GUCY2c antibodies and uses thereof

Abstract

Methods of treating cancer of the digestive system, preferably of the gastrointestinal system, in a patient using an anti-GUCY2CxCD3 antibody are disclosed.

Description

GUCY2C ANTIBODIES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 702,826 filed on October 3, 2024, and U.S. Provisional Application No. 63 / 754,685 filed on February 6, 2025, the disclosures of each of which are hereby incorporated by reference in their entireties.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on September 25, 2025, is named JBI6926PCT.xml and is 55,031 bytes in size.TECHNICAL FIELD

[0003] Disclosed are methods of treating cancer with anti-GUCY2CxCD3 bispecific antibodies.BACKGROUND

[0004] Cancer of the digestive system is among the most common cancer types worldwide. Colorectal cancer (CRC) is the fourth leading cause of cancer, and the second leading cause of cancer-related death in the United States and the world. Most colorectal carcinoma (CRC) result from the uncontrolled growth on the inner lining of the colon or rectum called polyps. Surgery to remove the cancerous growth is the most common treatment for colorectal cancer. Surgery procedures may include partial colectomy. Currently, surgery, chemotherapy, targeted drugs and immunotherapy are the mainstream treatment options for patient. While surgical excision of primary tumors can be curative, particularly at the earliest stages of disease, about 50% of patients with colorectal cancer ultimately die of distant metastases. While chemo-, radio-, and targeted therapies may extend survival to about 24 months, less than 15% of patients with metastatic CRC survive beyond 5 years, highlighting the unmet need for new therapeutic paradigms for this disease.SUMMARY OF THE INVENTION

[0005] Provided herein are methods of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprising, consisting of and / or consisting essentially of administering to the subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody.

[0006] In some embodiments, the disclosure provide a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti- GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein the antibody comprises a VH1 and a VL1 comprising amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0007] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti- GUCY2CxCD3 antibody comprising a first heavy chain (HC1) of SEQ ID NO: 9 and a first light chain (LC1) of SEQ ID NO: 10.

[0008] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti- GUCY2CxCD3 antibody comprising a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20.

[0009] In some embodiments, the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously or intravenously.

[0010] In some embodiments, the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously at a treatment dose of less than about 1000 mg.

[0011] In some embodiments, the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously at a treatment dose of less than about 500 mg.

[0012] In some embodiments, the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously at a treatment dose of about less than about 100 mg.

[0013] In some embodiments, the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously at a treatment dose ranging from about 1 mg to about 50 mg.

[0014] In some embodiments, the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously at a treatment dose ranging from about 2 mg to about 50 mg.

[0015] In some embodiments, the anti-GUCY2CxCD3 antibody is administered to the subject intravenously at a treatment dose of less than about 1000 mg.

[0016] In some embodiments, the anti-GUCY2CxCD3 antibody is administered to the subject intravenously at a treatment dose of less than about 500 mg.

[0017] In some embodiments, the anti-GUCY2CxCD3 antibody is administered to the subject intravenously at a treatment dose of less than about 100 mg.

[0018] In some embodiments, the anti-GUCY2CxCD3 antibody is administered to the subject intravenously at a treatment dose ranging from about 1 mg to about 50 mg.

[0019] In some embodiments, the anti-GUCY2CxCD3 antibody is administered to the subject intravenously at a treatment dose ranging from about 2 mg to about 50 mg.

[0020] In some embodiments, the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0021] In some embodiments, the treatment dose is administered once every 2 weeks,

[0022] In some embodiments, the method comprises one or more step-up doses of the anti- GUCY2CxCD3 antibody prior to administering any treatment dose of the anti- GUCY2CxCD3 antibody, wherein the one or more step-up doses are lower than the treatment dose.

[0023] In some embodiments, the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first administration of the treatment dose (or prior the first administration of the treatment dose).

[0024] In some embodiments, one or more step-up doses and the treatment dose are administered subcutaneously.

[0025] In some embodiments, one or more step-up doses and the treatment dose are administered intravenously.

[0026] In some embodiments, the metastatic colorectal cancer is unresectable metastatic colorectal cancer.DESCRIPTION OF DRAWINGS

[0027] FIG. 1 shows binding of GUCY2C_mAb to human and cynomolgus monkey GUCY2C.

[0028] FIG. 2 A shows binding of GUCY2CxCD3_biAb-l and 2 to T84 cell line. FIG. 2B shows binding of GUCY2CxCD3_biAb-l and 2 to T cells.

[0029] FIG. 3 shows competition flow cytometry between GUCY2C_biAb-l and reference bi Ab.

[0030] FIG. 4A, FIG. 4B, and FIG. 4C show T cell mediated cytotoxicity of GUCY2CxCD3_biAb-l, GUCY2CxCD3_biAb-2 and reference biAb. FIG. 4D, FIG 4E, and FIG. 4F show T-cell activation of GUCY2CxCD3_biAb-l, GUCY2CxCD3_biAb-2 and reference bi Ab.

[0031] FIG. 5 shows T cell-mediated cytotoxic activity of GUCY2CxCD3_biAb-l on WT and CRC tumor organoids. The upper panel shows live cell fluorescence images showing apoptosis (white) of CRC and WT organoids post treatment with GUCY2CxCD3_biAb-l and reference bi Ab. The lower panel shows dose normalized cytotoxicity dose response curves of GUCY2CxCD3_biAb-l and reference biAb. Abbreviations: CRC, colorectal cancer; EpCAM, epithelial cell adhesion molecule; HC, high control; LC, low control; WT, wild type.

[0032] FIG. 6 shows the effect of GUCY2CxCD3_biAb-l treatment on growth and T-cell infiltration in T-cell humanized NSG Mice bearing established SC HT55 tumors. Group tumor volumes were graphed as mean ± SEM. *Denotes significant difference of GUCY2CxCD3_biAb-l treated groups on Day 33 versus control group (p<0.05, n=10 / group). (DPBS=Dulbecco’s phosphate buffered saline; SEM=standard error of the mean.)

[0033] FIG. 7 shows representative IHC micrographs for HT55 tumors treated as indicated on Days 10, 13, and 17, followed by tumor harvest on Days 12 (48 hours post first dose), 14 (24 hours post second dose), and 18 (24 hours post third dose), and stained for CD8, GUCY2C, and pan-cytokeratin. Magnification bar denotes 200 pm. (DPBS=Dulbecco’s phosphate buffered saline).

[0034] FIG. 8 shows the effect of GUCY2CxCD3_biAb-l treatment on growth and T cell infiltration in NSG mice bearing SC LS1034 human colorectal CDX tumors. Group tumor volumes are graphed as the mean ± SEM (n=10 / group). * Denotes significant difference (p<0.05) of GUCY2CxCD3_biAb-l treated groups on Day 38 versus DPBS control group. (DPBS=Dulbecco’s phosphate buffered saline; SEM=standard error of the mean.)

[0035] FIG. 9A - FIG. 9C show cynomolgus monkey T-cell activation (CD25) induced by GUCY2CxCynoCD3 bispecific antibody (GUCY2CxCD3_biAb-3), NullxCD3, or reference GUCY2CxCD3 bispecific, in the presence of cynomolgus monkey GUCY2C-expressing CHO-K1, or parental untransduced CHO-K1, across 3 cynomolgus monkey donors.

[0036] FIG. 10 shows human T cell activation (CD25) induced by GUCY2CxHumanCD3 bispecific antibody (GUCY2CxCD3_biAb-l), NullxCD3, or reference GUCY2CxCD3 bispecific antibody, in the presence of human GUCY2C-expressing CHO-K1, or parental untransduced CHO-K1, across 1 human donor.

[0037] FIG. 11A shows individual GUCY2CxCynoCD3 antibody Serum Concentration- Time Profiles following SC Injection of 0.3 mg / kg GUCY2CxCynoCD3 antibody to Male and Female Cynomolgus Monkeys. FIG. 1 IB shows individual GUCY2CxCynoCD3 antibody Serum Concentration-Time Profiles following SC Injection of 1 mg / kg followed by 3 mgs / kg (day 8 onwards) of GUCY2CxCynoCD3 antibody to Male and Female Cynomolgus Monkeys.DETAILED DESCRIPTION OF THE INVENTION

[0038] Provided herein are methods of treating metastatic colorectal cancer in a subject, comprising, consisting of and / or consisting essentially of administering to a subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody.

[0039] The disclosed methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed methods are not limited to the specific methods described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods.

[0040] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

[0041] Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosedmethods are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.

[0042] Unless specifically stated otherwise, any term used herein is intended to have its ordinary and customary meanings in the field.

[0043] It is to be appreciated that certain features of the invention which are, for clarity, described herein in the context of separate embodiments may also be provided in combination in a single embodiment. That is, unless obviously incompatible or specifically excluded, each individual embodiment is deemed to be combinable with any other embodiment s) and such a combination is considered to be another embodiment.Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. Finally, although an embodiment may be described as part of a series of steps or part of a more general structure, each said step may also be considered an independent embodiment or may be combinable with others.

