen-binding fragment thereof. For example, the first and second polypeptides may be combined in a suitable buffer and the first and second protein monomers allowed to dimerize via a suitable interaction, such as a hydrophobic interaction. In some embodiments, the first and second polypeptides may be combined in a suitable buffer comprising an enzyme and/or cofactor that promotes dimerization of the first and second polypeptides. In some embodiments, the first and second polypeptides may be combined in a suitable vehicle and allowed to interact with each other in the presence of suitable reagents and/or catalysts.

Any suitable method may be used to collect the expressed polypeptide and/or polypeptide complex. The polypeptide and/or polypeptide complex may be expressed intracellularly, in the periplasmic space of the cell or secreted extracellularly into the medium. If the polypeptide and/or polypeptide complex is expressed intracellularly, the host cell containing the polypeptide and/or the polypeptide complex can be lysed and the polypeptide and/or the polypeptide complex can be separated from the lysate by centrifugation or ultrafiltration to remove unwanted debris. If the polypeptide and/or the polypeptide complex is secreted into the periplasmic space of E.coli, the cytoplasm may be thawed in the presence of reagents such as sodium acetate (pH3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) within about 30 minutes and cell debris removed by centrifugation (Carter et al, Biotechnology 10:163-167 (1992)). If the polypeptide and/or the polypeptide complex is secreted into the culture medium, the cell culture supernatant can be collected and concentrated using commercially available protein concentration filters (e.g., Amicon or Millipore Pellicon ultrafiltration devices). Protease inhibitors and/or antibiotics may be included in the collection and concentration steps to inhibit protein degradation and/or growth of contaminating microorganisms.

The expressed polypeptide and/or polypeptide complex may be further purified by suitable methods, including, for example, but not limited to: affinity chromatography, hydroxylapatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange separation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin agarose, chromatography on anion or cation exchange resins (e.g.polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation (see for review Bonner, P.L., Protein purification, Taylor & Francis, 2007; Janson, J.C., et al, Protein purification: printips, high resolution methods and applications, Wiley-VCH, 1998).

In some embodiments, the polypeptide and/or polypeptide dimer may be purified by affinity chromatography. In some embodiments, protein a chromatography or protein a/G (fusion protein of protein a and protein G) chromatography may be used to purify polypeptides and/or polypeptide complexes comprising components derived from the CH2 domain and/or CH3 domain of antibodies (Lindmark et al, j.immunol.meth.62:1-13 (1983)); zettlit, K.A., anti-body Engineering, Part V,531-535, 2010). In some embodiments, protein G chromatography can be used to purify polypeptides and/or polypeptide complexes comprising IgG γ 3 heavy chain (Guss et al, EMBO J.5: 15671575 (1986)). In some embodiments, protein L chromatography can be used to purify polypeptides and/or polypeptide complexes comprising kappa light chains (Sudhir, P., antibiotic engineering protocols, Chapter 26, Humana Press,1995, Nilson, B.H.K. et al, J.biol.chem.,267, 2234-one 2239 (1992)). The affinity ligand-attached matrix is most commonly agarose, but other matrices can be used. Mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene have faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX resin (J.T.Baker, Phillipsburg, N.J.) can be used for purification.

Pharmaceutical compositions, unit dosage forms, articles of manufacture and kits

The invention also provides a pharmaceutical composition comprising any one of the MSFP or anti-EpCAM antibodies (or antigen-binding fragments thereof) and a pharmaceutically acceptable carrier.

The pharmaceutical compositions can be used in a variety of methods of administration of the present disclosure, including, for example, systemic or local injection. In some embodiments, the pharmaceutical composition is formulated for intravenous injection. In some embodiments, the pharmaceutical composition is formulated for subcutaneous injection. In some embodiments, the pharmaceutical composition is formulated for local injection at a tumor site. In some embodiments, the pharmaceutical composition is formulated for intratumoral injection.

The "carrier" used includes pharmaceutically acceptable carriers, excipients or stabilizers which are non-toxic to the cells or mammals exposed to the dose or concentration used. A common physiologically acceptable carrier is a water-soluble pH buffered solution. Acceptable carriers, excipients are non-toxic to recipients exposed to the dosages or concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (e.g. for Octadecyl dimethyl benzyl ammonium chloride; (ii) hexanediamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG).

In some embodiments, the pharmaceutical composition is prepared at a pH in the range of about 4.5 to about 9.0, including, for example, any one of the ranges of about 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0. In some embodiments, the pharmaceutical composition is made isotonic to blood by the addition of a suitable strength-modifying agent, such as glycerol.

Pharmaceutical compositions for in vivo injection are typically prepared sterile, substantially isotonic, and in full compliance with Good Manufacturing Practice (GMP) regulations of the U.S. food and drug administration. Sterility can be accomplished by filtration using sterile filtration membranes. In some embodiments, the composition is pathogen-free. For injection, the pharmaceutical composition may be in liquid form, e.g., a physiologically compatible buffer such as Hank's solution or Ringer's solution. In addition, the pharmaceutical composition may be in solid form and reconstituted or resuspended immediately prior to use. Lyophilized compositions are also included in the present invention.

In some embodiments, the pharmaceutical composition is formulated according to conventional procedures to prepare a pharmaceutical composition suitable for intravenous, intraperitoneal, or intravitreal injection. Typically, the compositions for injection are sterile isotonic buffered solutions. If desired, the composition may also contain a solubilizing agent and a local anesthetic such as lignocaine to reduce injection site pain. Typically, the ingredients are provided separately or mixed together in unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate in a sealed container (e.g., an ampoule or sachet indicating the amount of active agent). When the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

In some embodiments, the pharmaceutical composition is suitable for administration to a human. In some embodiments, the pharmaceutical composition is stored in a single-use vial, e.g., a single-use sealed vial. In some embodiments, the pharmaceutical composition is stored in one multi-use vial. In some embodiments, the pharmaceutical composition is stored in a container in bulk form. In some embodiments, the pharmaceutical composition is cryopreserved.

The invention also provides unit dosage forms of MSFP, anti-EpCAM antibody (or antigen-binding fragment thereof), or a composition thereof. Each dose may comprise from about 0.01 μ g to about 10mg, including, for example, about 0.01 μ g to about 10mg, about 0.01 μ g to about 5mg, about 0.01 μ g to about 1mg, about 0.1 μ g to about 300 μ g, about 0.1 μ g to about 200 μ g, about 0.1 μ g to about 100 μ g, about 0.1 μ g to about 90 μ g, about 0.1 μ g to about 80 μ g, about 0.1 μ g to about 70 μ g, about 0.1 μ g to about 60 μ g, about 0.1 μ g to about 50 μ g, about 0.1 μ g to about 40 μ g, about 0.1 μ g to about 30 μ g, about 0.1 μ g to about 20 μ g, about 0.1 μ g to about 10 μ g, about 0.1 μ g to about 5 μ g, or about 0.1 μ g to about 1 μ g. In some embodiments, the unit dosage form of the MSFP, anti-EpCAM antibody (or antigen-binding fragment thereof), or composition thereof is in any one of the following ranges, with the upper limit of the range being 0.1. mu.g, 0.2. mu.g, 0.3. mu.g, 0.4. mu.g, 0.5. mu.g, 0.6. mu.g, 0.7. mu.g, 0.8. mu.g, 0.9. mu.g, 1. mu.g, 5. mu.g, 10. mu.g, 15. mu.g, 20. mu.g, 25. mu.g, 30. mu.g, 35. mu.g, 40. mu.g, 45. mu.g, 50. g, 55. mu.g, 60. mu.g, 65. g, 70. g, 75. mu.g, 80. mu.g, 85. g, 90. mu.g, 95. g, 100. g, 150. g, 200. mu.g, 250. g, 300. mu.g, 350. g, 400. mu.g, 450. g, 500. mu.g, 550. mu.g, 600. g, 650. g, 1500. mu.g, 800. mu.g, 3000. g, 3000. mu.g, etc, 6000 μ g, or 10000 μ g, the lower limit of the range being independently selected from 0.1 μ g, 0.2 μ g, 0.3 μ g, 0.4 μ g, 0.5 μ g, 0.6 μ g, 0.7 μ g, 0.8 μ g, 0.9 μ g, 1 μ g, 5 μ g, 10 μ g, 15 μ g, 20 μ g, 25 μ g, 30 μ g, 35 μ g, 40 μ g, 45 μ g, 50 μ g, 55 μ g, 60 μ g, 65 μ g, 70 μ g, 75 μ g, 80 μ g, any one of 85 μ g, 90 μ g, 95 μ g, 100 μ g, 150 μ g, 200 μ g, 250 μ g, 300 μ g, 350 μ g, 400 μ g, 450 μ g, 500 μ g, 550 μ g, 600 μ g, 650 μ g, 700 μ g, 750 μ g, 800 μ g, 850 μ g, 900 μ g, 1000 μ g, 1500 μ g, 2000 μ g, 2500 μ g, 3000 μ g, 6000 μ g, or 10000 μ g, and wherein the lower limit is less than the upper limit. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for individuals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent or excipient. These unit dosage forms may be stored in suitable packaging in single or multiple unit doses, and may further be sterilized and sealed.

The present invention also provides an article of manufacture comprising a composition of the present disclosure (e.g., a pharmaceutical composition) in a suitable package. Suitable packaging for the described compositions (e.g., such as MSFP or anti-EpCAM antibody compositions) are known in the art and include, for example, vials (e.g., sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed mylar or plastic bags), and the like. These articles may be further sterilized and/or sealed.

The invention also provides kits comprising the compositions (e.g., pharmaceutical compositions), and further comprises instructions for using the compositions, e.g., as described herein. The kits described herein may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any of the methods described herein.

Illustrative embodiments

The present invention provides the following embodiments:

1. a method of treating cancer in an individual comprising administering to the individual an effective amount of a multi-specific Fab fusion protein comprising: a Fab fragment that specifically binds CD3, and a binding domain that specifically binds EpCAM; wherein the binding domain is fused to the N-terminus of the Fab fragment; and wherein the dosage of administration of the multi-specific Fab fusion protein is from about 0.01 μ g/kg to about 250 μ g/kg.

2. The method of embodiment 1, wherein the binding domain is an scFv.

3. The method of embodiment 2, wherein the multi-specific Fab fusion protein comprises a first scFv that specifically binds EpCAM, and a second scFv that specifically binds 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.

4. The method of embodiment 3, wherein the first scFv and the second scFv have the same sequence.

5. The method according to any of embodiments 1 to 4, wherein the multi-specific Fab fusion protein is administered intravenously.

6. The method of any one of embodiments 1 to 5, wherein the multi-specific Fab fusion protein is administered at a low frequency.

7. The method of embodiment 6, wherein the multi-specific Fab fusion protein is administered twice weekly.

8. The method of any one of embodiments 1-7, wherein the multi-specific Fab fusion protein is administered at a dose equivalent to about 0.1 to about 100 μ g/kg for cynomolgus monkeys.

9. The method of any one of embodiments 1 to 8, wherein the multi-specific Fab fusion protein is administered in a dose that does not cause a cytokine storm.

10. The method of embodiment 9, wherein the multi-specific Fab fusion protein is administered at a dose equivalent to no more than about 30 μ g/kg for cynomolgus monkeys.

11. The method of any one of embodiments 1-10, wherein the multi-specific Fab fusion protein is administered to the individual at a first dose for a first period of time and, continuously, the multi-specific Fab fusion protein is administered to the individual at a second dose for a second period of time, and wherein the second dose exceeds the first dose.

12. The method of embodiment 11, wherein the second period of time exceeds the first period of time.

13. The method of embodiment 11 or embodiment 12, wherein the first period of time is at least about 7 days.

14. The method of any one of embodiments 11-13, wherein the second period of time is at least about 2 weeks.

15. The method of any one of embodiments 11-14, wherein the first dose is no more than about 1 μ g/kg.

16. The method of any of embodiments 11-14, wherein the second dose is about 0.1 μ g/kg to about 10 μ g/kg.

17. The method of any one of embodiments 1-16, wherein the method further comprises administering a glucocorticoid to the individual.

18. The method of embodiment 17, wherein the glucocorticoid is dexamethasone.

19. The method of embodiment 17 or embodiment 18, wherein the glucocorticoid is administered prior to the first dose of the multi-specific Fab fusion protein.

20. The method of any one of embodiments 17-19, wherein the glucocorticoid is administered in a dose of about 0.1mg/kg to about 5 mg/kg.

21. The method of any one of embodiments 1-20, wherein the subject is a human subject.

22. The method of any one of embodiments 1 to 21, wherein the Fab fragment specifically binds to the N-terminus of CD3 epsilon.

23. The method of embodiment 22 wherein the Fab fragment specifically binds to an epitope within amino acids 1-27 of CD3 epsilon.

24. The method of embodiment 23, wherein the VH of the Fab fragment comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1; HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2; and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3.

25. The method of embodiment 23 or embodiment 24, wherein the VL of the Fab fragment comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4; HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5; and HVR-L3 comprising the amino acid sequence of SEQ ID NO 6.

26. The method of any one of embodiments 23 to 25, wherein the VH of the Fab fragment comprises an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to an amino acid sequence selected from SEQ ID NOs 7 and 39-43.

27. The method of any one of embodiments 23 to 26, wherein the VL of said Fab fragment comprises an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 8 and 44-47.

28. The method of any one of embodiments 23 to 27, wherein said Fab fragment comprises a human immunoglobulin heavy chain constant region 1(CH1) comprising the amino acid sequence of SEQ ID NO. 9.

29. The method of any one of embodiments 23 to 27, wherein the Fab fragment comprises a human λ light chain constant region comprising the amino acid sequence of SEQ ID No. 10.

30. The method of any one of embodiments 23-29, wherein CH1 and CL of the Fab fragment are linked by one or more disulfide bonds.

31. The method of embodiment 30, wherein the Fab fragment comprises a first polypeptide comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID No. 11.

32. The method of embodiment 30 or embodiment 31, wherein the Fab fragment comprises a second polypeptide comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID No. 12.