[0044] Throughout this text, where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the invention as described herein. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value. It is not intended that the scope of the invention be limited to the specific values recited when defining a range. All ranges are inclusive and combinable.

[0045] When values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The term “about” is used to encompass variations of ± 10% or less, variations of ± 5% or less, variations of ± 1% or less, variations of ± 0.5% or less, or variations of ± 0.1% or less from the specified value.

[0046] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0047] The transitional terms “comprising,” “consisting essentially of,” and “consisting of’ are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of’ excludes any element, step, or ingredient not specified in the claim; and (iii) “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of’ and “consisting essentially of.” Embodiments described in terms of the phrase “consisting essentially of’ (or its equivalents) also provide as embodiments those independently described in terms of “consisting of.”

[0048] In an attempt to help the reader of the application, the description has been separated in various paragraphs or sections or is directed to various embodiments of the application. These separations should not be considered as disconnecting the substance of a paragraph or section or embodiments from the substance of another paragraph or section or embodiments. To the contrary, one skilled in the art will understand that the description has broad application and encompasses all the combinations of the various sections, paragraphs and sentences that can be contemplated. The discussion of any embodiment is meant only to be exemplary and is not intended to suggest that the scope of the disclosure, including the claims, is limited to these examples.Method of treatment

[0049] Any reference in the description to a method of treatment (comprising the administration of a compound, composition, medicament, etc.) equally refers to the compound, pharmaceutical composition, medicament, etc. for use in the method for treatment or for use in the manufacture of the medicament. For example, every embodiment or combination of embodiments, which has been described herein for methods for treating a disease, disorder or condition (comprising the administration of a compound, composition, medicament, combination, association etc.), is also applicable to the same compound, composition, medicament, combination, association, etc. for use in treating the same disease,disorder or condition as well as to the use of the same compound, composition, medicament, combination, association, etc. in the manufacture of a medicament for the treatment of the same disease, disorder or condition.

[0050] In some embodiments, the disclosure comprises a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3.

[0051] “ Treat,” “treating,” or “treatment” as used herein, include alleviating, abating or ameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting the disease or condition, e.g. arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, reducing the severity and / or duration of the disorder, limiting or preventing recurrence of the disease or condition in subjects that have previously had the disorder, limiting or preventing recurrence of symptoms in subjects that were previously symptomatic for the disorder or stopping the symptoms of the disease or condition either prophylactically and / or therapeutically.

[0052] “ Treat,” “treating,” or “treatment” as used herein also includes (a) delaying or slowing the development or progression of one or more symptoms or complications of the disease, condition or disorder; (b) delaying or slowing the development or progression of one or more new / additional symptoms or complications of the disease, condition or disorder; (c) inhibiting worsening of symptoms characteristic of the disorder being treated and / or (d) delaying or slowing the progression of the disease, condition or disorder to a later stage or more serious form of said disease, condition or disorder.

[0053] A “therapeutically effective amount”, “therapeutically effective dose” or “effective amount” refers to an amount effective, at doses and for periods of time necessary, to achieve a desired therapeutic result. An therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, the physical condition of the subject, the duration of the treatment, the nature of concurrent therapy (if any), the specific formulations employed, the structure of the antibody or antibody fragment or of its variants and the ability of a therapeutic or a combination of therapeutics to elicit a desired response in the individual. Example indicators of an effective therapeutic or combination of therapeutics that include, for example, improved wellbeing of the subject,reduction of a tumor burden, arrested or slowed growth of a tumor, and / or absence of metastasis of cancer cells to other locations in the body. An effective amount of the administered antibody or antibody fragment may depend on the type and severity of the cancer being treated, and the route of administration of the antibody polypeptide or the pharmaceutical composition of the antibody.

[0054] In some embodiments, the treatment efficacy can be assessed by radiographic image assessment, tumor markers, CT scans of the chest, abdomen, and pelvis (plus other areas of known disease involvement as appropriate), or magnetic resonance imaging.

[0055] In some embodiments, the treatment efficacy can be assessed by measuring the progression of soft tissue lesions by CT or MRI using the RECIST criteria. The term "Response Evaluation Criteria In Solid Tumors (RECIST)" refers to a set of published rules that define when cancer patients improve ("respond"), stay the same ("stable") or worsen ("progression") during treatments.

[0056] In the present disclosure, an amount or dose would be deemed therapeutically effective if it resulted in inhibition of tumor growth.

[0057] “Subject” or “patient” as used herein includes any human or non-human animal. “Non-human animal” includes all vertebrates, e.g., mammals and non-mammals. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, non-human primates, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc. The terms “subject” and “patient” can be used interchangeably herein. In some embodiments, the subject or patient is human. In some embodiments, the subject according to the invention has or is susceptible to have a cancer of the digestive system, preferably of the gastrointestinal system. In some embodiments, the subject according to the invention has or is susceptible to have a metastatic colorectal cancer (mCRC), e.g., unresectable metastatic colorectal cancer. In some embodiments, the subject according to the invention has or is susceptible to have a gastric cancer, e.g. a primary or a metastatic gastric cancer.

[0058] As used herein, the term “GUCY2C” refers to Guanylate cyclase 2C (GUCY2C) (GenBank Accession No. NM-004963 and GenPept Accession No. NP-004954), also known as guanylyl cyclase C (GC-C), intestinal guanylate cyclase, guanylate cyclase-C receptor, or the heat-stable enterotoxin receptor (hSTAR). GUCY2C is a member of the guanylyl cyclase (GC; heat-stable enterotoxin receptor [STaR]) family of enzymes that convert intracellular guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) upon ligand binding. GUCY2C is a 120 kDa transmembrane protein and has an extracellularligand-binding domain, a single transmembrane region, a region with sequence similar to that of protein kinases, and a C-terminal guanylate cyclase domain. GUCY2C is found in the luminal surface of intestinal epithelium cells and in dopamine neurons in the brain. GUCY2C’s cell-surface expression is confined to the apical surfaces of intestinal epithelial cells and exhibits limited expression in extra-intestinal tissues of humans and mice. It plays a role in gut fluid secretion, tumor suppression, inflammation, and intestine homeostasis though cGMP-regulated mechanisms. Dysregulation of the GUCY2C axis (through mutation or enterotoxigenic bacterial infection) results in fluid secretion disorders.

[0059] Of significance, GUCY2C is a cancer mucosa antigen, universally overexpressed by primary and metastatic human colorectal cancer and is ectopically expressed in esophageal and gastric cancers associated with intestinal dysplasia. Moreover, anatomical segregation of GUCY2C on the luminal surface of the intestinal epithelium limits access to systemically delivered GUCY2C-targeted molecules permitting diagnostic imaging and monoclonal antibody -based therapy of colorectal cancer metastasis without recognition of intestinal epithelium.

[0060] “ GUCY2C “is intended in accordance with its ordinary meaning in the field. Unless the context dictates otherwise, “GUCY2C “refers more specifically to human GUCY2C (e.g., Accession# P25092.2). The extracellular domain of human GUCY2C spans from amino acid residue 24 amino acid residue 430, which is followed by a transmembrane domain (amino acid residue 431-454) and a cytoplasmic domain (amino acid residue 455-1-73).

[0061] The term “CD3” is intended in accordance with its ordinary meaning in the field and refers to an antigen that is expressed on T cells as part of the multimeric T cell receptor (TCR) complex and which consists of a homodimer or heterodimer formed from the association of two or four receptor chains: CD3 epsilon, CD3 delta, CD3 zeta and CD3 gamma. In some embodiments, CD3 antibodies provided herein bind to the CD3-epsilon polypeptide, which together with CD3 -gamma, -delta and -zeta, and the T cell receptor alpha / beta and gamma / delta heterodimers, forms the T cell receptor-CD3 complex. This complex plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. The CD3 complex mediates signal transduction, resulting in T cell activation and proliferation. CD3 is required for the immune response. The term “CD3” includes any CD3 variant, isoform, and species homolog, which is naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding the polypeptide. In specific embodiments, the CD3 is a human CD3. Unless thecontext dictates otherwise, “CD3” refers to human CD3 epsilon (CD3s) (e.g., Accession# P07766) or cynomolgus monkey (Macaca fascicularis) CD3 epsilon (e.g., Accession # XP 065385109) unless explicitly specified as being from other species.

[0062] As used herein, “GUCY2CxCD3 antibody”, GUCY2CxCD3 bispecific antibody”, “GUCY2CxCD3 biAb”, and “anti-GUCY2CxCD3 antibody” refer to a bispecific antibody with a first antigen binding domain that specifically binds to GUCY2C and a second binding domain that specifically binds to CD3s. The domains specifically binding GUCY2C are typically referred herein as VH1 / VL1 pairs. The domains specifically binding CD3 epsilon are typically referred herein as VH2 / VL2 pairs. The VH2 and VL2 of the domains specifically binding CD3 epsilon can form a scFv or spFv. The heavy chain and light chain of the binding domain specifically binding GUCY2C are referred herein as HC1 and LC1, respectively. The heavy chain of the binding domain specifically binding CD3s is referred herein as HC2.