33. The method of any one of embodiments 1-32, wherein the cancer is EpCAM positive solid cancer.

34. The method of embodiment 33, wherein said EpCAM positive solid cancer is a carcinoma or an adenocarcinoma.

35. The method of any one of embodiments 1-34, wherein the cancer is selected from the group consisting of: small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer.

36. The method of embodiment 35, wherein the cancer is colorectal adenocarcinoma.

37. The method of embodiment 35, wherein the cancer is lung adenocarcinoma.

38. The method according to any one of embodiments 2-37, wherein the scFv comprises an N-VH-VL-C fusion polypeptide.

39. The method of any one of embodiments 2-38, wherein the VH of the scFv comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO 13; HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14; and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15.

40. The method of any one of embodiments 2-39, wherein the VL of said scFv comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO. 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO. 18.

41. The method of any one of embodiments 2-40, wherein the VH of the scFv comprises an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO 19.

42. The method of any one of embodiments 2-41, wherein the VL of the scFv comprises an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 20.

43. The method of embodiment 42, wherein the scFv comprises an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 21.

44. The method of any one of embodiments 1 to 43, wherein the multi-specific Fab fusion protein comprises a first polypeptide comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO. 22.

45. The method of any one of embodiments 1-44, wherein the multi-specific Fab fusion protein comprises a second polypeptide comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO. 23.

46. An anti-EpCAM antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising: (1) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, (2) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and (3) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15; the light chain variable region comprises: (1) HVR-L1 comprising the amino acid sequence of SEQ ID NO. 16, (2) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 17, and (3) HVR-L3 comprising the amino acid sequence of SEQ ID NO. 18.

47. The anti-EpCAM antibody or antigen-binding fragment thereof of embodiment 46, wherein said heavy chain variable domain sequence comprises a VH comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO. 19.

48. The anti-EpCAM antibody or antigen-binding fragment thereof of embodiment 46 or 47, wherein said light chain variable domain sequence comprises a VL comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID No. 20.

49. The anti-EpCAM antibody or antigen-binding fragment thereof of any one of embodiments 46-48, wherein said anti-EpCAM antibody comprises the Fc sequence of a human IgG.

50. The anti-EpCAM antibody of any one of embodiments 46-49, wherein said anti-EpCAM antibody is a multispecific antibody.

51. The antigen-binding fragment of an anti-EpCAM antibody of any one of embodiments 46-48, wherein said antigen-binding fragment is a single chain fv (scfv).

52. The antigen-binding fragment of an anti-EpCAM antibody of embodiment 51, wherein said scFv comprises an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 21.

53. A multi-specific Fab fusion protein comprising an anti-EpCAM antigen binding fragment of any one of embodiments 46-48 and 51-52.

54. The multi-specific Fab fusion protein of embodiment 53, comprising a Fab fragment that specifically binds to CD3, a first copy of an anti-EpCAM antigen-binding fragment, and a second copy of an anti-EpCAM antigen-binding fragment; wherein a first copy of the anti-EpCAM antigen-binding fragment is fused to the N-terminus of the VH of the Fab fragment; and wherein a second copy of the anti-EpCAM antigen-binding fragment is fused to the N-terminus of the VL of the Fab fragment.

55. The multi-specific Fab fusion protein of embodiment 54, wherein the Fab fragment specifically binds to the N-terminus of CD3 epsilon.

56. The multi-specific Fab fusion protein of embodiment 55, wherein the Fab fragment specifically binds to an epitope within amino acids 1-27 of CD3 epsilon.

57. The multi-specific Fab fusion protein of claim 56, wherein the VH of the Fab fragment comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1; HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2; and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3.

58. The multi-specific Fab fusion protein of embodiment 56 or embodiment 57, wherein the VL of the Fab fragment comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4; HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5; and HVR-L3 comprising the amino acid sequence of SEQ ID NO 6.

59. The multi-specific Fab fusion protein of any one of claims 56 to 58, wherein the VH of the Fab fragment comprises an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 7 and 39-43.

60. The multi-specific Fab fusion protein of any one of claims 56 to 59, wherein the VL of the Fab fragment comprises an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 8 and 44-47.

61. The multi-specific Fab fusion protein of any one of claims 53 to 60, wherein the Fab fragment comprises a human immunoglobulin heavy chain constant region 1(CH1) comprising the amino acid sequence of SEQ ID NO. 9.

62. The multi-specific Fab fusion protein of any one of claims 53 to 61, wherein the Fab fragment comprises a human lambda light chain constant region comprising the amino acid sequence of SEQ ID NO. 10.

63. The multi-specific Fab fusion protein of any one of claims 53 to 61, wherein the CH1 and CL of the Fab fragment are linked by one or more disulfide bonds.

64. The method of embodiments 56-63, wherein the Fab fragment comprises a first polypeptide comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 11.

65. The method of embodiments 56-64, wherein the Fab fragment comprises a second polypeptide comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO 12.

66. An isolated nucleic acid molecule encoding the anti-EpCAM antibody or antigen-binding fragment thereof or the multi-specific Fab fusion protein of any one of embodiments 46-65.

67. An expression vector comprising the isolated nucleic acid molecule of embodiment 66.

68. An isolated host cell comprising the expression vector of embodiment 67.

69. A method of producing an anti-EpCAM antibody or antigen-binding fragment thereof or a multi-specific Fab fusion protein comprising culturing the isolated host cell of embodiment 68 and recovering the anti-EpCAM antibody or antigen-binding fragment thereof or multi-specific Fab fusion protein from the cell culture.

70. A composition comprising an anti-EpCAM antibody or antigen-binding fragment thereof or a multi-specific Fab fusion protein of any one of embodiments 46-65, and a pharmaceutically acceptable carrier.

71. A method of treating cancer in an individual comprising administering to the individual an effective amount of the composition of embodiment 70.

72. Use of an anti-EpCAM antibody or antigen-binding fragment thereof or a multi-specific Fab fusion protein of any one of embodiments 46-65 in the manufacture of a medicament for treating cancer in an individual.

73. Use of a multi-specific Fab fusion protein in the manufacture of a medicament for treating cancer in an individual, wherein the multi-specific Fab fusion protein comprises a Fab fragment that specifically binds CD3 and a binding domain that specifically binds EpCAM; wherein the binding domain is fused to the N-terminus of the Fab fragment.

74. The use of embodiment 73, wherein the binding domain is an scFv.

75. The use of embodiment 74, wherein the multi-specific 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 the second scFv is fused to the N-terminus of the VL of the Fab fragment.

76. The use of embodiment 75, wherein said first scFv and said second scFv have the same sequence.

77. The use of any one of embodiments 73-76, wherein the multi-specific Fab fusion protein is administered intravenously.

78. The use of any one of embodiments 73-77, wherein the multi-specific Fab fusion protein is administered at a low frequency.

79. The method of embodiment 78, wherein the multi-specific Fab fusion protein is administered twice weekly.

80. The use of any one of embodiments 73-79, wherein the multi-specific Fab fusion protein is present at about 0.1 μ g/kg to about 250 μ g/kg.

81. The use of embodiment 80, wherein the multi-specific Fab fusion protein is administered at a dose equivalent to about 0.1 to about 100 μ g/kg for cynomolgus monkeys.

82. The use of any one of embodiments 63-81, wherein said multi-specific Fab fusion protein is to be administered in a dose that does not cause a cytokine storm.

83. The use of embodiments 63-82, wherein said multi-specific Fab fusion protein is administered to the individual at a first dose for a first period of time and, continuously, said multi-specific Fab fusion protein is administered to the individual at a second dose for a second period of time, and wherein said second dose exceeds said first dose.

84. The use of embodiment 83, wherein the second period of time exceeds the first period of time.

85. The use of embodiment 83 or embodiment 84, wherein the first period of time is at least about 7 days.

86. The use of embodiments 83-85, wherein the second period of time is at least about 2 weeks.

87. The use of embodiments 83-86, wherein the first dose is not more than about 1 μ g/kg.

88. The use of embodiments 83-87, wherein the second dose is about 0.1 μ g/kg to about 10 μ g/kg.

89. The use according to embodiments 83-88, further comprising administering a glucocorticoid to the subject.

90. The use of embodiment 89, wherein the glucocorticoid is dexamethasone.

91. The use of embodiment 89 or embodiment 90, wherein the glucocorticoid is administered prior to the first dose of the multi-specific Fab fusion protein.

92. The method of embodiments 89-91, wherein the glucocorticoid is administered at a dose of about 0.1mg/kg to about 5 mg/kg.

93. The use of embodiments 73-92, wherein the subject is a human subject.

94. The use according to any one of embodiments 73 to 93, wherein the Fab fragment specifically binds to the N-terminus of CD3 epsilon.

95. The use of embodiment 94, wherein said Fab fragment specifically binds to an epitope within amino acids 1-27 of CD3 epsilon.

96. The use of embodiment 95, wherein the VH of the Fab fragment comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 1; HVR-H2 comprising the amino acid sequence of SEQ ID NO. 2; and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 3.

97. The use of embodiment 95 or embodiment 96, wherein the VL of the Fab fragment comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO. 4; HVR-L2 comprising the amino acid sequence of SEQ ID NO. 5; and HVR-L3 comprising the amino acid sequence of SEQ ID NO 6.

98. The use of any one of embodiments 95 to 97, wherein the VH of the Fab fragment comprises an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 7 and 39-43.

99. The use of any one of embodiments 95 to 98, wherein the VL of said Fab fragment comprises an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs 8 and 44-47.

100. The use of any one of embodiments 95 to 99, wherein said Fab fragment comprises human immunoglobulin heavy chain constant region 1(CH1) comprising the amino acid sequence of SEQ ID No. 9.

101. The use of any one of embodiments 95 to 100, wherein said Fab fragment comprises a human λ light chain constant region comprising the amino acid sequence of SEQ ID No. 10.

102. The use of any one of embodiments 95 to 101, wherein CH1 and CL of the Fab fragment are linked by one or more disulfide bonds.

103. The use of embodiment 102, wherein the Fab fragment comprises a first polypeptide comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID No. 11.

104. The use of embodiment 102 or embodiment 103, wherein the Fab fragment comprises a second polypeptide comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID No. 12.

105. The use of any one of embodiments 73-104, wherein the cancer is an EpCAM positive solid cancer.

106. The use of embodiment 105, wherein the EpCAM-positive solid cancer is a carcinoma or adenocarcinoma.

107. The use of any one of embodiments 73-106, wherein the cancer is selected from the group consisting of: small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer.

108. The use of embodiment 107, wherein the cancer is colorectal adenocarcinoma.

109. The use of embodiment 107, wherein the cancer is lung adenocarcinoma.

110. The use according to any one of embodiments 74-109, wherein the scFv comprises an N-VH-VL-C fusion polypeptide.

111. The use of any one of embodiments 74 to 110, wherein the VH of the scFv comprises: HVR-H1 comprising the amino acid sequence of SEQ ID NO. 13, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 14, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 15.

112. The use of any one of embodiments 74-111, wherein the VL of the scFv comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO 16, HVR-L2 comprising the amino acid sequence of SEQ ID NO 17, and HVR-L3 comprising the amino acid sequence of SEQ ID NO 18.

113. The use according to any one of embodiments 74-112, wherein the VH of the scFv comprises an amino acid sequence that shares at least about 85% (e.g., about 100%) sequence identity with the amino acid sequence of SEQ ID NO: 19.

114. The use according to any one of embodiments 74-113, wherein the VL of the scFv comprises an amino acid sequence that shares at least about 85% (e.g., about 100%) sequence identity with the amino acid sequence of SEQ ID NO: 20.

115. The use of embodiment 114, wherein the scFv comprises an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID NO: 21.

116. The use of any one of embodiments 73-115, wherein the multi-specific Fab fusion protein comprises a first polypeptide comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID No. 22.

117. The use of any one of embodiments 73-116, wherein the multi-specific Fab fusion protein comprises a second polypeptide comprising an amino acid sequence having at least about 85% (e.g., about 100%) sequence identity to the amino acid sequence of SEQ ID No. 23.

Examples

The following examples are intended to be purely exemplary of the invention and therefore should not be considered as limiting the invention in any way. The following examples and detailed description of the invention are provided by way of illustration and not by way of limitation. For embodiments in which details of the experimental methods are not described, these methods are performed according to conventional conditions, such as those described by Sambrook et al in Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press,1989, N.Y.), or the experimental methods suggested by the manufacturer.

Example 1: expression and purification of exemplary bispecific Fab fusion proteins

1. Transient expression

The Fab fragments or Fab fusion proteins are expressed by conventional methods. Cloning of DNA fragments comprising light and heavy chains encoding Fab fusion proteins into an expression vector pcDNA, resulting in constructs for expression of the light and heavy chains, these being designed for secretory expression of the light and heavy chain proteinsThe construct also includes a signal peptide. DNA sequencing identified the inserted gene as correct. The construct plasmid was transformed into E.coli to obtain transfection grade plasmid DNA. In EXPI293^TMHEK293F cells were cultured in medium (Invitrogen). For transfection, 10mL of medium containing plasmid DNA and 25kD Polyethyleneimine (PEI) complex (DNA/linear 25kD PEI weight ratio of 1:3) was added to 90mL of cell culture. Transfecting the cells in CO ₂Incubator (37 ℃, 5% CO)₂125rpm) for about 6 days, and then collecting the supernatant.