[0063] “Bispecific” refers to a molecule (such as an antibody) that specifically binds two distinct antigens or two distinct epitopes within the same antigen. The bispecific molecule may have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca cynomolgus (cynomolgus, cyno) or Pan troglodytes, or may bind an epitope that is shared between two or more distinct antigens.

[0064] “Antibodies” is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antigen binding fragments, multispecific antibodies, such as bispecific, trispecific, tetraspecific, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity.

[0065] “Full length antibodies” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g. IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CHI, hinge, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino- to-carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa (K) and lambda (X), based on the amino acid sequences of their constant domains.

[0066] “Single chain Fv” or “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region (VL) and at least one antibody fragment comprising a heavy chain variable region (VH), wherein the VL and the VH are contiguously linked via a polypeptide linker, and capable of being expressed as a single chain polypeptide. Unless specified, as used herein, a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. The scFv can be fused to an Fc to create a scFv-Fc wherein the scFv is fused to the IgG CH2 and IgG CH3 domain of the Fc via the hinge domain.

[0067] “Stapled single chain Fv”, “stapled scFv” or “spFv” refers to a scFv that comprises one or more disulfide bonds between the VH and the linker or the VL and the linker. Typically, the spFv may comprise one disulfide bond between the VH and the linker, and one disulfide bond between the VL and the linker, or two disulfide bonds between the VH and the linker and the VL and the linker. The spFv can be fused to an Fc to create a spFv-Fc wherein the spFv is fused to the IgG CH2 and IgG CH3 domain of the Fc via the hinge domain.

[0068] “Complementarity determining regions” (CDR) are antibody regions that bind an antigen. There are three CDRs in the VH (HCDR1, HCDR2, HCDR3) and three CDRs in the VL (LCDR1, LCDR2, LCDR3). CDRs may be defined using various delineations such as Kabat (Wu et al. (1970) J Exp Med 132: 211-50; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al. (1987) J Mol Biol 196: 901-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27: 55-77) and AbM (Martin and Thornton J Bmol Biol 263: 800-15, 1996). The correspondence between the various delineations and variable region numbering is described (see e.g., Lefranc et al. (2003) Dev Comp Immunol 27: 55-77; Honegger and Pluckthun, J Mol Biol (2001) 309:657-70; International ImMunoGeneTics (IMGT) database). Available programs such as abYsis by UCL Business PLC may be used to delineate CDRs. The term “CDR”, “HCDR1”, “HCDR2”, “HCDR3”,“LCDR”, “LCDR2” and “LCDR3” as used herein includes CDRs defined by any of the methods described supra, Kabat, Chothia, IMGT or AbM, unless otherwise explicitly stated in the specification.

[0069] Unless indicated otherwise, the numbering of amino acid residues in the antibody constant region throughout the specification is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), unless otherwise explicitly stated.

[0070] Within the present disclosure, the terms “Specifically binds,” “specific binding,” “specifically binding”, and “binds” are used interchangeably and refer to a proteinaceous molecule binding to an antigen or an epitope within the antigen with greater affinity than for other antigens. Typically, the proteinaceous molecule binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (KD) of about I * 107M or less, for example about 5x 10-8M or less, about 1 x 10-8M or less, about 1 x109M or less, about 1 x1010M or less, about 1 x10- 1 1M or less, or about 1 x1012M or less, typically with the KD that is at least one hundred fold less than its KD for binding to a non-specific antigen (e.g., BSA, casein).

[0071] “Epitope” refers to a portion of an antigen to which an antibody specifically binds. Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. For a discontinuous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3- dimensional space through the folding of the protein molecule. Antibody “epitope” depends on the methodology used to identify the epitope.

[0072] An antibody that “binds to GUCY2C” or that “specifically binds to GUCY2C” refers to an antibody that binds to GUCY2C, preferably human GUCY2C, with a KD of 1 x107M or less, preferably 1 x108M or less, more preferably 5x109M or less, 1 x 109M or less, 5 / 1010M or less, or 1 x1010M, 5 / 10- 1 1M, U 10- 1 1M, 5 / 10l 2M, or U I O12M or less.

[0073] An antibody that “binds to CD3” or that “specifically binds to CD3” refers to an antibody that binds to CD3ε, preferably human CD3s, with a KD of 1 x107M or less, preferably 1 x108M or less, more preferably 5x109M or less, 1 x109M or less, 5x 1010M or less, or x1010M, 5x 10- 1 1M, U 10- 1 1M, 5 / 1012M, or U I O12M or less.

[0074] In some embodiments, the anti-GUCY2CxCD3 antibody of the disclosure specifically binds to human GUCY2C with a KD of 1 x107M or less, preferably 1 x 10-8M or less, more preferably 5 x | 09M or less, 1 * 1 CT9M or less, 5 x1010M or less, or 1 x1010M, 5 x101 1M, 1 x101 1M, 5x1012M, or 1 x1012M or less, and specifically binds to human CD3s, with a KD of 1 x107M or less, preferably 1 x108M or less, more preferably 5x109M or less, 1 x109M or less, 5 x1010M or less, or 1 x1010M, 5 x101 1M, 1 x1011M, 5 / 1012M, or 1 / I O12M or less.

[0075] In some embodiments, the anti-GUCY2CxCD3 antibody of the disclosure specifically binds to human and cynomolgus monkey CD3 with a KD of 1 x10-7 M or less, preferably 1 x108M or less, more preferably 5x109M or less, 1 x109M or less, 5x1010M or less, or 1 x 1010M, 5x10-11M, x10 -11 M, 5x 1012M, or 1x 1012M or less.

[0076] In some embodiments, the anti-GUCY2CxCD3 antibody of the disclosure specifically binds to human CD3 with a KD of 1 x107M or less, preferably 1 x108M or less, more preferably 5 x109M or less, 1 x109M or less, 5 x1010M or less, or 1 x1010M, 5 x101 1M, 1 x101 1M, 5 x1012M, or 1 x1012M or less or less but does not bind to cynomolgus monkey CD3.

[0077] In some embodiments, the disclosure provide a method of treating a metastatic colorectal cancer in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein the antibody comprises a VH1 and a VL1 comprising amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0078] In some embodiments, the VH1 and VL1 of the GUCY2CxCD3 antibody used in the method of the disclosure form a Fab or are comprised in a Fab.

[0079] In some embodiments, the first antigen binding domain of the GUCY2CxCD3 antibody used in the method of the disclosure is (covalently) linked to a first Ig constant region or to a fragment of a first Ig constant region.

[0080] In some embodiments, the first Ig constant region or the fragment of the first Ig constant region comprises(i) mutations that reduce Fc binding to a Fey receptor (such as the mutations L234A, L235A, D265S (EU numbering)); and(ii) mutations that promote antibody heterodimerization such as the hole mutations T366S, L368A, Y407V (EU numbering) or the knob mutation T366W (EU numbering).

[0081] In some embodiments, the first Ig constant region or fragment of first Ig constant region comprises the L234A, L235A, D265S mutations (EU numbering), and the T366S, L368A, Y407V mutations (EU numbering) and the mutations H435R and Y436F (EU numbering).

[0082] In some embodiments, the first Ig constant region or fragment of first Ig constant region comprises the L234A, L235A, D265S mutations (EU numbering) and the T366W mutation (EU numbering).

[0083] In some embodiments, the first Ig constant region or fragment of first Ig constant is of the Igl isotype.

[0084] In some embodiments, the disclosure provide a method of treating a metastatic colorectal cancer in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein the first antigen binding domain comprises a first binding domain that specifically binds to GUCY2C comprising a first heavy chain (HC1) and a first light chain (LC1), comprising amino acid sequences of SEQ ID NO: 9 and 10, respectively.

[0085] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the invention comprises a VH2 and a VL2 comprising amino acid sequences of SEQ ID NO: 17 and 18.

[0086] In some embodiments, the VH2 and VL2 of the second antigen binding domain form a scFv or a spFv, particularly a spFv, or are comprised in a scFv or a spFv, particularly a spFv.

[0087] In some embodiments, the VH2 and the VL2 are linked to via linker and form a scFv or a spFv.

[0088] In some embodiments, the VH2 and the VL2 are linked to via linker in the VL2- Linkr-VH2 format and form a scFv or a spFv, particularly as spFv.

[0089] In some embodiments, the VH2 and VL2 of the GUCY2CxCD3 antibody used in the method of the disclosure form a spFv or are comprised in a spFv.

[0090] In some embodiments, the VH2 and the VL2 are linked to via linker and form a scFv or a spFv and the linker comprises an amino acid sequence of SEQ ID NO: 31.

[0091] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the invention comprises a VL2-Linker-VH2 (spFv2) comprising an amino acid sequence of SEQ ID NO: 19.

[0092] In some embodiments, the second antigen binding domain of the anti- GUCY2CxCD3 antibody is (covalently) linked to a second Ig constant region or a fragment of a second Ig constant region.

[0093] In some embodiments, the second Ig constant region or fragment of second Ig constant region comprises mutations that reduce Fc binding to a Fey receptor; and mutations that promote antibody heterodimerization.