2. Stable expression

The DNA fragment (SEQ ID No.32) encoding the light chain comprising the EpCAM × CD3Fab fusion protein (designated ITAB1002) was cloned into an expression vector carrying the hygromycin resistance gene and the DNA fragment comprising the heavy chain (SEQ ID No.31) encoding the Fab fusion protein was cloned into an expression vector carrying the puromycin resistance gene. The DNA fragments encoding the light or heavy chain genes of the Fab fusion proteins also each included a Kozak sequence, a signal peptide (amino acid sequence shown in SEQ ID No.33 and nucleotide sequence shown in SEQ ID No. 34), and were cloned between HindIII and Not I restriction enzyme sites to generate constructs expressing the light and heavy chains, respectively. 40. mu.g each of plasmid DNA containing heavy and light chain genes, respectively, were transfected into CHO-S suspension cells of logarithmic growth phase by electroporation (MaxCyte) at a cell number of about 8X 10⁸One, 24 hours later cell count, transfected cells at about 1.5X 10⁶Cell density of cells/mL was seeded in CD OptiCHO Medium (Invitrogen) containing 5. mu.g/mL puromycin (Invitrogen) and 200. mu.g/mL hygromycin B (Hyclone) in CO₂Incubator (37 ℃, 5% CO)₂125 rpm). Changing fresh selective culture medium every 2-3 days until the transfected cells are restored to normal growth rate, then collecting cells in logarithmic growth phase, preserving stable transfected cell population, and obtaining single cell clone by limiting dilution method. The stably transfected cell population was cultured (37 ℃, 5% CO) in CD OptiCHO Medium (Invitrogen) supplemented with 1 Xglutamic acid and 1g/L PF68 ₂125rpm) when the cell concentration reaches at least 3x10⁶At cell/mL, cells were transferred to 32 ℃ culture (HF 5112.5%, HF 5020.2% MedinaBio) every 2 daysThe culture medium was changed, and the cell supernatant was collected after 2-4 medium changes.

The culture supernatant was purified using IgG-CH1 affinity chromatography (Thermo Fisher Scientific) for the desired protein. The culture supernatant was filtered through a 0.22 μ M sterile membrane, loaded onto an IgG-CH1 affinity matrix equilibrated with 150mM NaCl, 10mM phosphate buffer (PBS, pH7.5), and eluted with 150mM NaCl, 50mM sodium acetate buffer (pH3.5), the pH of the eluate was adjusted to 7.2 with 2M Tris, the protein concentration was concentrated with a 10KD molecular weight cut-off coil (Sartorius), and the purified protein was stored at 4 ℃. Proteins were typically analyzed by 4-20% SDS-PAGE (polyacrylamide gel electrophoresis) under non-reducing and/or reducing conditions (5% beta-mercaptoethanol), the analysis being shown in FIGS. 2A and 2B. The purified Fab fusion proteins were analyzed by non-reducing and/or reducing capillary electrophoresis (CE-SDS, Beckman Coulter, PA800plus) and the results are shown in FIGS. 3A and 3B. HPLC analysis indicated that the purity of the purified Fab fusion protein was > 90%.

Figures 2A and 2B show SDS-PAGE (polyacrylamide gel electrophoresis) electrophoresis of purified EpCAM x CD3Fab fusion proteins. Wherein FIG. 2A shows an SDS-PAGE under non-reducing conditions, and lane 1 shows a protein molecular weight standard PageRuler ^TMThe molecular weight of the Unstabained Protein Ladder (Thermo Scientific) is 200, 150, 120, 100, 85, 70, 60, 50, 40, 30, 25, 20, 15 and 10KD from top to bottom in sequence, the Lane 2 is a purified Protein, the molecular weight is 100KD and is consistent with the theoretical molecular weight of EpCAM multiplied by CD3Fab fusion Protein; FIG. 2B shows an SDS-PAGE electrophoresis under reducing conditions, Lane 1 is a protein molecular weight standard Pierce^TMUnstanated Protein MW Marker (Thermo Scientific) with molecular weights of 116, 66.2, 45, 35, 25, 18.4, 14.4kD from top to bottom, and lane 2 is a purified Protein with molecular weight of 45-66 kD.

FIGS. 3A and 3B show the CE-SDS (capillary electrophoresis) electrophoresis results of the purified EpCAM × CD3Fab fusion proteins. Wherein, FIG. 3A depicts the non-reduced CE-SDS results, showing a single protein peak at migration time 21.59 min; FIG. 3B depicts the results of reducing CE-SDS to present two single protein peaks at migration times 18.37 min and 18.84 min, corresponding to the light and heavy chains of the Fab fusion protein, respectively.

Example 2: binding Activity assay for EpCAM × CD3Fab fusion proteins

Antigen binding affinity

Octet QK with anti-human IgG Fc (AHC) capture sensor was used ^eThe affinity of the anti-EpCAM and anti-CD 3 domains of an exemplary EpCAM x CD3Fab fusion protein (e.g., ITAB1002) to the corresponding human and cynomolgus monkey antigens was examined. The human EpCAM antigen construct (huepcam. Fc) and cynomolgus EpCAM antigen construct (cynoepcam. Fc) have a full-length EpCAM protein fused to a human IgG Fc. The human CD3 antigen construct (CD3 epsilon AA1-27.Fc) and the cynomolgus CD3 antigen construct (cynoCD3 epsilon AA1-27.Fc) have a polypeptide consisting of amino acids 1-27 of the N-terminus of CD3 epsilon fused to human IgG Fc. Expression of the antigenic construct is described in us patent 8,846,042. Diluted with diluent (PBS, 0.1% BSA, 0.02% Tween-20, 0.05% NaN₃) The antigen construct was diluted to 0.02mg/mL and the diluted antigen construct was immobilized on an anti-hIgG Fc capture (AHC) biosensor. ITAB1002 was diluted to different concentrations and added to black microplates at 200 μ L/well. Control wells containing only PBS were also set. The results of the detection were analyzed using ForteBio Data Acquisition and ForteBio Data Analysis software.

As shown in table 1, the anti-EpCAM and anti-CD 3 domains have high in vitro binding affinity for both the EpCAM and CD3 constructs in human and cynomolgus monkey, respectively, indicating that the EpCAM × CD3Fab fusion protein has cross-reactivity in human and cynomolgus monkey.

TABLE 1 in vitro binding affinity (Kd)

Structural domains	Antigens	Kd(M)
			anti-EpCAM	huEpCAM.Fc	3.49x 10-9
anti-EpCAM	cynoEpCAM.Fc	4.71x 10-9
			anti-CD 3	CD3εAA 1-27.Fc	1.26x 10-8
anti-CD 3	cyno CD3εAA 1-27.Fc	1.56x 10-8

Cell binding affinity

The following binding assay was performed to determine the binding affinity of an exemplary EpCAM x CD3Fab fusion protein (i.e., ITAB1002) to cells expressing the target antigen.

Fluorescence Activated Cell Sorting (FACS) was used to determine the binding affinity of ITAB1002 to human and cynomolgus peripheral blood mononuclear cells (hPBMC or cynoPBMC).

Preparation of hBMC: healthy adult leukocyte concentrates were diluted with PBS buffer (Gibco), centrifuged by density gradient centrifugation (Ficoll-Paque, GE Healthcare) to obtain PBMC, washed twice with PBS, and then centrifuged at 1000g for 10 minutes at room temperature, and the cells were collected and resuspended in RPMI-1640 medium (Gibco) containing 10% FBS.

Preparation of cynomolgus PBMC: cynomolgus monkey whole blood was diluted with PBS buffer (Gibco), centrifuged by density gradient centrifugation (Ficoll-Paque, GE Healthcare) to obtain PBMC, washed twice with PBS, and then centrifuged at 1000g for 10 minutes at room temperature, and the cells were collected and resuspended in RPMI-1640 medium (Gibco) containing 10% FBS.

ITAB1002 was diluted with FACS buffer (PBS containing 1% FBS) to various concentrations, and approximately 3.6X10⁵Individual PBMCs were mixed and incubated for 45 minutes at room temperature while negative controls (1% FBS/PBS + hPBMC) without ITAB1002 were set. Cells were washed once with FACS buffer, resuspended in 50. mu.L FACS buffer, and supplemented with CD4 antibody (RFT-4g) Conjugate (Invitrogen) and PE Mouse Anti-Human Light Chain lambda (BD Pharmingen)^TM) And incubated at room temperature for 45 minutes. Finally 150. mu.L FACS buffer was added and usedC6(BD Bioscience) analyzes the samples. The results of the detection are shown in FIG. 4A.

ITAB1002 was diluted to different concentrations in 50. mu.L FACS buffer (1% FBS in PBS), and approximately 4X 10⁵Individual cynomolgus PBMCs were mixed and incubated at room temperature for 60 minutes while a negative control without ITAB1002 was set. The cells were treated with CD4 antibody (RFT-4g)Conjugate (Invitrogen) and PE Mouse Anti-Human Light Chain lambda (BD Pharmingen)^TM) Staining and incubation at room temperature for 45 minutes. Add 150. mu.L of FACS buffer to the final solutionC6(BD Bioscience) analyzed the samples. The detection results are shown in FIG. 4B.

As shown in fig. 4A and 4B, EpCAM × CD3Fab fusion proteins have strong binding affinity to human and cynomolgus PBMC, while negative controls have substantially no binding affinity to human and cynomolgus PBMC.

In addition, the following binding assay was performed to determine the binding affinity of EpCAM x CD3Fab fusion protein (i.e., ITAB1002) to EpCAM + cells.

Human colon cancer SW480 cell (typical of Chinese academy of sciences)Culture collection committee cell bank) and Chinese Hamster Ovary (CHO) cells transfected with cynomolgus monkey EpCAM (designated CyEpCAM-CHO) determined the binding affinity of ITAB1002 for EpCAM. SW480 cells or CyEpCAM-CHO cells were digested with 0.25% Trypsin-EDTA (Gibco) and resuspended in FACS buffer. ITAB1002 was diluted to different concentrations in FACS buffer and equal volumes of about 1X10 ⁵The SW480 cells or CyEpCAM-CHO cells were mixed and then incubated at 4 ℃ for 30 minutes. Then, the cells were washed with FACS buffer and resuspended in 50. mu.L FACS buffer, Mouse anti-Human IgG FabSecondary Antibody PE conjugate (Invitrogen) was added, and allowed to incubate at 4 ℃ for 30 minutes. Finally, 150. mu.L of FACS buffer was added and usedC6(BD Bioscience) analyzes the samples. The results of the detection are shown in FIG. 5.

As shown in figure 5, EpCAM × CD3Fab fusion proteins showed potent binding affinity for both SW480 cells expressing human EpCAM (EC50 ═ 411.2ng/mL) and CHO cells expressing cynomolgus monkey EpCAM on the cell surface (CyEpCAM-CHO, EC50 ═ 107.6 ng/mL).

Thus, the exemplary EpCAM × CD3Fab fusion protein ITAB1002 showed cross-reactivity to human and cynomolgus monkey antigens in vitro. The cross-reactivity of the bispecific Fab fusion proteins may facilitate the extrapolation of toxicity and efficacy studies in cynomolgus monkeys to human clinical studies.

Example 3: tumor-dependent activation of human PBMC cells by EpCAM × CD3Fab fusion proteins

CD4, CD8 are marker surface antigens of T cells, and thus can divide T cells into two major subsets, CD4+ and CD8+, and CD69 is a cell surface receptor that is upregulated upon T cell activation. The percentage of subtypes CD4+ CD69+, CD8+ CD69+ can be a useful indicator of the activation state of T cells. FACS-based T cell activation assays were performed to determine the ability of an exemplary EpCAM x CD3Fab fusion protein (e.g., ITAB1002) to activate T cells.

Human PBMC were prepared as described in example 2 and resuspended in RPMI-1640 medium (Gibco) containing 10% FBS. SW480 cells were treated with 0.25% Trypsin-EDTA (Gi)bco) and resuspended in RPMI-1640 medium containing 10% FBS. 50 μ L/well of the cell mixture was added to each well of a 96-well plate at a final density of 10,000 SW480 cells/well and 100,000 PBMCs/well. ITAB1002 or OKT3(Sigma) was diluted to different concentrations with RPMI-1640 medium and 50 μ L of diluted ITAB1002 or OKT3 was added to each well. Control wells without SW480 cells (PBMC + ITAB1002) were also set. All samples were incubated at 37 ℃ for 24 hours. Thereafter, the supernatant was discarded, the cells were resuspended in 50. mu.L of FACS buffer, and the antibody CD4 antibody (RFT-4g) was addedConjugate (Invitrogen), CD8 antibody (3B5) RPE conjugate (Invitrogen), and FITC Mouse Anti-Human CD69(BD Pharmingen)^TM) Added to resuspended cells and allowed to incubate at room temperature for 30 minutes. Add 150. mu.L FACS buffer and useThe samples were analyzed by C6 cytometry (BD BIOSCIENCE). The detection results are shown in fig. 6A and 6B.

As shown in fig. 6A and 6B, EpCAM x CD3Fab fusion protein up-regulated CD4 in a dose-dependent manner in the presence of SW480 cells⁺And CD8 ⁺Expression of CD69 in T cell populations (in CD 4)⁺EC50 ═ 149.6ng/mL in T cells; and in CD8⁺EC50 ═ 68.78ng/mL in T cells). In the absence of SW480 cells, EpCAM × CD3Fab fusion proteins did not significantly up-regulate CD69 expression by T cells, on the other hand, OKT3 up-regulated CD4⁺And CD8⁺CD69 expression in a population of T cells.

Furthermore, Ki-67 is an indicator of cell proliferation on the cell surface. CD4⁺Ki-67⁺Subtype is CD4⁺Percentage in cells, and CD8⁺Ki-67⁺Subtype is CD8⁺The percentage in the cells can be an effective indicator of the proliferative state of the T cells. FACS-based T cell proliferation assays were performed to determine the capacity of an exemplary EpCAM x CD3Fab fusion protein (e.g., ITAB1002) in T cell proliferation.

Human PBMCs were prepared as described in example 2, andresuspended in RPMI-1640 medium (Gibco) containing 10% FBS. SW480 cells were digested with 0.25% Trypsin-EDTA (Gibco) and resuspended in RPMI-1640 medium containing 10% FBS. The cell mixture was added to each well of a 96-well plate at a final density of 20,000 SW480 cells/well and 300,000 PBMCs/well. ITAB1002 was diluted to different concentrations with RPMI-1640 medium containing 10% FBS and added to the cells. Wells containing PBMC and ITAB1002 only (PBMC + ITAB1002), PBMC and SW480 only (PBMC + SW480) and PBMC only were set as controls. Incubation was carried out at 37 ℃ for 72 hours. Thereafter, the supernatant was discarded, and the cells were fixed and permeabilized at a temperature of 4 ℃ for 20 minutes in a Fixation/Permeabilization Solution (BD Pharmingen), and then resuspended in 50. mu.L of BD Perm/Wash ^TMAnd (4) a buffer solution. The cells were treated with CD4 antibody (RFT-4g)Conjugate (Invitrogen), CD8 antibody (3B5) RPE conjugate (Invitrogen), FITC Mouse Anti-Ki-67Set (BD Pharmingen)^TM) Staining was carried out in the dark for 30 minutes. Use ofSamples were analyzed by C6 cytometry (BD Biosciences). The detection results are shown in fig. 6C and 6D.

As shown in fig. 6C and 6D, EpCAM x CD3Fab fusion protein up-regulated CD4 in a dose-dependent manner in the presence of SW480 cells⁺And CD8⁺Ki-67 expression in T cell populations. In the absence of SW480 cells, the EpCAM x CD3Fab fusion protein did not significantly up-regulate Ki-67 expression of T cells.

The above results demonstrate that EpCAM x CD3Fab fusion proteins are specific for T cell activation and dependent on their tumor antigen target.

Example 4: EpCAM × CD 3-mediated cytotoxicity of PBMCs against tumor cells (cytotoxicity assay)

Human and cynomolgus PBMCs were prepared as described in example 2 and resuspended in RPMI-1640 medium (Gibco) containing 10% FBS (Gibco).

SW480 cells (target cells) were digested with 0.25% Trypsin-EDTA (Gibco) and resuspended in R containing 10% FBSPMI-1640 medium. 50 μ L/well of the cell mixture was added to each well of a 96-well plate at a final density of 10,000 SW480 cells/well and 100,000 PBMC/well. An exemplary EpCAM x CD3Fab fusion protein (i.e., ITAB1002) was added to the cells at various concentrations according to the experimental design. Wells without drug (PBMC + target cells), wells containing only target cells, wells containing only PBMC and wells containing only culture medium were set as controls. At 37 ℃ with 5% CO ₂Culturing for about 18 hr, and performing CYTOTOXNon-Radioactive cytoxicity Assay (Promega) to measure Lactate Dehydrogenase (LDH) release, and OD490 using a microplate reader (Molecular device, Versa Max). EpCAM × CD3Fab fusion protein-mediated cytotoxicity was calculated using the formula:

mortality rate (OD)_{Sample well}-OD_{No drug control wells})/(OD_{Control wells of target cells with maximal lysis}-OD_{Control wells with spontaneous release of target cells})×100％

Data were analyzed using GraphPad Prism 6.0 software with mortality as the ordinate and drug concentration as the abscissa. Curves were fitted using a 4-parameter logistic model to determine EC 50. The measurement results are shown in FIG. 7. ITB1002 mediated cytotoxicity of human PBMCs against SW480 cells with an EC50 of 41.4 ng/mL. ITB 1002-mediated cytotoxicity of cynomolgus monkey PBMC against SW480 cells EC50 was 35.5 ng/mL.

As shown in figure 7, EpCAM × CD3Fab fusion proteins are capable of mediating the killing of tumor cells, such as tumor cell SW480, by cynomolgus monkey PBMC or human PBMC. Cytotoxicity against tumor cells was comparable for human and cynomolgus PBMC.

Example 5: EpCAM × CD 3-mediated cytotoxicity of human PBMCs against tumor cells (cytotoxicity assay)

Human PBMCs were prepared as described in example 2 and resuspended in RPMI-1640 medium (Gibco) containing 10% FBS. Tumor cells (target cells) were digested with 0.25% Trypsin-EDTA (Gibco) and resuspended in RPMI-1640 medium containing 10% FBS. 50 μ L/well of the cell mixture was processed at a final density of 10,000 tumor cells/well and 100,000 PBMC/well Aliquots were added to each well of a 96-well plate. An exemplary EpCAM x CD3Fab fusion protein (i.e., ITAB1002) was added to the cells at various concentrations according to the experimental design. Wells without drug (PBMC + target cells), wells containing only target cells, wells containing only PBMC and wells containing only culture medium were set as controls. At 37 ℃ with 5% CO₂An incubation of about 18 hours was performed (48 hours for H1975 and N87 cells). Carrying out CytoToxNon-Radioactive cytoxicity Assay (Promega) to measure LDH release and OD490 using a microplate reader (Molecular device, Versa Max). The EpCAM × CD3Fab fusion protein-mediated cytotoxicity can be calculated using the formula:

mortality rate (OD)_{Sample well}-OD_{No drug control wells})/(OD_{Control wells of target cells with maximal lysis}-OD_{Control wells with spontaneous release of target cells})×100％

Data were analyzed using GraphPad Prism 6.0 software, using mortality as the ordinate and drug concentration as the abscissa, and curves were fitted according to a 4-parameter logistic model to determine EC 50. The measurement results are shown in table 2 and fig. 8.

TABLE 2 EC50 values for EpCAM × CD3Fab fusion protein-mediated cytotoxicity of human PBMCs against tumor cells

Example 6: determination of efficacy of EpCAM x CD3Fab fusion protein in killing subcutaneous human colon tumor xenografts in immunodeficient mice

To examine the growth inhibitory effect of an exemplary EpCAM x CD3Fab fusion protein (i.e., ITAB1002) on human colon tumor xenografts, in vivo drug efficacy assays were performed on immunodeficient mice transplanted with SW480 tumor cells.

Female immunodeficient mouse NOD SCID (NOD. CB17-Prkdc)^scidNcrl) were purchased from Shanghai Ling Chang Biotechnology Ltd in SPF grade animal facilities.

Human colon cancer SW480 cells were cultured and harvested in vitro, resuspended in ice-precooled serum-free L-15 medium (Gibco) and kept on ice until use. Leukocyte concentrates from healthy human donors were collected, centrifuged by density gradient centrifugation (Ficoll-Paque, GE Healthcare) to obtain PBMCs, resuspended in ice-pre-chilled medium RPMI-1640(Gibco), and kept on ice until use. Tumor cells and human PBMC were mixed in equal volumes and inoculated subcutaneously into NOD SCID mice (approximately 5.0X 10 per animal)⁶SW480 cells and 5.0X 10⁶Human PBMC).

1 hour after cell inoculation of mice, random group administration was performed, and animals were divided into 5 groups of 6 animals each, namely, vehicle control group, 2.5. mu.g/kg ITAB 1002-treated group, 25. mu.g/kg ITAB 1002-treated group, 250. mu.g/kg ITAB 1002-treated group, and Cetuximab (Cetuximab)30mg/kg control group. The day of vaccination grouping was defined as D0. The drug to be tested, ITAB1002, was diluted to the desired concentration with vehicle (0.05% Tween-80/PBS buffer), administered intravenously to the animal's tail at doses of 2.5. mu.g/kg, 25. mu.g/kg and 250. mu.g/kg, respectively, in a volume of 0.1mL/10g body weight once a day for 5 consecutive days (D0 to D4). Animals in the vehicle control group were given an equal volume of vehicle. The reference drug Cetuximab (Cetuximab) (Merck Serono) was diluted with vehicle to the desired concentration and administered intravenously to the animal tails at a dose of 30mg/kg in a volume of 0.1mL/10g body weight 2 times a week for 3 consecutive weeks.

Meanwhile, a control treatment group not inoculated with human PBMC in a mixed manner was set, and NOD SCID mice were inoculated subcutaneously with about 5.0X 10⁶SW480 tumor cells were inoculated for 1 hour and randomly grouped and intravenously administered to the animal tail at different doses of ITAB 1002.

Animals were examined weekly for body weight and tumor size. Tumor volume (mm) according to the formula³) Tumor volume was calculated as long diameter (mm) x wide diameter (mm) x 0.5. The effect of the drug therapy was evaluated by the tumor growth inhibition ratio (TGI%), TGI% ([ 1- (avT) ]_i-avT₀)/(avCi-avC₀)]X 100, wherein avT_i-avT₀Mean tumor volume at day i minus mean tumor volume at day 0 for the treatment groups avC_i-avC₀Mean tumor volume at day i minus mean tumor volume at day 0 for vehicle control groups. The results of tumor volume measurements for each group of animals are shown in FIG. 9A, and photographs of SW480 tumors isolated from mice at the end of the experiment are shown in FIG. 9B.

As shown in FIGS. 9A and 9B, in the vehicle control group, SW480 tumor cells and human PBMC were inoculated in mixture, the tumor cells grew normally, and the volume of the average tumor reached 1414.06mm at 56 th day after inoculation³. ITAB1002 administration was effective to inhibit the in vivo growth of SW480 tumors in a dose-dependent manner. The% TGI at day 56 in the 2.5. mu.g/kg, 25. mu.g/kg and 250. mu.g/kg ITAB1002 treatment groups were 46.93%, 89.80% and 99.35%, respectively. The TGI% at day 56 in the 30mg/kg Cetuximab (Cetuximab) treated group was-16.97%, failing to inhibit the in vivo growth of SW480 tumors on the surface. In contrast, the treatment groups that were not co-inoculated with human PBMC grew normally and showed no signs of tumor regression (data not shown). Thus, cytotoxicity of exemplary EpCAM x CD3Fab fusion proteins against SW480 xenografts in mice was dependent on human PBMCs.

The results indicate that EpCAM x CD3Fab fusion protein can redirect immune cell killing of tumor cells and significantly inhibit tumor in vivo growth in a dose-dependent manner.

Example 7: determination of efficacy of EpCAM x CD3Fab fusion protein in killing subcutaneous human colon tumor xenografts in immune reconstituted mouse model

To examine the inhibitory effect of an exemplary EpCAM x CD3Fab fusion protein (i.e., ITAB1002) on the growth of human colon tumor xenografts, in vivo drug efficacy assays were performed on immunodeficient mice with an immune system reconstituted with human lymphocytes and transplanted with SW480 tumor cells.

Female immunodeficient mouse NOG (NOD. Cg-Prkdc)^scidII2rg^tm1Sug/Jiccrl) from Experimental animals technology Ltd of Viton-Rihua, Beijing, raised on SPF scaleIn the facility.

The experiment was started when NOG mice reached a body weight of 20 g. Mice were first treated with busulfan (Sigma) to eliminate bone marrow cells. The following day, in vitro cultured human colon cancer SW480 cells were harvested, thoroughly mixed and resuspended in ice-precooled serum-free L-15 medium (Gibco), and NOG mice were subcutaneously inoculated (approximately 2.5X 10 per animal)⁶Individual tumor cells), defined as D0 on the day of tumor cell inoculation; after 2 weeks, leukocyte concentrates from healthy human donors were collected, centrifuged by density gradient centrifugation (Ficoll-Paque, GE Healthcare) to obtain PBMCs, resuspended and mixed in ice-chilled medium RPMI-1640(Gibco), and subcutaneously inoculated into NOG mice (about 3.0X 10 for each animal) ⁶Individual tumor cells); when the tumor volume grows to 150-³The animals were randomized and administered with the drugs, 40 animals divided into 5 groups (8 animals per group), a model group, 2.5 μ g/kg ITAB 1002-treated group, 25 μ g/kg ITAB 1002-treated group, 250 μ g/kg ITAB 1002-treated group, 30mg/kg Cetuximab (Cetuximab) control group, respectively.

The test drug ITAB1002 was diluted to different concentrations with sterile-filtered media (PBS + 0.05% Tween-80) and administered intraperitoneally at 2.5. mu.g/kg, 25. mu.g/kg and 250. mu.g/kg, respectively, in a volume of 0.1mL/10g of body weight once a day for 25 consecutive days. The reference drug Cetuximab (Cetuximab) (Merck Serono) was diluted to the desired concentration with sterile filtered medium (PBS + 0.05% Tween-80) and administered intraperitoneally at a dose of 30mg/kg 2 times a week for 25 consecutive days. Animals in the model group were given an equal volume of vehicle.

Animals were examined weekly for body weight and tumor size. Tumor volume (mm) according to formula³) Tumor volume was calculated as long diameter (mm) x wide diameter (mm) x 0.5. The effect of the drug therapy was evaluated by the tumor growth inhibition ratio (TGI%), TGI% ([ 1- (avT) ]_i-avT₀)/(avC_i-avC₀)]X 100; avT therein_i-avT₀Mean tumor volume at day i minus mean tumor volume at start of cohort avC for treatment group _i-avC₀The mean tumor volume at the start of the cohort was subtracted from the mean tumor volume at day i of the model group. End of experimentIn this case, anticoagulated whole blood was collected and used in PE-Cy^TM5Mouse Anti-Human CD3(BD Pharmingen), lysed erythrocytes, and blood samples analyzed by FACS for Human CD3⁺T cell ratio. Fig. 10A shows the results of tumor volume assessment. Figure 10B shows photographs of SW480 tumors isolated from mice at the end of the experiment.

Fig. 10A and 10B show the growth inhibitory effect of ITAB1002 on subcutaneous SW480 transplants of NOG mice inoculated in an immune reconstitution with human PBMC. The results showed that in the model group, SW480 tumor cells were inoculated subcutaneously into NOG mice which were reconstituted by immunization with human PBMC, the tumor cells grew normally, and the volume of the average tumor reached 738.78mm at 46 days after inoculation³. ITAB1002 administration is effective to inhibit the in vivo growth of SW480 tumors and lead to tumor regression. In the 2.5 μ g/kgITAB1002 treatment group, the TGI% was 13.29% at day 40; in the 25. mu.g/kg ITAB1002 treated group, the TGI% was 68.55% at day 46. In the 250. mu.g/kg ITAB1002 treated group, the TGI% was 131.65% at day 46. In the 30mg/kg Cetuximab (Cetuximab) treated group, Cetuximab failed to inhibit the in vivo growth of SW480 tumors, and the TGI% was 8.91% on day 46. As shown in fig. 10C, the average proportion of CD3+ human T cells to total blood leukocytes was 26.84%, indicating successful reconstitution of the human T cell immune system in NOG mice.