[0094] In some embodiments, the second Ig constant region or fragment of second Ig constant region comprises the mutations L234A, L235A, D265S (EU numbering) and the mutation T366W (EU numbering). In some embodiments, the second Ig constant region or fragment of second Ig constant region is of the Igl isotype.

[0095] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the invention comprises a second heavy chain (HC2) comprising an amino acid sequence of SEQ ID NO: 20.

[0096] In some embodiments, the second binding domain of the anti-GUCY2CxCD3 antibody used in the method of the invention specifically binds to human CD3 epsilon but does not bind to cynomolgus monkey CD3 epsilon.

[0097] In some embodiments, the disclosure comprises a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, a second antigen binding domain that binds specifically to CD3, wherein the anti-GUCY2CxCD3 antibody comprises a first heavy chain variable region (VH1) of SEQ ID NO: 7 and a first light chain variable region (VL1) of SEQ ID NO: 8, a second heavy chain variable region (VH2) of SEQ ID NO: 17 and a second light chain variable region (VL2) of SEQ ID NO: 18.

[0098] In some embodiments, the disclosure comprises a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein the anti-GUCY2CxCD3 antibody comprises a first heavy chain variable region (VH1) of SEQ ID NO: 7 and a first light chain variable region (VL1) of SEQ ID NO: 8, and a spFv2 of SEQ ID NO: 19.

[0099] In some embodiments, the disclosure comprises a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein the anti-GUCY2CxCD2 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20.

[0100] In some embodiments, the disclosure provides methods of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, comprising administering to the subject an anti- GUCY2CxCD3 antibody that comprises a first binding domain that binds GUCY2C and a second binding domain that binds CD3, wherein the anti- GUCY2CxCD3 antibody comprises(i) a VH1 having an amino acid sequence of SEQ ID NO: 7 and a VL1 having an amino acid sequence of SEQ ID NO: 8;(ii) a HC1 having an amino acid sequence of SEQ ID NO: 9 and a LC1 having an amino acid sequence of SEQ ID NO: 10;(iii) a VH2 having an amino acid sequence of SEQ ID NO: 17 and a VL1 having an amino acid sequence of SEQ ID NO: 18;(iv) a spFv having an amino acid sequence of SEQ ID NO: 19; and / or(v) a HC2 having an amino acid sequence of SEQ ID NO: 20.

[0101] In certain embodiments, the anti-GUCY2CxCD3 antibody used in the method of the invention comprises sequences as shown in Tables 1-7 and 9-13.

[0102] In some embodiments, the method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject comprises administering to the subject an anti-GUCY2CxCD3 antibody comprising a VH1, VL1, HC1, LC1, VH2, VL2, spFv2, or HC2 having at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NO: 7, 8, 9, 10, 17, 18, 19, or 20, respectively.

[0103] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences (e.g., anti-GUCY2CxCD3 bispecific antibodies and polynucleotides that encode them, anti-GUCY2CxCD3 bispecific antibodies and polynucleotides that encode them), refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Optimal alignment of sequences for comparison can be conducted by sequence comparison algorithm that calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.

[0104] In some embodiments, the disclosure provide a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti- GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein the anti-GUCY2CxCD3 antibody comprises a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1) comprising amino acid sequences at least 90%, preferably 100%, identical to SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0105] In some embodiments, the disclosure provide a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti- GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) and a first light chain (LC1) comprising amino acid sequences at least 90%, preferably 100% identical to SEQ ID NO: 9 and 10, respectively.

[0106] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the invention comprises a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2), wherein the VH2 and the VL2 comprise amino acid sequences at least 90% identical to SEQ ID NO: 17 and 18.

[0107] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the invention comprises comprises a spFv2 comprising an amino acid sequence at least 90% identical, preferably 100%, to SEQ ID NO: 19.

[0108] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the invention comprises a second heavy chain (HC2) comprising an amino acid sequence at least 90%, preferably 100%, identical to SEQ ID NO: 20.

[0109] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the invention comprises a HC1 and LC1 comprising amino acid sequences at least 90%, preferably 100%, identical to SEQ ID NOs: 9 and 10, respectively, and comprises a HC2 comprising an amino acid sequence at least 90%, preferably 100%, identical to SEQ ID NO: 20.

[0110] In some embodiments, the disclosure provides methods of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that specifically binds to GUCY2C and a second antigen binding domain that specifically binds to CD3, wherein the anti- GUCY2CxCD3 antibody comprises(i) a VH1 having at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 7, and / or a VL1 having at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 8; and / or(ii) a HC1 having at least 90%, at least 95%, or at least 98% sequence identity to an amino acid sequence of SEQ ID NO: 9 and a LC1 having at least 90%, at least 95%, or at least 98% sequence identity to an amino acid sequence of SEQ ID NO: 10.

[0111] In some embodiments, the disclosure provides methods of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject an anti-GUCY2CxCD3 antibody that comprises a first binding domain that binds GUCY2C and a second binding domain that binds CD3, wherein the anti- GUCY2CxCD3 antibody comprises(i) a VH1 and VL1 having an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 7 and SEQ ID NO: 8, respectively; and / or(ii) a HC1 and LC1 having an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 9 and SEQ ID NO: 10, respectively;(iii) a VH2 and VL2 having an amino acid sequence at least 90% identical to SEQ ID NO: 17 and 18, respectively;(iv) a spFv2 having an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 19; and / or(v) a HC2 having an amino acid sequence at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 20.Route of administration

[0112] The methods of the instant invention include any route of administration of the antibody of the present invention that achieves the intended purpose. Any suitable route of administration considered appropriate by a person of skill in the art can be used to administer the antibody or a pharmaceutical composition including the antibody used in the method of treatment of the disclosure. For example, administration may be accomplished by a number of different routes including, subcutaneous route (for example subcutaneous injection), or intravenous route (for example intravenous injection).

[0113] In some embodiments, the anti-GUCY2CxCD3 antibody used in the methods of the disclosure is administered using the subcutaneous route of administration.

[0114] In some embodiments, the anti-GUCY2CxCD3 antibody used in the methods of the disclosure is administered using the intravenous route of administration.

[0115] In some embodiments, the disclosure comprises a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9 and a first light chain (LC1) of SEQ ID NO: 10; and wherein the anti- GUCY2CxCD3 antibody is administered to the subject subcutaneously.

[0116] In some embodiments, the disclosure comprises a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9 a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; and wherein the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously.

[0117] In some embodiments, the disclosure comprises a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that bindsspecifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9 and a first light chain (LC1) of SEQ ID NO: 10; and wherein the anti- GUCY2CxCD3 antibody is administered to the subject intravenously.

[0118] In some embodiments, the disclosure comprises a method of treating a cancer of the digestive system, preferably of the gastrointestinal system in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9 a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; and wherein the anti-GUCY2CxCD3 antibody is administered to the subject intravenously.Dosages

[0119] Dosages to be administered depend on individual needs, on the desired effect and the chosen route of administration. It is understood that the dosage administered will be dependent upon the age, sex, health, and weight of the recipient, concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The total dose required for each treatment may be administered by multiple doses or in a single dose. The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

[0120] As used herein, the term “dose” refers to a treatment dose or to a step-up dose unless otherwise specified.Treatment dose

[0121] In some embodiment, the method of the disclosure comprises administering to the subject a treatment dose of the anti-GUCY2CxCD3 antibody.

[0122] A “treatment dose” may refer to a dose of anti-GUCY2CxCD3 antibody that prevents disease progression.

[0123] In some embodiments, the anti-GUCY2CxCD3 antibody is administered at a treatment dose that is (greater than zero and) less than about 1000 mg (per dose of administration).

[0124] In some embodiments, the anti-GUCY2CxCD3 antibody is administered at a treatment dose that is (greater than zero and) less than about 500 mg (per dose of administration).

[0125] In some embodiments, the anti-GUCY2CxCD3 antibody is administered at a treatment dose that is (greater than zero and) less than about 100 mg (per dose of administration).

[0126] In some embodiments, the anti-GUCY2CxCD3 antibody is administered at a treatment dose ranging from about 1 mg to about 50 mgs (per dose of administration).

[0127] In some embodiments, the anti-GUCY2CxCD3 antibody is administered at a treatment dose ranging from about 2 mg to about 50 mgs (per dose of administration).

[0128] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the disclosure is administered subcutaneously at a treatment dose of (greater than zero and) less than about 1000 mg.

[0129] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the disclosure is administered subcutaneously at a treatment dose of (greater than zero and) less than about 500 mg.

[0130] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the disclosure is administered subcutaneously at a treatment dose of (greater than zero and) less than about 100 mg.

[0131] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the disclosure is administered subcutaneously at a treatment dose ranging from about 2 mg to about 50 mgs.

[0132] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the disclosure is administered subcutaneously at a treatment dose ranging from about 1 mg to about 50 mgs.

[0133] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the disclosure is administered intravenously at a treatment dose of (greater than zero and) less than 1000 mg.

[0134] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the disclosure is administered intravenously at a treatment dose of (greater than zero and) less than 500 mg.

[0135] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the disclosure is administered intravenously at a treatment dose of (greater than zero and) less than 100 mg.