Thus, after busulfan-depleted NOG mouse bone marrow reconstitution of the immune system with human PBMCs, administration of EpCAM x CD3Fab fusion protein was effective in inhibiting the growth of human colon cancer cells SW480 in mice, indicating that EpCAM x CD3Fab fusion protein can mediate killing of tumor cells in immune cells and significantly inhibit tumor growth in a dose-dependent manner.

Example 8: determination of efficacy of EpCAM x CD3Fab fusion protein in killing subcutaneous human lung tumor xenografts in immune reconstituted mouse model

To examine the inhibitory effect of an exemplary EpCAM × CD3Fab fusion protein (i.e., ITAB1002) on the growth of human lung tumor xenografts, in vivo drug efficacy assays were performed on immunodeficient mice bearing an immune system reconstituted with human PBMC and transplanted with human lung cancer tumor cells (NCI-H1975).

Female immunodeficient mouse NOG (NOD. Cg-Prkdc)^scidII2rg^tm1Sug/jicrl) was purchased from experimental animal technology ltd, viton, beijing, and was housed in an animal facility of SPF grade.

The experiment was started when NOG mice reached a body weight of 20 g. Treatment of mice with busulfan (Sigma) eliminated bone marrow cells. The following day, in vitro cultured human lung adenocarcinoma NCI-H1975 cells were collected, resuspended in ice-precooled serum-free L-15 medium (Gibco), mixed well, and inoculated subcutaneously with NOG mice (approximately 2.5X 10 per animal) ⁶Individual tumor cells), D0 on the day of tumor cell inoculation. After 2 days, leukocyte concentrates from healthy human donors were collected, centrifuged by density gradient centrifugation (Ficoll-Paque, GE Healthcare) to obtain PBMCs, resuspended and mixed in ice-chilled medium RPMI-1640(Gibco), and subcutaneously inoculated into NOG mice (about 3.0X 10 for each animal)⁶PBMC cells, except animals in the control group). When the tumor volume reaches 100-150mm³18 mice were randomly divided into 3 groups and administered drugs, including a control group (tumor cell inoculation, n ═ 6), a model group (tumor cell inoculation and PBMC, n ═ 5), and a 250 μ g/kg ITAB1002 treatment group (tumor cell inoculation and PBMC, n ═ 7).

The test drug ITAB1002 was diluted to the desired concentration with sterile filtered medium (0.05% Tween-80/PBS buffer) and administered intraperitoneally at a dose of 250. mu.g/kg in a volume of 0.1mL/10g body weight once a day for 24 consecutive days. The control and model groups were given equal volumes of vehicle. Animals were examined weekly for body weight and tumor size.

FIG. 11 shows the growth inhibitory effect of ITAB1002 on subcutaneous NCI-H1975 transplants of immunodeficient mice inoculated for immune reconstitution with human PBMC. The tumor cells of the animals of the control group and the model group grow normally, and the 28-day-average tumor volume after inoculation is 1382.63mm respectively ³、1432.15mm³(ii) a The 250. mu.g/kg ITAB1002 treated group completely inhibited the growth of SW480 tumor and resulted in tumor regression with an average tumor volume of 7.77mm on day 34³。

Thus, administration of the exemplary EpCAM x CD3Fab fusion protein was effective in inhibiting the growth of human lung adenocarcinoma cells NCI-H1975 in NOG mice that were immunoregulated with human PBMC, indicating that the EpCAM x CD3Fab fusion protein can mediate immune cell killing of tumor cells, significantly inhibiting tumor growth in vivo.

Example 9: EpCAM x CD3Fab fusion protein induces T cell redistribution, cytokine release and pharmacokinetics in cynomolgus monkeys

Cynomolgus monkey (grade: general grade), age: the exemplary EpCAM × CD3Fab fusion protein (e.g., ITAB1002) was administered in four groups of 16 cynomolgus monkeys aged 3-5 years, weighing 3.0-5.0kg, at 0.5 μ g/kg dose group (n ═ 3), 5 μ g/kg dose group (n ═ 4), 15 μ g/kg dose group (n ═ 6), and 50 μ g/kg dose group (n ═ 3), respectively.

The test drug ITAB1002 was diluted with vehicle (0.5% monkey serum/PBS buffer) to different concentrations, administered in a volume of 2.0mL/kg body weight at doses of 0.5. mu.g/kg, 5. mu.g/kg, 15. mu.g/kg, 50. mu.g/kg, and the forelimb was infused intravenously for 1 hour for a single administration. The animal signs, behaviors, mental states, feces and the like are observed every day.

Whole blood collection: coated with EDTA-K2Anticoagulant collection tubes (BD Bioscience) collect blood samples 0.3mL separately at different time points before and after the start of infusion of the test drug; the collected anticoagulated blood was divided into two portions, one portion of blood 100. mu.L was collected by a blood cell automatic counter (Siemens,2120) determining lymphocyte subclasses; another 200. mu.L portion of blood was subjected to CD4and CD8 antigens (APC Mouse Anti-Human CD4and PE Mouse Anti-Human CD8, BD PHARMINGEN)^TM) Staining, CD4+ and CD8+ T cell subsets were analyzed using FACS (Beckman Coulter, Cytomics FC 500).

Serum collection: simultaneously using a Non-anticoagulation tube, 1.2mL of blood samples were collected at different time points before and after the start of infusion of the test drug, and after the blood coagulation, serum was collected by centrifugation, stored at-80 ℃ and administered with Non-human prime Th1/Th2Cytokine Kit (BD Pharmi)ngen^TM) Detecting the secretion of IL-2, IL-4, IL-5, IL-6, TNF and IFN-gamma cell factors in serum, determining the drug concentration in the serum (enzyme linked immunosorbent assay), and calculating the pharmacokinetic parameters by using PK Solver2.0 software.

During the experiment, none of the monkeys in each group died or moribund. Each group of monkeys had normal activity, good mental status, clean skin and hair, and no red swelling, ulceration and other obvious abnormal symptoms at the injection site. The EpCAM × CD3Fab fusion protein showed good safety and tolerability in cynomolgus monkeys.

FIG. 12A is CD4 in cynomolgus monkey blood following single intravenous infusion administration of different doses of EpCAM x CD3Fab fusion protein⁺T cell number, time-dependent curve (H: H, D: day in abscissa). After administration, CD4 in the blood of the animals⁺The number of T cells decreased, decreased to a minimum at about 8 hours after administration, and then gradual recovery began.

FIG. 12B is CD8 in cynomolgus monkey blood following single intravenous infusion administration of different doses of EpCAM x CD3Fab fusion protein⁺T cell number, time-dependent curve (H: H, D: day in abscissa). After administration, CD8 in the blood of the animals⁺The number of T cells decreased, decreased to a minimum at about 8 hours after administration, and then gradual recovery began.

FIGS. 13A-13F are the concentrations of IL-2 (FIG. 13A), IL-4 (FIG. 13B), IL-5 (FIG. 13C), IL-6 (FIG. 13D), TNF (FIG. 13E), and IFN-. gamma. (FIG. 13F) in cynomolgus monkey sera following single intravenous infusion administration of different doses of EpCAM × CD3Fab fusion protein. The results show that after single intravenous infusion administration of EpCAM multiplied by CD3Fab fusion protein, the concentrations of IL-6, IL-2, TNF and IFN-gamma in cynomolgus monkey serum are increased to different degrees, and the cynomolgus monkey serum returns to the basic level after reaching the peak concentration and presents a dose dependent relationship; there was no significant change in the serum concentrations of IL-4 and IL-5.

Figure 14 shows the time course of drug concentration in cynomolgus monkey serum following a single intravenous infusion administration of different doses of EpCAM x CD3Fab fusion protein.

The results show that in the three dose groups (5. mu.g/kg, 15. mu.g/kg, 50. mu.g/kg), the drug concentration in the serum peaked and declined stepwise after a single intravenous infusion administration of the EpCAM x CD3Fab fusion protein for 1 hour. The EpCAM × CD3Fab fusion protein showed prolonged in vivo half-life in cynomolgus monkeys.

Specific pharmacokinetic parameters for the EpCAM × CD3 fusion protein are shown in table 3.

TABLE 3 exemplary EpCAM × CD3Fab fusion proteins pharmacokinetic parameters in vivo in cynomolgus monkeys (mean. + -. SD)

Example 10: comparison of ITAB1002 and ITAB 1012-mediated PBMC cytotoxicity against tumor cells

ITAB1012 is an EpCAM x CD3Fab fusion protein comprising an EpCAM scFv having a different HVR than the HVR in the EpCAM scFv of ITAB 1002. Transient expression and purification of ITAB1012 was as described in example 1. The heavy chain of ITAB1012 has the amino acid sequence of SEQ ID NO. 35 and is encoded by the nucleic acid sequence of SEQ ID NO. 37. The light chain of ITAB1012 has the amino acid sequence of SEQ ID NO. 36 and is encoded by the nucleic acid sequence of SEQ ID NO. 38. The HVRs of the EpCAM scFv fragment are underlined in the following sequences. The EpCAM scFv of ITAB1012 has been described in U.S. patent No. 8,846,042.

35(ITAB1012 heavy chain)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQDGSEKFYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDMAVYYCARVGGAWELGYWGQGTLVTVSAGGGGSGGGGSGGGGSGAQSVLTQPPSVSGAPGQRVTISCTGSSSNIGSYYGVHWYQQLPGTAPKLLIYSDTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGRVFGGGTKLTVLGGEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCPPCS

SEQ ID NO:36(ITAB1012 light chain)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMSWVRQAPGKGLEWVANIKQDGSEKFYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDMAVYYCARVGGAWELGYWGQGTLVTVSAGGGGSGGGGSGGGGSGAQSVLTQPPSVSGAPGQRVTISCTGSSSNIGSYYGVHWYQQLPGTAPKLLIYSDTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGRVFGGGTKLTVLGGQAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECPPCS

37 (nucleic acid encoding ITAB1012 heavy chain)

gaggtgcagctggtggagtcagggggaggcttggtccagcctgggggatcactgagactctcctgtgcagcctctggattcacctttagtaattattggatgagctgggtccgccaggctccagggaaggggctggagtgggtggccaacataaagcaagatggaagtgagaaattctatgtggactctgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaagacatggctgtctattactgtgcgagagtggggggggcgtgggagctaggctactggggccagggaaccctggtcactgtctcggccggtggcggtggcagcggcggtggtgggtccggtggcggcggatctggcgcgcagtctgtactgactcaaccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcgggtcttattatggtgtgcactggtaccagcagcttccaggaacagcccccaaactcctcatctattctgacactaatcgaccctcaggggtccctgaccgattctctggctccaagtctggcacctcggcctccctggccatcactgggctccaggctgaggatgaggctgattattactgccagtcgtatgacagcagcctgagtggccgggtgttcggcggagggaccaagctgacagtactaggtggcgaggtgcagctggtggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcacctttaacacctacgccatgaactgggtccgccaggctccagggaaggggctggagtgggtcgcacgcataagaagtaaatataataattatgcaacatattatgccgattcagtgaaagaccggttcaccatctccagagacgattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgtgagacatgggaacttcggtaatagctacgtttcctggtttgcttactggggccaagggacaatggtcaccgtctcttcagctagcaccaagggcccatccgtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtccaccgtgctcatga

38 (encoding ITAB1012 light chain)

gaggtgcagctggtggagtcagggggaggcttggtccagcctgggggatcactgagactctcctgtgcagcctctggattcacctttagtaattattggatgagctgggtccgccaggctccagggaaggggctggagtgggtggccaacataaagcaagatggaagtgagaaattctatgtggactctgtgaagggccgattcaccatctccagagacaacgccaagaactcactgtatctgcaaatgaacagcctgagagccgaagacatggctgtctattactgtgcgagagtggggggggcgtgggagctaggctactggggccagggaaccctggtcactgtctcggccggtggcggtggcagcggcggtggtgggtccggtggcggcggatctggcgcgcagtctgtactgactcaaccgccctcagtgtctggggccccagggcagagggtcaccatctcctgcactgggagcagctccaacatcgggtcttattatggtgtgcactggtaccagcagcttccaggaacagcccccaaactcctcatctattctgacactaatcgaccctcaggggtccctgaccgattctctggctccaagtctggcacctcggcctccctggccatcactgggctccaggctgaggatgaggctgattattactgccagtcgtatgacagcagcctgagtggccgggtgttcggcggagggaccaagctgacagtactaggtggccaggctgtggtgactcaggagccctcactgactgtgtccccaggagggacagtcactctcacctgtcgctcatccactggggctgttacaactagtaactatgccaactgggtccagcagaaacctggacaagcacccaggggtctgattggtggtaccaacaagcgagctccaggtacccctgcccggttctcaggctccctccttgggggcaaagctgccctgacactgtcaggtgtgcagcctgaggacgaggctgagtattactgcgctctatggtacagcaacctctgggtgttcggcggagggaccaagctgaccgtccta ggccaaccgaaagcggcgccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaa tgtccaccgtgctcatga

ITAB1002 and ITAB1012 were tested in cytotoxicity assays using human PBMC as described in example 5. The results are shown in FIG. 15. The ITAB 1002-mediated cytotoxicity of human PBMCs against SW480 cells had an EC50 of about 3.54 ng/mL. In contrast, ITAB 1012-mediated cytotoxicity of human PBMCs against SW480 cells had an EC50 of about 223.3 ng/mL. Thus, ITAB1002 exhibited significantly higher activity in mediating cytotoxicity of human PBMCs against tumor cells compared to ITAB 1012.

In addition, the binding affinity of the anti-EpCAM and anti-CD 3 domains of the exemplary EpCAM x CD3Fab fusion proteins (i.e., ITAB1002 and ITAB1012) to human antigens was tested using the methods described in example 2. As shown in table 4, ITAB1002 exhibited a stronger binding affinity to EpCAM, while it had a weaker binding affinity to CD3 compared to ITAB 1012.

TABLE 4 in vitro binding affinities (KD)

Example 11: EpCAM xCD 3Fab fusion protein mediated cytotoxicity of human PBMCs against tumor cells in the presence of steroids

In vitro exploratory assays were performed to evaluate the effect of steroid pretreatment on tumor cell killing activity and cytokine release of human T cells induced by ITAB 1002.