[0136] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the disclosure is administered intravenously at a treatment dose ranging from about 1 mg to about 50 mgs.

[0137] In some embodiments, the anti-GUCY2CxCD3 antibody used in the method of the disclosure is administered intravenously at a treatment dose ranging from about 2 mg to about 50 mgs.

[0138] In some embodiments, the treatment dose is a flat dose.

[0139] In some embodiments, the method of the invention comprises more than one administration of the treatment dose to the subject. In some embodiments, the method of the instant invention comprises at least one administration of the treatment dose to the subject.

[0140] In some embodiments, the treatment dose is administered more than once.

[0141] In some embodiments, the treatment dose is administered subcutaneously more than once.

[0142] In some embodiments, the treatment dose is administered intravenously more than once.

[0143] In some embodiments, the treatment dose is administered several times at a regular frequency.

[0144] In some embodiments, the treatment dose is administered subcutaneously, several times at a regular frequency.

[0145] In some embodiments, the treatment dose is administered intravenously, several times at a regular frequency.

[0146] In some embodiments, the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 9 weeks.

[0147] In some embodiments, the treatment dose is administered subcutaneously, once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 9 weeks.

[0148] In some embodiments, the treatment dose is administered intravenously, once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 9 weeks

[0149] In some embodiments, the treatment dose is administered once every 2 weeks.

[0150] In some embodiments, the treatment dose is administered subcutaneously, once every 2 weeks.

[0151] In some embodiments, the treatment dose is administered intravenously, once every 2 weeks.

[0152] In some embodiments, the method of the disclosure comprises administering to a human subject a treatment dose of the anti-GUCY2CxCD3 antibody, wherein the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 9 weeks.

[0153] In some embodiments, the method of the disclosure comprises administering a treatment dose of the anti-GUCY2CxCD3 antibody, wherein the treatment dose is administered subcutaneously once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 9 weeks.

[0154] In some embodiments, the method of the disclosure comprises administering a treatment dose of the anti-GUCY2CxCD3 antibody, wherein the treatment dose is administered intravenously once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 9 weeks.

[0155] In some embodiments, the treatment dose of the anti-GUCY2CxCD3 antibody is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 9 weeks, over a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks or, alternatively or additionally, at least one of 1, 2, 3, 4, 5, 6,, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years.

[0156] In some embodiments, the treatment dose of the anti-GUCY2CxCD3 antibody used in the method of the disclosure is administered subcutaneously once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 9 weeks over a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks or, alternatively or additionally, at least one of 1, 2, 3, 4, 5, 6„ 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years.

[0157] In some embodiments, the treatment dose of the anti-GUCY2CxCD3 antibody used in the method of the disclosure is administered intravenously once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 9 weeks over a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks or, alternatively or additionally, at least one of 1, 2, 3, 4, 5, 6„ 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years.

[0158] In some embodiments, the treatment dose prevents disease progression in the subject.

[0159] In some embodiments the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein: a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose is less than about 1000 mg; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0160] In some embodiments the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, ,in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein: a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose is less than about 500 mg; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0161] In some embodiments the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein: a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose is less than about 100 mg; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0162] In some embodiments the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein: a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose ranges from about 2 mgs to about 50 mgs; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0163] In some embodiments the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein: a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose ranges from about 1 mgs to about 50 mgs; and / ord. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0164] In some embodiments the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein: a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject intravenously; c. the treatment dose is less than about 1000 mg; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0165] In some embodiments the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein: a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject intravenously; c. the treatment dose is less than about 500 mg; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0166] In some embodiments the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3T1antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein: a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject intravenously; c. the treatment dose is less than about 100 mg; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0167] In some embodiments the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein: a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject intravenously; c. the treatment dose ranges from about 2 mgs to about 50 mgs; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0168] In some embodiments the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein: a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject intravenously; c. the treatment dose ranges from about 1 mgs to about 50 mgs; and / ord. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.Step-up dose

[0169] The disclosure also provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering one or more step-up doses of the anti-GUCY2CxCD3 antibody prior to administering any treatment dose of the anti-GUCY2CxCD3 antibody, wherein the one or more step-up doses are lower than the treatment dose.

[0170] The step-up doses are designed to prevent, reduce or ameliorate cytokine storm or cytokine release syndrome (CRS) in a subject. Reducing "cytokine storm" in a subject refers to reducing the effect of a cytokine cascade or hypercytokinemia, wherein a negative immune reaction may be caused by, but is not limited to, a positive feedback loop between cytokines and white blood cells, and / or highly elevated levels of various cytokines triggered by the administration of the antibody.

[0171] In some embodiments, the one or more step-up doses are (greater than zero and) less than about 1000 mg but are lower than the treatment dose.

[0172] In some embodiments, the one or more step-up doses are (greater than zero and) less than about 500 mg but are lower than the treatment dose.

[0173] In some embodiments, the one or more step-up doses are (greater than zero and) less than about 100 mg but are lower than the treatment dose.

[0174] In some embodiments, the one or more step-up doses range between about 1 mg and about 50 mg but are lower than the treatment dose.

[0175] In some embodiments, the one or more step-up doses range between about 2 mg and about 50 mg but are lower than the treatment dose.

[0176] In some embodiments, the one or more step-up doses range between about 1 mg and about 50 mg and the treatment dose ranges from about 1 mg to about 50 mg or from about 2 mg to about 50 mg.

[0177] In some embodiments, the one or more step-up doses lower the risk of cytokine release syndrome (CRS) in the subject.

[0178] In some embodiments, the one or more step-up dose lower the risk of cytokine release syndrome (CRS) and are (greater than zero and) less than about 1000 mgs.

[0179] In some embodiments, the one or more step-up dose lower the risk of cytokine release syndrome (CRS) and are (greater than zero and) less than about 500 mgs.

[0180] In some embodiments, the one or more step-up dose lower the risk of cytokine release syndrome (CRS) and are (greater than zero and) less than about 100 mgs.

[0181] In some embodiments, the one or more step-up dose lower the risk of cytokine release syndrome (CRS) and ranges from about 1 mg to about 50 mg.

[0182] In some embodiments, the one or more step-up dose lower the risk of cytokine release syndrome (CRS) and range from about 2 mg to about 50 mg.

[0183] In some embodiments, each step-up dose is about 1.2 to about 30 times greater than the prior step-up dose.

[0184] In certain embodiments, the each step-up dose is about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 7 , about 8 , about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 times greater than the prior step-up dose.

[0185] In certain embodiments, each step-up dose is about 3.2 fold greater than the prior step-up dose.

[0186] In some embodiments, each step up dose is about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, 5 about 5%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200%, about 500%, about 1000%, about 1500%, about 2000%, about 2500% or about 3000% greater than the prior step-up dose.

[0187] In some embodiments, the one or more step-up doses are each administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times). In some embodiments, the one or more step-up doses are administered before the treatment dose, and the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 9 weeks.

[0188] In some embodiments, the one or more step-up doses are administered before the treatment dose, and the treatment dose is administered every once every 2 weeks.

[0189] In some embodiments, the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as1 week, before the first administration of the treatment dose (or prior the first administration of the treatment dose).

[0190] In some embodiments, the method of the disclosure comprises administering one or more step-up doses of the anti-GUCY2CxCD3 antibody before administering the treatment dose of the anti-GUCY2CxCD3 antibody, wherein the first step-up dose is administered 2-15 days, such as 1 week, before the second step-up dose; wherein each subsequent step-up doses are administered 2-15 days, such as 1 week, from each other, and wherein the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose.

[0191] In some embodiments, the method of the disclosure comprises administering one or more step-up doses of the anti-GUCY2CxCD3 antibody before administering the treatment dose of the anti-GUCY2CxCD3 antibody, wherein the first step-up dose is administered 1 week before the second step-up dose; wherein each subsequent step-up doses are administered 1 week from each other, and wherein the last step-up dose is administered 1 week before the first treatment dose.

[0192] In some embodiments, the method of the disclosure comprises administering one or more step-up doses of the anti-GUCY2CxCD3 antibody before administering the treatment dose of the anti-GUCY2CxCD3 antibody, wherein the first step-up dose is administered 2 weeks before the second step-up dose; wherein each subsequent step-up doses are administered 2 weeks from each other, and wherein the last step-up dose is administered 2 weeks before the first treatment dose.

[0193] In some embodiments, the method of the disclosure comprises administering one or more step-up doses of the anti-GUCY2CxCD3 antibody before administering the treatment dose of the anti-GUCY2CxCD3 antibody, wherein the first step-up dose is administered 2-15 days, such as 1 week, before the second step-up dose; wherein each subsequent step-up doses are administered 2-15 days, such as 1 week, from each other, wherein the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; and wherein the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0194] In some embodiments, the method of the disclosure comprises administering one or more step-up doses of the anti-GUCY2CxCD3 antibody before administering the treatment dose of the anti-GUCY2CxCD3 antibody, wherein the first step-up dose is administered 2-15 days, such as 1 week, before the second step-up dose; wherein eachsubsequent step-up doses are administered 2-15 days, such as 1 week, from each other, wherein the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; wherein the treatment dose is administered once every 2 weeks.

[0195] In some embodiments, the one or more step-up doses are administered once per week or once every two weeks, and the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, or once every 9 weeks.

[0196] In some embodiments, the one or more step-up doses are administered once per week or once every two weeks, and the treatment dose is administered once every 2 weeks.

[0197] In some embodiments, the method of the disclosure comprises administering one or more step-up doses of the anti-GUCY2CxCD3 antibody before administering the treatment dose of the anti-GUCY2CxCD3 antibody, wherein the one or more step-up doses are administered once per week or once every two weeks and the treatment dose is administered every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 week over a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks or, alternatively or additionally, at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years.

[0198] In some embodiments, the method of the disclosure comprises administering one or more step-up doses of the anti-GUCY2CxCD3 antibody before administering the treatment dose of the anti-GUCY2CxCD3 antibody, wherein the one or more step-up doses are administered once per week or once every two weeks and the treatment dose is administered once every 2 weeks over a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks or, alternatively or additionally, at least one of 1, 2, 3, 4, 5, 6„ 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years.

[0199] In some embodiments, the one or more step-up doses are administered subcutaneously.

[0200] In some embodiments, the one or more step-up doses are administered intravenously.

[0201] In some embodiments, the disclosure provides a method treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up dose of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and treatment dose are administered subcutaneously; c. the one or more step-up doses and the treatment dose are (greater than zero and) less than about 1000 mg; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step up doses lower the risk of cytokine release syndrome (CRS) in the subject; f. the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0202] In some embodiments, the disclosure provides a method treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up dose of the anti- GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and treatment dose are administered subcutaneously; c. the one or more step-up doses and the treatment dose are (greater than zero and) less than about 500 mg; d. the one or more step-up doses are lower than the treatment dose;e. the one or more step up doses lower the risk of cytokine release syndrome (CRS) in the subject; f. the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0203] In some embodiments, the disclosure provides a method treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up dose of the anti- GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and treatment dose are administered subcutaneously; c. the one or more step-up doses and the treatment dose are (greater than zero and) less than about 100 mg; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step up doses lower the risk of cytokine release syndrome (CRS) in the subject; f. the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0204] In some embodiments, the disclosure provides a method treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up dose of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and treatment dose are administered subcutaneously; c. the one or more step-up doses and the treatment dose range from about 2 mg to about 50 mg; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step up doses lower the risk of cytokine release syndrome (CRS) in the subject; f. the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0205] In some embodiments, the disclosure provides a method treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up dose of the anti- GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and treatment dose are administered subcutaneously; c. the one or more step-up doses and the treatment dose range from about 1 mg to about 50 mg; d. the one or more step-up doses are lower than the treatment dose;e. the one or more step up doses lower the risk of cytokine release syndrome (CRS) in the subject; f. the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0206] In some embodiments, the disclosure provides a method treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up dose of the anti- GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and treatment dose are administered subcutaneously; c. the one or more step-up doses range from about 1 mg to about 50 mg and the treatment dose ranges from about 1 mg to about 50 mg or from about 2 mg to about 50 mg; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step up doses lower the risk of cytokine release syndrome (CRS) in the subject; f. the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0207] In some embodiments, the disclosure provides a method treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up dose of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and treatment dose are administered intravenously; c. the one or more step-up doses and the treatment are (greater than zero and) less than about 1000 mg; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step up doses lower the risk of cytokine release syndrome (CRS) in the subject; f. the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0208] In some embodiments, the disclosure provides a method treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up dose of the anti- GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and treatment dose are administered intravenously; c. the one or more step-up doses and the treatment are (greater than zero and) less than about 500 mg; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step up doses lower the risk of cytokine release syndrome (CRS) in the subject;f. the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0209] In some embodiments, the disclosure provides a method treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up dose of the anti- GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and treatment dose are administered intravenously; c. the one or more step-up doses and the treatment are (greater than zero and) less than about 100 mg; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step up doses lower the risk of cytokine release syndrome (CRS) in the subject; f. the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0210] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up dose of the anti- GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, whereina. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and treatment dose are administered intravenously; c. the one or more step-up doses and the treatment range from about 2 mg to about 50 mg; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step up doses lower the risk of cytokine release syndrome (CRS) in the subject; f. the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0211] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up dose of the anti- GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and treatment dose are administered intravenously; c. the one or more step-up doses and the treatment range from about 1 mg to about 50 mg; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step up doses lower the risk of cytokine release syndrome (CRS) in the subject; f. the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; andg. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0212] In some embodiments, the disclosure provides a method treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up dose of the anti- GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and treatment dose are administered intravenously; c. the one or more step-up doses range from about 1 mg to about 50 mg and the treatment dose ranges from about 1 mg to about 50 mg or from about 2 mg to about 50 mg; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step up doses lower the risk of cytokine release syndrome (CRS) in the subject; f. the one or more step-up doses are administered 2-15 days, such as 1 week from each other and the last step-up dose is administered 2-15 days, such as 1 week, before the first treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0213] In some embodiments, the method comprises administering one or two step-up doses of the anti-GUCY2CxCD3 antibody prior to administering any treatment dose of the anti-GUCY2CxCD3 antibody, wherein the one or two step-up doses are lower than the treatment dose.

[0214] In some embodiments, the method of the disclosure comprises administering two step-up doses and a treatment dose.

[0215] In some embodiments, the method comprises administering two step-up doses of the anti-GUCY2CxCD3 antibody prior to administering any treatment dose of the anti- GUCY2CxCD3 antibody, wherein the two step-up doses are lower than the treatment dose.

[0216] In some embodiments, the two step-up doses are less than about 1000 mg.

[0217] In some embodiments, the two step-up doses are less than about 500 mg.

[0218] In some embodiments, the two step-up doses are less than about 100 mg.

[0219] In some embodiments, the two step-up doses range from about 1 mg to about 50 mg.

[0220] In some embodiments, the two step-up doses range from about 2 mg to about 50 mg.

[0221] In some embodiments, the two step-up doses lower the risk of cytokine release syndrome (CRS) in the subject.

[0222] In some embodiments, the two step-up doses lower the risk of cytokine release syndrome (CRS) in the subject, are less than about 1000 mgs and are lower than the treatment dose.

[0223] In some embodiments, the two step-up doses lower the risk of cytokine release syndrome (CRS) in the subject, are less than about 500 mgs and are lower than the treatment dose.

[0224] In some embodiments, the two step-up doses lower the risk of cytokine release syndrome (CRS) in the subject, are less than about 100 mgs and are lower than the treatment dose.

[0225] In some embodiments, the two step-up doses lower the risk of cytokine release syndrome (CRS) in the subject, range from about 1 mg to about 50 mg and are lower than the treatment dose.

[0226] In some embodiments, the two step-up doses lower the risk of cytokine release syndrome (CRS) in the subject, range from about 2 mg to about 50 mg and are lower than the treatment dose.

[0227] In some embodiments, the first step up dose is about 1.2 to about 30 times lower than the second the step-up dose.

[0228] In certain embodiments, the first step-up dose is about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 7 , about 8 , about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 times lower than the second step-up dose.

[0229] In certain embodiments, the first step-up dose is about 3.2 times lower than the second step-up dose.

[0230] In some embodiments, the first step-up dose is about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, 5 about 5%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200%, about 500%, about 1000%, about 1500%, about 2000%, about 2500% or about 3000% lower than the second step-up dose.

[0231] In some embodiments, the two step-up doses and the treatment dose are (greater than zero and) less than about 1000 mgs and the step-up doses are lower than the treatment dose.

[0232] In some embodiments, the two step-up doses and the treatment dose are (greater than zero and) less than about 500 mgs and the step-up doses are lower than the treatment dose.

[0233] In some embodiments, the two step-up doses and the treatment dose are (greater than zero and) less than about 100 mgs and the step-up doses are lower than the treatment dose.

[0234] In some embodiments, the two step-up doses and the treatment dose range from about 1 mg to about 50 mg and the step-up doses are lower than the treatment dose.

[0235] In some embodiments, the two step-up doses and the treatment dose range from about 2 mg to about 50 mg and the step-up doses are lower than the treatment dose.

[0236] In some embodiments, the two step-up doses range from about 1 mg to about 50 mg and the treatment dose ranges from about 1 mg to about 50 mg or from about 2 mg to about 50 mg.

[0237] In some embodiments, the two step-up doses are administered subcutaneously.

[0238] In some embodiments, the two step-up doses are administered intravenously.

[0239] In some embodiments, the method of the disclosure comprises administering a first step-up dose of the anti-GUCY2CxCD3 antibody and a second step-up dose of the anti- GUCY2CxCD3 antibody, wherein the first step-dose is lower than the second step-up dose and is administered 2-15 days, such as 1 week, prior to the second step-up dose and wherein the second step-up dose is lower than the treatment dose and is administered 2-15 days, such as 1 week, prior to the treatment dose (or prior the first administration of the treatment dose).