Cytotoxicity assays were performed as described in example 5. PBMCs were isolated from healthy donors and incubated with Dexamethasone (DXM) at a concentration of 3 μ M for 1 hour, and then added to wells of a 96-well plate at final densities of 30,000 SW480 cells and 300,000 PBMCs per well. Mixing the cells at 37 deg.C, 5% CO₂The following incubation took about 18 hours with a DXM final concentration of 0.15 μ M. Cell killing was measured by Lactate Dehydrogenase (LDH) assay and calculated. Cytokine (i.e., IL-6) release was analyzed by human IL-6ELISA kit (BD Biosciences) in the same assay.

Figure 16 shows the effect of Dexamethasone (DXM) on ITAB 1002-mediated SW480 cell killing activity. In the presence of dexamethasone, ITAB1002 showed comparable killing activity against SW480 cells in vitro compared to ITAB1002 alone.

Figure 17 shows the results of the cytokine release assay. IL-6 is released from ITAB 1002-induced activated T cells. However, in the presence of dexamethasone, IL-6 release was almost completely inhibited, suggesting that DXM treatment may be an effective pretreatment strategy to control Cytokine Release Syndrome (CRS) in patients receiving ITAB 1002.

Example 12: preclinical study of EpCAM × CD3Fab fusion proteins in cynomolgus monkeys

An exploratory study was performed to evaluate the effect of dexamethasone pretreatment to mitigate the potential toxicity of cynomolgus monkeys receiving ITAB1002 treatment. During the first week, 6 monkeys (ITAB1002+ DXM) were treated twice weekly by Intravenous (IV) infusion with 0.5 μ g/kg ITAB1002 (day 1 and day 4). During the second week, animals were treated by IV infusion of 1.0 μ g/kg ITAB1002 twice weekly (day 8 and day 11). During week 3, animals were treated by IV infusion of 2.0 μ g/kg ITAB1002 twice weekly (day 15 and day 18). Animals were treated by IV infusion of 4.0 μ g/kg ITAB1002 twice weekly (days 22, 25, 29 and 32) during weeks 4 and 5. Dexamethasone was administered to each animal at a 1mg/kg dose by intravenous Injection (IV) approximately 1 hour prior to the first infusion of each dose level. At week 5, the dose of dexamethasone was increased to 2 mg/kg. In the group without DXM pretreatment (n-10), each monkey was administered 2.0 μ g/kg ITAB1002 by intravenous infusion twice weekly without any DXM pretreatment. Blood samples were collected at different time points and analyzed for serum chemistry parameters. Serum IL-6 levels were analyzed by the human IL-6ELISA kit (BD Biosciences).

Pretreatment with DXM prior to ITAB1002 significantly reduced serum ALT (fig. 18A-18B), Tbil (fig. 19A-19B), and ALP levels (fig. 20A-20B), as well as inhibited IL-6 release (fig. 21A-21B), compared to the treatment group without DXM pretreatment. These results demonstrate that early induction of IL-6 release and side effects in the liver in monkeys treated with ITAB1002 can be ameliorated by DXM pretreatment.

Example 13: clinical testing of ITAB1002 in human cancer patients

The safety and efficacy of ITAB1002 was evaluated in clinical trials on human cancer patients. The purpose of this assay was to assess the safety, tolerability, pharmacokinetics, immunogenicity and antitumor activity of ITAB1002 in adult subjects with locally advanced or metastatic solid tumors for which standard therapy was either not present or has been shown to be ineffective or intolerant. Five dose levels of 0.3, 0.6, 1.2, 2.4 and 3.6 μ g/kg were evaluated by dose escalation. The conventional 3+3 design (3 patients per dose group, possibly with an additional 3 patients added to the same group to further assess toxicity) was used for dose escalation and MTD assays. 5 groups of approximately 6 patients per group were recruited.

All patients received a compliant dose of 0.3 μ g/kg ITAB1002 by Intravenous (IV) infusion twice (day 1 and day 4) during the first week. Over the next three weeks, patients in group 5 received ITAB1002 by intravenous infusion twice weekly at the respective doses: 0.3. mu.g/kg, 0.6. mu.g/kg, 1.2. mu.g/kg, 2.4. mu.g/kg and 3.6. mu.g/kg. Dexamethasone was given at a dose of 20mg 1 hour prior to the first compliant dose of ITAB1002 and the first ramp dose (0.3. mu.g/kg to 3.6. mu.g/kg). Dexamethasone was administered at 20mg or 10mg prior to the subsequent ITAB1002 dose, according to clinical assessment. Table 4 below shows the administration schedule for the five groups in this study.

TABLE 4 administration protocol for human clinical trials

Dosage unit of ITAB1002 was μ g/kg.

All references mentioned in this disclosure are incorporated herein by reference as if each had been individually incorporated by reference. Although the description refers to particular embodiments, it will be apparent to those skilled in the art that the present invention may be practiced with modification of these specific details. Accordingly, the present invention should not be construed as limited to the embodiments set forth herein.

Sequence listing

<110> Jianneng pharmaceutical technology (Shanghai) Co., Ltd (GENERON (SHANGHAI) CORPORATION LTD.)

<120> multi-specificity Fab fusion protein and application thereof

<130> 720622000841

<140> not yet allocated

<141> accompanying this application

<150> CN 201610147227.8

<151> 2016-03-15

<160> 47

<170> FastSEQ for Windows version 4.0

<210> 1

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 HVR-H1

<400> 1

Thr Tyr Ala Met Asn

1 5

<210> 2

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 HVR-H2

<400> 2

Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser

1 5 10 15

Val Lys Asp

<210> 3

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 HVR-H3

<400> 3

His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr

1 5 10

<210> 4

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 HVR-L1

<400> 4

Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 HVR-L2

<400> 5

Gly Thr Asn Lys Arg Ala Pro

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 HVR-L3

<400> 6

Ala Leu Trp Tyr Ser Asn Leu Trp Val

1 5

<210> 7

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 VH

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 8

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 VL

<400> 8

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 9

<211> 102

<212> PRT

<213> human (Homo sapiens)

<400> 9

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser

100

<210> 10

<211> 104

<212> PRT

<213> human (Homo sapiens)

<400> 10

Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu

100

<210> 11

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 heavy chain

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser

225

<210> 12

<211> 213

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 light chain

<400> 12

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu

115 120 125

Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro

130 135 140

Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala

145 150 155 160

Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala

165 170 175

Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg

180 185 190

Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr

195 200 205

Val Ala Pro Thr Glu

210

<210> 13

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> EpCAM HVR-H1

<400> 13

Asn Tyr Trp Met Ser

1 5

<210> 14

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> EpCAM HVR-H2

<400> 14

Asn Ile Lys Gln Asp Gly Ser Glu Lys Phe Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 15

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> EpCAM HVR-H3

<400> 15

Val Gly Pro Ser Trp Glu Gln Asp Tyr

1 5

<210> 16

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> EpCAM HVR-L1

<400> 16

Thr Gly Ser Ser Ser Asn Ile Gly Ser Tyr Tyr Gly Val His

1 5 10

<210> 17

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> EpCAM HVR-L2

<400> 17

Ser Asp Thr Asn Arg Pro Ser

1 5

<210> 18

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> EpCAM HVR-L3

<400> 18

Gln Ser Tyr Asp Lys Gly Phe Gly His Arg Val

1 5 10

<210> 19

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> EpCAM VH

<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Pro Ser Trp Glu Gln Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 20

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> EpCAM VL

<400> 20

Gly Ala Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro

1 5 10 15

Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly

20 25 30

Ser Tyr Tyr Gly Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ser Asp Thr Asn Arg Pro Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr

65 70 75 80

Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp

85 90 95

Lys Gly Phe Gly His Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val

100 105 110

Leu

<210> 21

<211> 246

<212> PRT

<213> Artificial sequence

<220>

<223> EpCAM scFv

<400> 21

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Pro Ser Trp Glu Gln Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Ala Gln Ser Val Leu Thr Gln Pro Pro Ser

130 135 140

Val Ser Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser

145 150 155 160

Ser Ser Asn Ile Gly Ser Tyr Tyr Gly Val His Trp Tyr Gln Gln Leu

165 170 175

Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asp Thr Asn Arg Pro

180 185 190

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala

195 200 205

Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr

210 215 220

Cys Gln Ser Tyr Asp Lys Gly Phe Gly His Arg Val Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Thr Val Leu

245

<210> 22

<211> 480

<212> PRT

<213> Artificial sequence

<220>

<223> ITAB1002 HC

<400> 22

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Pro Ser Trp Glu Gln Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Ala Gln Ser Val Leu Thr Gln Pro Pro Ser

130 135 140

Val Ser Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser

145 150 155 160

Ser Ser Asn Ile Gly Ser Tyr Tyr Gly Val His Trp Tyr Gln Gln Leu

165 170 175

Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asp Thr Asn Arg Pro

180 185 190

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala

195 200 205

Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr

210 215 220

Cys Gln Ser Tyr Asp Lys Gly Phe Gly His Arg Val Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Thr Val Leu Gly Gly Glu Val Gln Leu Val Glu Ser Gly

245 250 255

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

260 265 270

Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala

275 280 285

Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn

290 295 300

Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile

305 310 315 320

Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

325 330 335

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe

340 345 350

Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Met

355 360 365

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

370 375 380

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

385 390 395 400

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

405 410 415

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

420 425 430

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

435 440 445

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

450 455 460

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Pro Pro Cys Ser

465 470 475 480

<210> 23

<211> 466

<212> PRT

<213> Artificial sequence

<220>

<223> ITAB1002 LC

<400> 23

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Phe Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Pro Ser Trp Glu Gln Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Ala Gln Ser Val Leu Thr Gln Pro Pro Ser

130 135 140

Val Ser Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser

145 150 155 160

Ser Ser Asn Ile Gly Ser Tyr Tyr Gly Val His Trp Tyr Gln Gln Leu

165 170 175

Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asp Thr Asn Arg Pro

180 185 190

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala

195 200 205

Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr

210 215 220

Cys Gln Ser Tyr Asp Lys Gly Phe Gly His Arg Val Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Thr Val Leu Gly Gly Gln Ala Val Val Thr Gln Glu Pro

245 250 255

Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser

260 265 270

Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln

275 280 285

Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg

290 295 300

Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys

305 310 315 320

Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr

325 330 335

Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr

340 345 350

Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu

355 360 365

Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val

370 375 380

Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys

385 390 395 400

Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser

405 410 415

Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr

420 425 430

Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His

435 440 445

Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Pro Pro

450 455 460

Cys Ser

465

<210> 24

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 24

Gly Gly Ser Gly

1

<210> 25

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 25

Gly Gly Ser Gly Gly

1 5

<210> 26

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 26

Gly Ser Gly Ser Gly

1 5

<210> 27

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 27

Gly Ser Gly Gly Gly

1 5

<210> 28

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 28

Gly Gly Gly Ser Gly

1 5

<210> 29

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 29

Gly Ser Ser Ser Gly

1 5

<210> 30

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic construct

<400> 30

Pro Leu Gly Leu Ala Gly

1 5

<210> 31

<211> 1443

<212> DNA

<213> Artificial sequence

<220>

<223> ITAB1002 HC

<400> 31

gaggtgcagc tggtggagtc agggggaggc ttggtccagc ctgggggatc cctgagactc 60

tcctgtgcag cctctggatt cacctttagt aattattgga tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtggccaac ataaagcaag atggaagtga gaaattctat 180

gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240

ctgcaaatga acagcctgag agccgaagac acggctgtct attactgtgc gagagtgggg 300

ccgtcctggg agcaggacta ctggggccag ggaaccctgg tcactgtctc ggccggtggc 360

ggtggcagcg gcggtggtgg gtccggtggc ggcggatctg gcgcgcagtc tgtactgact 420

caaccgccct cagtgtctgg ggccccaggg cagagggtca ccatctcctg cactgggagc 480

agctccaaca tcgggtctta ttatggtgtg cactggtacc agcagcttcc aggaacagcc 540

cccaaactcc tcatctattc tgacactaat cgaccctcag gggtccctga ccgattctct 600

ggctccaagt ctggcacctc ggcctccctg gccatcactg ggctccaggc tgaggatgag 660

gctgattatt actgccagtc gtatgacaag ggcttcgggc accgggtgtt cggcggaggg 720

accaagctga ccgtcctagg gggcgaggtg cagctggtgg agtctggggg aggcttggta 780

cagcctgggg ggtccctgag actctcctgt gcagcctctg gattcacctt taacacctac 840

gccatgaact gggtccgcca ggctccaggg aaggggctgg agtgggtcgc acgcataaga 900

agtaaatata ataattatgc aacatattat gccgattcag tgaaagaccg gttcaccatc 960

tccagagacg attccaagaa cacgctgtat ctgcaaatga acagcctgag agccgaggac 1020

acggccgtat attactgtgt gagacatggg aacttcggta atagctacgt ttcctggttt 1080

gcttactggg gccaagggac aatggtcacc gtctcttcag ctagcaccaa gggcccatcc 1140

gtcttccccc tggcaccctc ctccaagagc acctctgggg gcacagcggc cctgggctgc 1200

ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc 1260

agcggcgtgc acaccttccc ggctgtccta cagtcctcag gactctactc cctcagcagc 1320

gtggtgaccg tgccctccag cagcttgggc acccagacct acatctgcaa cgtgaatcac 1380

aagcccagca acaccaaggt ggacaagaaa gttgagccca aatcttgtcc accgtgctca 1440

tga 1443

<210> 32

<211> 1401

<212> DNA

<213> Artificial sequence

<220>

<223> ITAB1002 LC

<400> 32

gaggtgcagc tggtggagtc agggggaggc ttggtccagc ctgggggatc cctgagactc 60

tcctgtgcag cctctggatt cacctttagt aattattgga tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtggccaac ataaagcaag atggaagtga gaaattctat 180

gcggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240

ctgcaaatga acagcctgag agccgaagac acggccgtct attactgtgc gagagtgggg 300

ccgtcctggg agcaggacta ctggggccag ggaaccctgg tcactgtctc ggccggtggc 360

ggtggcagcg gcggtggtgg gtccggtggc ggcggatctg gcgcgcagtc tgtactgact 420

caaccgccct cagtgtctgg ggccccaggg cagagggtca ccatctcctg cactgggagc 480

agctccaaca tcgggtctta ttatggtgtg cactggtacc agcagcttcc aggaacagcc 540

cccaaactcc tcatctattc tgacactaat cgaccctcag gggtccctga ccgattctct 600

ggctccaagt ctggcacctc ggcctccctg gccatcactg ggctccaggc tgaggatgag 660

gctgattatt actgccagtc gtatgacaag ggcttcgggc accgggtgtt cggcggaggg 720

accaagctga ccgtcctagg gggccaggct gtggtgactc aggagccctc actgactgtg 780

tccccaggag ggacagtcac tctcacctgt cgctcaagta ctggggctgt tacaactagt 840

aactatgcca actgggtcca gcagaaacct ggacaagcac ccaggggtct gattggtggt 900

accaacaagc gagctccagg tacccctgcc cggttctcag gctccctcct tgggggcaaa 960

gctgccctga cactgtcagg tgtgcagcct gaggacgagg ctgagtatta ctgcgctcta 1020

tggtacagca acctctgggt gttcggcgga gggaccaagc tgaccgtcct aggccaaccg 1080

aaagcggcgc cctcggtcac tctgttcccg ccctcctctg aggagcttca agccaacaag 1140

gccacactgg tgtgtctcat aagtgacttc tacccgggag ccgtgacagt ggcctggaag 1200

gcagatagca gccccgtcaa ggcgggagtg gagaccacca caccctccaa acaaagcaac 1260

aacaagtacg cggccagcag ctatctgagc ctgacgcctg agcagtggaa gtcccacaga 1320

agctacagct gccaggtcac gcatgaaggg agcaccgtgg agaagacagt ggcccctaca 1380

gaatgtccac cgtgctcatg a 1401

<210> 33

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Signal peptide

<400> 33

Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser

<210> 34

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Signal peptide

<400> 34

atggaatgga gctgggtctt tctcttcttc ctgtcagtaa cgactggtgt ccactcc 57

<210> 35

<211> 480

<212> PRT

<213> Artificial sequence

<220>

<223> ITAB1012 HC

<400> 35

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Phe Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Gly Ala Trp Glu Leu Gly Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Ala Gln Ser Val Leu Thr Gln Pro Pro Ser

130 135 140

Val Ser Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser

145 150 155 160

Ser Ser Asn Ile Gly Ser Tyr Tyr Gly Val His Trp Tyr Gln Gln Leu

165 170 175

Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asp Thr Asn Arg Pro

180 185 190

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala

195 200 205

Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr

210 215 220

Cys Gln Ser Tyr Asp Ser Ser Leu Ser Gly Arg Val Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Thr Val Leu Gly Gly Glu Val Gln Leu Val Glu Ser Gly

245 250 255

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

260 265 270

Ser Gly Phe Thr Phe Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala

275 280 285

Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn

290 295 300

Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile

305 310 315 320

Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

325 330 335

Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe

340 345 350

Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Met

355 360 365

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

370 375 380

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

385 390 395 400

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

405 410 415

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

420 425 430

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

435 440 445

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

450 455 460

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Pro Pro Cys Ser

465 470 475 480

<210> 36

<211> 466

<212> PRT

<213> Artificial sequence

<220>

<223> ITAB1012 LC

<400> 36

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Phe Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Gly Ala Trp Glu Leu Gly Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Ala Gln Ser Val Leu Thr Gln Pro Pro Ser

130 135 140

Val Ser Gly Ala Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly Ser

145 150 155 160

Ser Ser Asn Ile Gly Ser Tyr Tyr Gly Val His Trp Tyr Gln Gln Leu

165 170 175

Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asp Thr Asn Arg Pro

180 185 190

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala

195 200 205

Ser Leu Ala Ile Thr Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr

210 215 220

Cys Gln Ser Tyr Asp Ser Ser Leu Ser Gly Arg Val Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Thr Val Leu Gly Gly Gln Ala Val Val Thr Gln Glu Pro

245 250 255

Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Arg Ser

260 265 270

Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Gln

275 280 285

Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly Thr Asn Lys Arg

290 295 300

Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys

305 310 315 320

Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp Glu Ala Glu Tyr

325 330 335

Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly Thr

340 345 350

Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu

355 360 365

Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val

370 375 380

Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys

385 390 395 400

Ala Asp Ser Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser

405 410 415

Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr

420 425 430

Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His

435 440 445

Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Pro Pro

450 455 460

Cys Ser

465

<210> 37

<211> 1443

<212> DNA

<213> Artificial sequence

<220>

<223> ITAB1012 HC

<400> 37

gaggtgcagc tggtggagtc agggggaggc ttggtccagc ctgggggatc actgagactc 60

tcctgtgcag cctctggatt cacctttagt aattattgga tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtggccaac ataaagcaag atggaagtga gaaattctat 180

gtggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240

ctgcaaatga acagcctgag agccgaagac atggctgtct attactgtgc gagagtgggg 300

ggggcgtggg agctaggcta ctggggccag ggaaccctgg tcactgtctc ggccggtggc 360

ggtggcagcg gcggtggtgg gtccggtggc ggcggatctg gcgcgcagtc tgtactgact 420

caaccgccct cagtgtctgg ggccccaggg cagagggtca ccatctcctg cactgggagc 480

agctccaaca tcgggtctta ttatggtgtg cactggtacc agcagcttcc aggaacagcc 540

cccaaactcc tcatctattc tgacactaat cgaccctcag gggtccctga ccgattctct 600

ggctccaagt ctggcacctc ggcctccctg gccatcactg ggctccaggc tgaggatgag 660

gctgattatt actgccagtc gtatgacagc agcctgagtg gccgggtgtt cggcggaggg 720

accaagctga cagtactagg tggcgaggtg cagctggtgg agtctggggg aggcttggta 780

cagcctgggg ggtccctgag actctcctgt gcagcctctg gattcacctt taacacctac 840

gccatgaact gggtccgcca ggctccaggg aaggggctgg agtgggtcgc acgcataaga 900

agtaaatata ataattatgc aacatattat gccgattcag tgaaagaccg gttcaccatc 960

tccagagacg attccaagaa cacgctgtat ctgcaaatga acagcctgag agccgaggac 1020

acggccgtat attactgtgt gagacatggg aacttcggta atagctacgt ttcctggttt 1080

gcttactggg gccaagggac aatggtcacc gtctcttcag ctagcaccaa gggcccatcc 1140

gtcttccccc tggcaccctc ctccaagagc acctctgggg gcacagcggc cctgggctgc 1200

ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc 1260

agcggcgtgc acaccttccc ggctgtccta cagtcctcag gactctactc cctcagcagc 1320

gtggtgaccg tgccctccag cagcttgggc acccagacct acatctgcaa cgtgaatcac 1380

aagcccagca acaccaaggt ggacaagaaa gttgagccca aatcttgtcc accgtgctca 1440

tga 1443

<210> 38

<211> 1401

<212> DNA

<213> Artificial sequence

<220>

<223> ITAB1012 LC

<400> 38

gaggtgcagc tggtggagtc agggggaggc ttggtccagc ctgggggatc actgagactc 60

tcctgtgcag cctctggatt cacctttagt aattattgga tgagctgggt ccgccaggct 120

ccagggaagg ggctggagtg ggtggccaac ataaagcaag atggaagtga gaaattctat 180

gtggactctg tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240

ctgcaaatga acagcctgag agccgaagac atggctgtct attactgtgc gagagtgggg 300

ggggcgtggg agctaggcta ctggggccag ggaaccctgg tcactgtctc ggccggtggc 360

ggtggcagcg gcggtggtgg gtccggtggc ggcggatctg gcgcgcagtc tgtactgact 420

caaccgccct cagtgtctgg ggccccaggg cagagggtca ccatctcctg cactgggagc 480

agctccaaca tcgggtctta ttatggtgtg cactggtacc agcagcttcc aggaacagcc 540

cccaaactcc tcatctattc tgacactaat cgaccctcag gggtccctga ccgattctct 600

ggctccaagt ctggcacctc ggcctccctg gccatcactg ggctccaggc tgaggatgag 660

gctgattatt actgccagtc gtatgacagc agcctgagtg gccgggtgtt cggcggaggg 720

accaagctga cagtactagg tggccaggct gtggtgactc aggagccctc actgactgtg 780

tccccaggag ggacagtcac tctcacctgt cgctcatcca ctggggctgt tacaactagt 840

aactatgcca actgggtcca gcagaaacct ggacaagcac ccaggggtct gattggtggt 900

accaacaagc gagctccagg tacccctgcc cggttctcag gctccctcct tgggggcaaa 960

gctgccctga cactgtcagg tgtgcagcct gaggacgagg ctgagtatta ctgcgctcta 1020

tggtacagca acctctgggt gttcggcgga gggaccaagc tgaccgtcct aggccaaccg 1080

aaagcggcgc cctcggtcac tctgttcccg ccctcctctg aggagcttca agccaacaag 1140

gccacactgg tgtgtctcat aagtgacttc tacccgggag ccgtgacagt ggcctggaag 1200

gcagatagca gccccgtcaa ggcgggagtg gagaccacca caccctccaa acaaagcaac 1260

aacaagtacg cggccagcag ctatctgagc ctgacgcctg agcagtggaa gtcccacaga 1320

agctacagct gccaggtcac gcatgaaggg agcaccgtgg agaagacagt ggcccctaca 1380

gaatgtccac cgtgctcatg a 1401

<210> 39

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 1VH

<400> 39

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 40

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 2VH

<400> 40

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 41

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 3VH

<400> 41

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 42

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 4VH

<400> 42

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 43

<211> 125

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 11VH

<400> 43

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 44

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 2VL

<400> 44

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 45

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 3VL

<400> 45

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 46

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 4VL

<400> 46

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 47

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> CD3 5VL

<400> 47

Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val

65 70 75 80

Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

Claims (133)

1. Use of a multi-specific Fab fusion protein in the manufacture of a medicament for treating cancer in an individual, wherein the multi-specific Fab fusion protein comprises a Fab fragment that specifically binds to CD3 ("anti-CD 3Fab fragment") and a binding domain that specifically binds to EpCAM ("anti-EpCAM binding domain"), wherein the anti-EpCAM binding domain is fused to the N-terminus of the anti-CD 3Fab fragment;

Wherein the anti-EpCAM binding domain comprises: hypervariable region 1 of heavy chain (HVR-H1) shown as SEQ ID NO. 13, HVR-H2 shown as SEQ ID NO. 14, and HVR-H3 shown as SEQ ID NO. 15; and

wherein the anti-EpCAM binding domain comprises: hypervariable region 1 of the light chain (HVR-L1) shown in SEQ ID NO: 16, HVR-L2 shown in SEQ ID NO: 17, and HVR-L3 shown in SEQ ID NO: 18;

wherein the medicament is suitable for administration at a dose of 0.01 to 250 mug/kg.

2. The use of claim 1, wherein the anti-EpCAM binding domain comprises: 19, and/or wherein the anti-EpCAM binding domain comprises: a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 20.

3. The use of claim 1, wherein the anti-EpCAM binding domain is an anti-EpCAM single chain fv (scfv).

4. The use of claim 2, wherein the anti-EpCAM binding domain is an anti-EpCAM single chain fv (scfv).

5. The use of claim 3, wherein the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO 21.

6. The use of claim 4, wherein the anti-EpCAM scFv comprises the amino acid sequence of SEQ ID NO 21.

7. The use of claim 3, wherein the multi-specific Fab fusion protein comprises a first anti-EpCAM scFv and a second anti-EpCAM scFv, wherein the first anti-EpCAM scFv is fused to the N-terminus of the VH of the anti-CD 3Fab fragment, and wherein the second anti-EpCAMscFv is fused to the N-terminus of the VL of the anti-CD 3Fab fragment.

8. The use of claim 5, wherein the multi-specific Fab fusion protein comprises a first anti-EpCAM scFv and a second anti-EpCAM scFv, wherein the first anti-EpCAM scFv is fused to the N-terminus of the VH of the anti-CD 3Fab fragment, and wherein the second anti-EpCAMscFv is fused to the N-terminus of the VL of the anti-CD 3Fab fragment.

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

10. The use of claim 8, wherein the first anti-EpCAM scFv and the second anti-EpCAM scFv have the same sequence.

11. The use of claim 1, wherein the medicament is suitable for intravenous administration.

12. The use of claim 1, wherein the medicament is suitable for administration at low frequency.

13. The use of claim 1, wherein the medicament is suitable for twice weekly administration.

14. The use of claim 1, wherein the medicament is adapted for administration to the individual at a first dose for a first period of time and, continuously, the medicament is adapted for administration to the individual at a second dose for a second period of time, and wherein the second dose exceeds the first dose.

15. The use of claim 14, wherein the second period of time exceeds the first period of time.

16. The use of claim 14, wherein the first period of time is at least about 7 days.

17. The use of claim 14, wherein the second period of time is at least about 2 weeks.

18. The use of claim 14, wherein the first dose is no more than about 1 μ g/kg.

19. The use of claim 14, wherein the second dose is 0.1 μ g/kg to 10 μ g/kg.

20. The use according to claim 1, wherein the medicament is suitable for administration in combination with a glucocorticoid.

21. The use of claim 20, wherein the glucocorticoid is dexamethasone.

22. The use of claim 20, wherein the glucocorticoid is suitable for administration prior to the first dose of the medicament.

23. The use of claim 22, wherein the glucocorticoid is suitable for further administration after the first dose of the medicament and before the second dose of the medicament.

24. The use of claim 20, wherein the glucocorticoid is suitable for administration at a dose of 0.1 mg/kg to 5 mg/kg.

25. The use of claim 1, wherein the subject is a human subject.

26. The use of any one of claims 1 to 25, wherein the anti-CD 3Fab fragment specifically binds to the N-terminus of CD3 epsilon.

27. The use of claim 26, wherein the anti-CD 3Fab fragment specifically binds to an epitope within amino acids 1-27 of CD3 epsilon.