[0240] In some embodiments, the method comprises administering two step-up doses, wherein the two step-up doses are administered before the treatment dose, and the treatmentdose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0241] In some embodiments, the method comprises administering two step-up doses, wherein the two step-up doses are administered before the treatment dose, and the treatment dose is administered once every 2 weeks.

[0242] In some embodiments, the method of the disclosure comprises administering a first step-up dose and a second step-up dose of the anti-GUCY2CxCD3 antibody, wherein the first step-dose is lower than the second step-up dose and is administered 2-15 days, such as 1 week, prior to the second step-up dose; wherein the second step-up dose is lower than the treatment dose and administered 2-15 days, such as 1 week, prior to the treatment dose (or prior the first administration of the treatment dose); and wherein the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0243] In some embodiments, the method of the disclosure comprises administering a first step-up dose and a second step-up dose of the anti-GUCY2CxCD3 antibody, wherein the first step-dose is lower than the second step-up dose and is administered 2-15 days, such as 1 week, prior to the second step-up dose; wherein the second step-up dose is lower than the treatment dose and is administered 2-15 days, such as 1 week, prior to the treatment dose (or prior the first administration of the treatment dose); and wherein the treatment dose is administered once every 2 weeks.

[0244] In some embodiments, the method of the disclosure comprises administering a first step-up dose and a second step-up dose of the anti-GUCY2CxCD3 antibody, wherein the first step-dose is lower than the second step-up dose and is administered 2-15 days, such as 1 week, prior to the second step-up dose; wherein the second step-up dose is lower than the treatment dose and is administered 2-15 days, such as 1 week, prior to the treatment dose (or prior the first administration of the treatment dose); and wherein the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks over a period of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks or, alternatively or additionally, at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 years.

[0245] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein themethod comprises administering to the subject two step-up doses of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the two step-up doses and treatment dose are administered subcutaneously; b. the two step-up doses and the treatment dose are (greater than zero and) lower than about 1000 mgs; c. the first step-up dose is lower than the second step up dose and the second step- up dose is lower than the treatment dose; d. the two step up doses lower the risk of cytokine release syndrome (CRS) in the subject; e. the first step-up dose is administered 2-15 days, such as 1 week prior to the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week prior to the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0246] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the two step-up doses and treatment dose are administered subcutaneously; b. the two step-up doses and the treatment dose are (greater than zero and) lower than about 500 mgs; c. the first step-up dose is lower than the second step up dose and the second step-up dose is lower than the treatment dose; d. the two step up doses lower the risk of cytokine release syndrome (CRS) in the subject; e. the first step-up dose is administered 2-15 days, such as 1 week prior to the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week prior to the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0247] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the two step-up doses and treatment dose are administered subcutaneously; b. the two step-up doses and the treatment dose are (greater than zero and) lower than about 100 mgs; c. the first step-up dose is lower than the second step up dose and the second step-up dose is lower than the treatment dose; d. the two step up doses lower the risk of cytokine release syndrome (CRS) in the subject; e. the first step-up dose is administered 2-15 days, such as 1 week prior to the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week prior to the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0248] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the two step-up doses and treatment dose are administered subcutaneously; b. the two step-up doses and the treatment dose range from about 2 mg to about 50 mg; c. the first step-up dose is lower than the second step up dose and the second step-up dose is lower than the treatment dose; d. the two step up doses lower the risk of cytokine release syndrome (CRS) in the subject; e. the first step-up dose is administered 2-15 days, such as 1 week prior to the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week prior to the treatment dose; and / orf. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0249] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein g. the two step-up doses and treatment dose are administered subcutaneously; h. the two step-up doses and the treatment dose range from about 1 mg to about 50 mg; i. the first step-up dose is lower than the second step up dose and the second step-up dose is lower than the treatment dose; j . the two step up doses lower the risk of cytokine release syndrome (CRS) in the subject; k. the first step-up dose is administered 2-15 days, such as 1 week prior to the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week prior to the treatment dose; and / or l. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0250] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein m. the two step-up doses and treatment dose are administered subcutaneously; n. the two step-up doses range from about 1 mg to about 50 mg and the treatment dose ranges from about 1 mg to about 50 mg or from about 2 mg to about 50 mg; o. the first step-up dose is lower than the second step up dose and the second step-up dose is lower than the treatment dose; p. the two step up doses lower the risk of cytokine release syndrome (CRS) in the subject;q. the first step-up dose is administered 2-15 days, such as 1 week prior to the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week prior to the treatment dose; and / or r. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0251] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the two step-up doses and treatment dose are administered intravenously; b. the two step-up doses and the treatment dose are (greater than zero and) less than about 1000 mgs; c. the first step-up dose is lower than the second step up dose and the second step- up dose is lower than the treatment dose; d. the two step up doses lower the risk of cytokine release syndrome (CRS) in the subject; e. the first step-up dose is administered 2-15 days, such as 1 week prior to the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week prior to the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0252] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the two step-up doses and treatment dose are administered intravenously; b. the two step-up doses and the treatment dose are (greater than zero and) less than about 500 mgs; c. the first step-up dose is lower than the second step up dose and the second step- up dose is lower than the treatment dose;d. the two step up doses lower the risk of cytokine release syndrome (CRS) in the subject; e. the first step-up dose is administered 2-15 days, such as 1 week prior to the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week prior to the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0253] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the two step-up doses and treatment dose are administered intravenously; b. the two step-up doses and the treatment dose are (greater than zero and) less than about 100 mgs; c. the first step-up dose is lower than the second step up dose and the second step- up dose is lower than the treatment dose; d. the two step up doses lower the risk of cytokine release syndrome (CRS) in the subject; e. the first step-up dose is administered 2-15 days, such as 1 week prior to the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week prior to the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0254] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein a. the two step-up doses and treatment dose are administered intravenously; b. the two step-up doses and the treatment dose range from about 2 mg to about 50 mg;c. the first step-up dose is lower than the second step up dose and the second step- up dose is lower than the treatment dose; d. the two step up doses lower the risk of cytokine release syndrome (CRS) in the subject; e. the first step-up dose is administered 2-15 days, such as 1 week prior to the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week prior to the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0255] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein g. the two step-up doses and treatment dose are administered intravenously; h. the two step-up doses and the treatment dose range from about 1 mg to about 50 mg; i. the first step-up dose is lower than the second step up dose and the second step- up dose is lower than the treatment dose; j . the two step up doses lower the risk of cytokine release syndrome (CRS) in the subject; k. the first step-up dose is administered 2-15 days, such as 1 week prior to the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week prior to the treatment dose; and / or l. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0256] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses of the anti-GUCY2CxCD3 antibody, and a treatment dose of the anti-GUCY2CxCD3 antibody, wherein m. the two step-up doses and treatment dose are administered intravenously;n. the two step-up doses range from about 1 mg to about 50 mg and the treatment dose range ranges from about 1 mg to about 50 mg or from about 1 mg to about 50 mg (per dose of administration); o. the first step-up dose is lower than the second step up dose and the second step- up dose is lower than the treatment dose; p. the two step up doses lower the risk of cytokine release syndrome (CRS) in the subject; q. the first step-up dose is administered 2-15 days, such as 1 week prior to the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week prior to the treatment dose; and / or r. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0257] In some embodiments, the method comprises administering only one step-up dose of the anti-GUCY2CxCD3 antibody prior to administering any treatment dose of the anti- GUCY2CxCD3 antibody, wherein the only one dose is lower than the treatment dose.

[0258] In some embodiments, the only one step-up dose is (greater than zero and) less than about 1000 mg.

[0259] In some embodiments, the only one step-up dose is (greater than zero and) less than about 500 mg.

[0260] In some embodiments, the only one step-up dose is (greater than zero and) less than about 100 mg.

[0261] In some embodiments, the only one step-up dose ranges from about 1 mg to about 50 mg.

[0262] In some embodiments, the only one step-up dose ranges from about 2 mg to about 50 mg.

[0263] In some embodiments, the only one step-up dose lowers the risk of cytokine release syndrome (CRS) in the subject.

[0264] In some embodiments, the only one step-up dose lowers the risk of cytokine release syndrome (CRS) in the subject and is (greater than zero and) less than about 1000 mgs.

[0265] In some embodiments, the only one step-up dose lowers the risk of cytokine release syndrome (CRS) in the subject and is (greater than zero and) less than about 500 mgs.

[0266] In some embodiments, the only one step-up dose lowers the risk of cytokine release syndrome (CRS) in the subject and is (greater than zero and) less than about 100 mgs.

[0267] In some embodiments, the only one step-up dose lowers the risk of cytokine release syndrome (CRS) in the subject and ranges from about 1 mg to about 50 mg.

[0268] In some embodiments, the only one step-up dose lowers the risk of cytokine release syndrome (CRS) in the subject and ranges from about 2 mg to about 50 mg.

[0269] In some embodiments, the only one step-up dose is administered subcutaneously.

[0270] In some embodiments, the only one step-up dose is administered intravenously.

[0271] In some embodiments, the method comprises administering only one step-up dose, preferably 2-15 days, such as 1 week, prior to the treatment dose (or prior the first administration of the treatment dose).