28. The use of claim 27, wherein the VH of the anti-CD 3Fab fragment comprises: HVR-H1 shown as SEQ ID NO. 1, HVR-H2 shown as SEQ ID NO. 2, and HVR-H3 shown as SEQ ID NO. 3; and wherein the VL of the anti-CD 3Fab fragment comprises: HVR-L1 shown as SEQ ID NO. 4, HVR-L2 shown as SEQ ID NO. 5, and HVR-L3 shown as SEQ ID NO. 6.

29. The use of claim 28, wherein the VH of the anti-CD 3Fab 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-CD 3Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: SEQ ID NO 8 and 44-47.

30. The use of claim 28, wherein said anti-CD 3Fab fragment comprises human immunoglobulin heavy chain constant region 1 (CH1), said heavy chain constant region 1 (CH1) comprising the amino acid sequence of SEQ ID NO 9.

31. The use of claim 28, wherein the anti-CD 3Fab fragment comprises a human lambda light chain constant region comprising the amino acid sequence of SEQ ID No. 10.

32. The use of claim 28, wherein CH1 and CL of the anti-CD 3Fab fragment are linked by one or more disulfide bonds.

33. The use of any one of claims 1 to 25, wherein the anti-CD 3Fab 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.

34. The use of claim 28, wherein the anti-CD 3Fab 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.

35. The use of any one of claims 1-25, wherein the multi-specific Fab fusion protein comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO 22; and/or

The multi-specific Fab fusion protein comprises: a second polypeptide comprising the amino acid sequence of SEQ ID NO. 23.

36. The use of claim 28, wherein the multi-specific Fab fusion protein comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO 22; and/or

The multi-specific Fab fusion protein comprises: a second polypeptide comprising the amino acid sequence of SEQ ID NO. 23.

37. The use of claim 33, wherein the multi-specific Fab fusion protein comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO 22; and/or

The multi-specific Fab fusion protein comprises: a second polypeptide comprising the amino acid sequence of SEQ ID NO. 23.

38. The use of any one of claims 1-25, wherein the cancer is EpCAM positive solid cancer.

39. The use of claim 38, wherein the EpCAM-positive solid cancer is a carcinoma (carcinosoma) or an adenocarcinoma (adenocarinoma).

40. The use of claim 28, wherein the cancer is EpCAM positive solid cancer.

41. The use of claim 40, wherein the EpCAM-positive solid cancer is a carcinoma (carcinosoma) or an adenocarcinoma (adenocarinoma).

42. The use of claim 33, wherein the cancer is EpCAM positive solid cancer.

43. The use of claim 42, wherein the EpCAM-positive solid cancer is a carcinoma or adenocarcinoma (adenocarinoma).

44. The use of claim 35, wherein the cancer is EpCAM positive solid cancer.

45. The use of claim 44, wherein said EpCAM-positive solid cancer is a carcinoma or adenocarcinoma (adenocarinoma).

46. The use of any one of claims 1-25, wherein the cancer is selected from the group consisting of: small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer.

47. The use of claim 46, wherein the cancer is colorectal adenocarcinoma.

48. The use of claim 46, wherein the cancer is lung adenocarcinoma.

49. The use of claim 28, wherein the cancer is selected from the group consisting of: small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer.

50. The use of claim 49, wherein the cancer is colorectal adenocarcinoma.

51. The use of claim 49, wherein the cancer is lung adenocarcinoma.

52. The use of claim 33, wherein the cancer is selected from the group consisting of: small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer.

53. The use of claim 52, wherein the cancer is colorectal adenocarcinoma.

54. The use of claim 52, wherein the cancer is lung adenocarcinoma.

55. The use of claim 35, wherein the cancer is selected from the group consisting of: small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer.

56. The use of claim 55, wherein the cancer is colorectal adenocarcinoma.

57. The use of claim 55, wherein the cancer is lung adenocarcinoma.

58. An anti-EpCAM antibody or antigen-binding fragment thereof comprising: HVR-H1 shown as SEQ ID NO. 13, HVR-H2 shown as SEQ ID NO. 14, HVR-H3 shown as SEQ ID NO. 15; HVR-L1 shown as SEQ ID NO 16, HVR-L2 shown as SEQ ID NO 17, and HVR-L3 shown as SEQ ID NO 18.

59. The anti-EpCAM antibody or antigen-binding fragment thereof of claim 58, wherein the anti-EpCAM antibody or antigen-binding fragment thereof comprises a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO 19; and/or wherein the VL comprises the amino acid sequence of SEQ ID NO 20.

60. The anti-EpCAM antibody of claim 58, wherein the anti-EpCAM antibody is a multispecific antibody.

61. The anti-EpCAM antibody of claim 59, wherein the anti-EpCAM antibody is a multispecific antibody.

62. An antigen-binding fragment of an anti-EpCAM antibody of claim 58, wherein said antigen-binding fragment is an anti-epCAMscFv.

63. The antigen-binding fragment of an anti-EpCAM antibody of claim 62, wherein the anti-EpCAMscFv comprises the amino acid sequence of SEQ ID NO 21.

64. An antigen-binding fragment of an anti-EpCAM antibody of claim 59, wherein said antigen-binding fragment is an anti-epcammscfv.

65. The antigen-binding fragment of an anti-EpCAM antibody of claim 64, wherein the anti-EpCAMscFv comprises the amino acid sequence of SEQ ID NO 21.

66. A multi-specific Fab fusion protein comprising the anti-EpCAM antigen-binding fragment of any one of claims 58, 59, 62, and 65.

67. The multi-specific Fab fusion protein of claim 66, comprising: an anti-CD 3Fab fragment, a first copy of the anti-EpCAM antigen-binding fragment, and a second copy of the anti-EpCAM antigen-binding fragment; wherein a first copy of the anti-EpCAM antigen-binding fragment is fused to the N-terminus of the VH of the anti-CD 3Fab fragment; and wherein a second copy of the anti-EpCAM antigen-binding fragment is fused to the N-terminus of the VL of the anti-CD 3Fab fragment.

68. The multi-specific Fab fusion protein of claim 67, wherein the anti-CD 3 Fab fragment specifically binds to the N-terminus of CD3 epsilon.

69. The multi-specific Fab fusion protein of claim 67, wherein the anti-CD 3 Fab fragment specifically binds to an epitope within amino acids 1-27 of CD3 epsilon.

70. The multi-specific Fab fusion protein of claim 67, wherein the VH of the anti-CD 3 Fab fragment comprises: HVR-H1 as shown in SEQ ID NO. 1, HVR-H2 as shown in SEQ ID NO. 2, and HVR-H3 as shown in SEQ ID NO. 3; and wherein the VL of the anti-CD 3 Fab fragment comprises: HVR-L1 shown as SEQ ID NO. 4, HVR-L2 shown as SEQ ID NO. 5, and HVR-L3 shown as SEQ ID NO. 6.

71. The multi-specific Fab fusion protein of claim 67, wherein the VH of the anti-CD 3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO 7 and 39-43; and/or wherein the VL of the anti-CD 3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO 8 and 44-47.

72. The multi-specific Fab fusion protein of claim 70, wherein the VH of the anti-CD 3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO 7 and 39-43; and/or wherein the VL of the anti-CD 3 Fab fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO 8 and 44-47.

73. The multi-specific Fab fusion protein of claim 70, wherein the anti-CD 3 Fab fragment comprises human CH1, and the human CH1 comprises the amino acid sequence of SEQ ID NO 9.

74. The multi-specific Fab fusion protein of claim 70, wherein the anti-CD 3 Fab fragment comprises a human lambda light chain constant region comprising the amino acid sequence of SEQ ID No. 10.

75. The multi-specific Fab fusion protein of claim 70, wherein the CH1 and CL of the anti-CD 3 Fab fragment are linked by one or more disulfide bonds.

76. The multi-specific Fab fusion protein of claim 67, wherein the 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.

77. The multi-specific Fab fusion protein of claim 70, wherein the 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.

78. The multi-specific Fab fusion protein of claim 71, wherein the 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.

79. The multi-specific Fab fusion protein of claim 67, wherein 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 scFv.

80. The multi-specific Fab fusion protein of claim 70, wherein 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 scFv.

81. The multi-specific Fab fusion protein of claim 71, wherein 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 scFv.

82. The multi-specific Fab fusion protein of claim 76, wherein 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 scFv.

83. The multi-specific Fab fusion protein of claim 67, 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.

84. The multi-specific Fab fusion protein of claim 83, wherein 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 scFv comprising the amino acid sequence of SEQ ID NO. 21.

85. The multi-specific Fab fusion protein of claim 70, 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.

86. The multi-specific Fab fusion protein of claim 85, wherein 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 scFv comprising the amino acid sequence of SEQ ID NO. 21.

87. The multi-specific Fab fusion protein of claim 71, 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.

88. The multi-specific Fab fusion protein of claim 87, wherein 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 scFv comprising the amino acid sequence of SEQ ID NO. 21.

89. The multi-specific Fab fusion protein of claim 76, 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.

90. The multi-specific Fab fusion protein of claim 89, wherein 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 scFv comprising the amino acid sequence of SEQ ID NO. 21.

91. The multi-specific Fab fusion protein of claim 80, 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.

92. The multi-specific Fab fusion protein of claim 67, wherein the multi-specific Fab fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO. 22; the second polypeptide comprises the amino acid sequence of SEQ ID NO 23.

93. The multi-specific Fab fusion protein of claim 70, wherein the multi-specific Fab fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO. 22; the second polypeptide comprises the amino acid sequence of SEQ ID NO 23.

94. The multi-specific Fab fusion protein of claim 76, wherein the multi-specific Fab fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO 22; the second polypeptide comprises the amino acid sequence of SEQ ID NO 23.

95. The multi-specific Fab fusion protein of claim 79, wherein the multi-specific Fab fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO 22; the second polypeptide comprises the amino acid sequence of SEQ ID NO. 23.

96. The multi-specific Fab fusion protein of claim 84, wherein the multi-specific Fab fusion protein comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO. 22; the second polypeptide comprises the amino acid sequence of SEQ ID NO 23.

97. An isolated nucleic acid encoding the anti-EpCAM antibody or antigen-binding fragment thereof of any one of claims 58-65.

98. An isolated nucleic acid encoding the multi-specific Fab fusion protein of claim 66.

99. An isolated nucleic acid encoding the multi-specific Fab fusion protein of any one of claims 67-96.

100. An expression vector comprising the isolated nucleic acid of claim 97.

101. An expression vector comprising the isolated nucleic acid of claim 98.

102. An expression vector comprising the isolated nucleic acid of claim 99.

103. An isolated host cell comprising the expression vector of claim 100.

104. An isolated host cell comprising the expression vector of claim 101.

105. An isolated host cell comprising the expression vector of claim 102.

106. A method of producing an anti-EpCAM antibody or antigen-binding fragment thereof, comprising culturing the isolated host cell of claim 103, and recovering said anti-EpCAM antibody or antigen-binding fragment thereof from the cell culture.

107. A method of producing a multi-specific Fab fusion protein comprising culturing the isolated host cell of claim 104, and recovering the multi-specific Fab fusion protein from the cell culture.

108. A method of producing a multi-specific Fab fusion protein comprising culturing the isolated host cell of claim 105, and recovering the multi-specific Fab fusion protein from the cell culture.

109. A composition comprising the anti-EpCAM antibody or antigen-binding fragment thereof of any one of claims 58-65, and a pharmaceutically acceptable carrier.

110. A composition comprising the multi-specific Fab fusion protein of claim 66, and a pharmaceutically acceptable carrier.

111. A composition comprising the multi-specific Fab fusion protein of any one of claims 67-96, and a pharmaceutically acceptable carrier.

112. Use of a multi-specific Fab fusion protein of any one of claims 67-96 in the manufacture of a medicament for treating cancer in an individual.

113. The use of claim 112, wherein the medicament is suitable for administration at a dose of 0.01 μ g/kg to 250 μ g/kg.

114. The use of claim 112, wherein the medicament is suitable for intravenous administration.

115. The use of claim 112, wherein the medicament is suitable for administration at a low frequency.

116. The use of claim 112, wherein the medicament is suitable for twice weekly administration.

117. The use of claim 112, wherein the medicament is suitable for administration to the individual at a first dose for a first period of time and, continuously, the medicament is suitable for administration to the individual at a second dose for a second period of time, and wherein the second dose exceeds the first dose.

118. The use of claim 117, wherein the second period of time exceeds the first period of time.

119. The use of claim 117, wherein said first period of time is at least about 7 days.

120. The use of claim 117, wherein said second period of time is at least about 2 weeks.

121. The use of claim 117, wherein the first dose is not more than about 1 μ g/kg.

122. The use of claim 117, wherein the second dose is 0.1 μ g/kg to 10 μ g/kg.

123. The use of claim 112, wherein the medicament is suitable for administration in combination with a glucocorticoid.

124. The use of claim 123, wherein the glucocorticoid is dexamethasone.

125. The use of claim 123, wherein the glucocorticoid is suitable for administration prior to the first dose of the medicament.

126. The use of claim 123, wherein the glucocorticoid is suitable for further administration after the first dose of the medicament and before the second dose of the medicament.

127. The use of claim 123, wherein the glucocorticoid is suitable for administration at a dose of 0.1 mg/kg to 5 mg/kg.

128. The use of claim 112, wherein the subject is a human subject.

129. The use of claim 112, wherein the cancer is EpCAM positive solid cancer.

130. The use of claim 129, wherein the EpCAM positive solid cancer is a carcinoma (carcinosoma) or an adenocarcinoma (adenocarinoma).

131. The use of claim 112, wherein the cancer is selected from the group consisting of: small intestine cancer, colorectal cancer, lung cancer, cervical cancer, liver cancer, stomach cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, thyroid cancer, prostate cancer, ovarian cancer, endometrial cancer, breast cancer, bile duct cancer, and head and neck cancer.

132. The use of claim 131, wherein the cancer is colorectal adenocarcinoma.

133. The use of claim 131, wherein the cancer is lung adenocarcinoma.