[0272] In some embodiments, the only one step up dose is administered preferably 2-15 days, such as 1 week, prior to the treatment dose (or prior the first administration of the treatment dose).

[0273] In some embodiments, the method comprises administering only one step-up dose, wherein the only one step-up dose is administered before the treatment dose, preferably 2-15 days, such as 1 week, prior to the treatment dose and the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0274] In some embodiments, the method comprises administering only one step-up dose, wherein the only one step-up dose is administered before the treatment dose, and the treatment dose is administered once every 2 weeks.

[0275] In some embodiments, the method of the disclosure comprises administering only one step-up dose of the anti-GUCY2CxCD3 antibody before administering the treatment dose of the anti-GUCY2CxCD3 antibody, and wherein the one step-up dose is administered 2 weeks before the first treatment dose.

[0276] In some embodiments, the method of the disclosure comprises administering only one step-up dose of the anti-GUCY2CxCD3 antibody before administering the treatment dose of the anti-GUCY2CxCD3 antibody, wherein the one step-up dose is administered 2weeks before the first treatment dose, and wherein the treatment dose is administered once every 2 weeks.

[0277] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose are less than about 1000 mgs; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0278] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose are less than about 500 mgs; d. the only one step-up is lower than the treatment dose;e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0279] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose are less than about 100 mgs; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0280] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, whereina. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose range from about 2 mg to about 50 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0281] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose range from about 1 mg to about 50 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / org. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0282] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose ranges from about 1 mg to about 50 mg and the treatment dose ranges from about 1 mg to about 50 mg or from about 2 mg to about 50 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0283] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20;b. the only one step-up dose and the treatment dose are administered intravenously; c. the only one step-up dose and the treatment dose are less than about 1000 mgs; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0284] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered intravenously; c. the only one step-up dose and the treatment dose are less than about 500 mgs; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0285] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose ofan anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered intravenously; c. the only one step-up dose and the treatment dose are less than about 100 mgs; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0286] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered intravenously; c. the only one step-up dose and the treatment dose range from about 2 mg to about 50 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times);f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0287] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered intravenously; c. the only one step-up dose and the treatment dose range from about 1 mg to about 50 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0288] In some embodiments, the disclosure provides a method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, whereina. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered intravenously; c. the only one step-up dose ranges from about 1 mg to about 50 mg and the treatment dose ranges from about 1 mg to about 50 mg or from about 2 mg to about 50 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.Cancer of the digestive system

[0289] “ Cancer” refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. A “cancer” or “cancer tissue” can include a tumor.

[0290] “ Cancer” is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathology type or stage of invasiveness. Examples of cancers include solid tumors, hematological malignancies, soft tissue tumors, and metastatic lesions. Exemplary solid tumors include malignancies, e.g., sarcomas, and carcinomas (including adenocarcinomas and squamous cell carcinomas) of the various organ systems, such as those affecting prostate, liver, lung, breast, lymphoid, gastrointestinal (e.g., colon), genitourinary tract (e.g., renal, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, a rectal cancer, a renal-cell carcinoma, a liver cancer, a non-small cell carcinoma of the lung, a cancer of the small intestine and a cancer of the esophagus.Squamous cell carcinomas include malignancies, e.g., in the lung, esophagus, skin, head and neck region, oral cavity, anus, and cervix.

[0291] The cancer can be a hyperproliferative condition or disorder, a solid tumor, a neovasculature, a soft tissue tumor, or a metastatic lesion.

[0292] “ Cancer of the digestive system” is meant to include all types of cancer that start in any part of the digestive tract.

[0293] “ Gastrointestinal cancer” is meant to include all types of cancer that start in the digestive system.

[0294] “ Colorectal cancer” is meant to include all types of cancer that start in the colon or rectum.

[0295] “Metastatic colorectal cancer” is understood in accordance with its ordinary meaning in the field and typically refers to an advanced colorectal cancer that has spread to other areas outside the colon and rectum. Commonly, this type of cancer spreads to the lymph nodes and liver, but it may spread to other parts of the body, such as the brain.

[0296] “ Gastric cancer” is understood in accordance with its ordinary meaning in the field and typically refers to a cancer that originates in the stomach, preferably in the lining of the stomach.

[0297] “Metastatic gastric cancer” is understood in accordance with its ordinary meaning in the field and typically refers to an advanced gastric cancer that has spread from the stomach to other parts of the body. Common sites of metastasis for gastric cancer include the liver, lungs, and peritoneum (the lining of the abdominal cavity).

[0298] In some embodiments, the cancer is a cancer of the digestive system, preferably of the gastrointestinal system.

[0299] In some embodiments, the cancer of the digestive system, preferably of the gastrointestinal system, is a colorectal cancer.

[0300] In some embodiments, the cancer of the digestive system, preferably of the gastrointestinal system, is a metastatic colorectal cancer.

[0301] In some embodiments, the cancer of the digestive system, preferably of the gastrointestinal system, is unresectable metastatic colorectal cancer.

[0302] In some embodiments, the cancer of the digestive system, preferably of the gastrointestinal system, is a gastric cancer.

[0303] In some embodiments, the cancer of the digestive system, preferably of the gastrointestinal system, is a primary or metastatic gastric cancer.

[0304] In certain embodiments, the administration of the anti-GUCY2CxCD3 antibody does not trigger the risk of a treatment emergent adverse event.

[0305] As used herein, the term “adverse event” (AE) refers to any untoward medical occurrence in a patient administered a pharmaceutical product and which does not necessarily have a causal relationship with the treatment. According to embodiments of the invention, AEs are rated on a 3-point scale of increasing severity using the following definitions: mild (grade 1), referring to an AE that is easily tolerated by the subject, which causes minimal discomfort and does not interfere with everyday activities; moderate (grade 2), referring to an AE that is sufficiently discomforting to interfere with normal everyday activities and intervention may be needed; severe (grade 3), referring to an AE that prevents normal everyday activities, and treatment or other intervention is usually needed. Adverse events include cytokine release syndrome (CRS), immune effector cell associated neurotoxicity syndrome (ICANS), systemic administration related reactions (sARRs), immune-related adverse events (irAEs).

[0306] A serious AE (SAE) can be any AE occurring at any dose that results in any of the following outcomes: death, where death is an outcome, not an event; life-threatening, referring to an event in which the patient is at risk of death at the time of the event; it does not refer to an event which could hypothetically have caused death had it been more severe; in patient hospitalization, i.e., an unplanned, overnight hospitalization, or prolongation of an existing hospitalization; persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions; congenital anomaly / birth defect; important medical that may jeopardize the patients or may require medical or surgical intervention to prevent one of the other outcomes listed above (e.g., intensive treatment in an emergency room or at home for allergic bronchospasm or blood dyscrasias or convulsions that do not result in hospitalization).

[0307] In some embodiments, the human subject does not have one or more of the following a. Active (new or progressive) brain metastases, leptomeningeal disease, or untreated spinal cord compression. b. Toxicity from prior anticancer therapy that has not resolved to Grade <1 (except alopecia, vitiligo, Grade <= 2 peripheral neuropathy, or endocrinopathies that are stable on hormone replacement)c. Prior or concurrent second malignancy (other than mCRC) unless natural history or treatment is unlikely to interfere with any study endpoints of safety or the efficacy of the study treatment. c. Received glucocorticoids (doses >10 mg / day prednisone or equivalent) within 7 days prior to the first dose of the anti-GUCY2CxCD3 antibody. e. Received or plans to receive any live, attenuated vaccine within 4 weeks before the dose of the antibody or within 4 weeks after the last step-up dose or last treatment dose of the antibody.

[0308] In some embodiments, the subject does not have one or more of the following: a. Active (new or progressive) brain metastases, leptomeningeal disease, or untreated spinal cord compression. b. Toxicity from prior anticancer therapy that has not resolved to Grade <1 (except alopecia, vitiligo, Grade <= 2 peripheral neuropathy, or endocrinopathies that are stable on hormone replacement) c. A history of Grade >2 immune-related adverse event (AE) on prior immunotherapy. d. Prior or concurrent second malignancy (other than mCRC) unless natural history or treatment is unlikely to interfere with any study endpoints of safety or the efficacy of the study treatment. e. Prior treatment with any other GUCY2C targeted anti-cancer therapy. f. Prior anti-cancer treatments discontinued for less than 3 weeks or 5 half-lives (whichever is shorter) prior to the first dose of study treatment. i. Prior immune checkpoint inhibitors and / or T-cell redirecting bispecific antibodies (eg, CD3 targeting or other bispecific antibodies) discontinued for less than 6 weeks before first dose of study treatment. ii. Prior Car-T therapies less than 12 weeks prior to first dose of the anti- GUCY2CxCD3 antibody iii. Palliative radiation less than 14 days before prior to first dose of the anti GUCY2CxCD3 antibody g. Received glucocorticoids (doses >10 mg / day prednisone or equivalent) within 7 days prior to the first dose of an anti-GUCY2CxCD3 antibody.h. Received or plans to receive any live, attenuated vaccine within 4 weeks before the first dose of the anti-GUCY2CxCD3 antibody or within 4 weeks after the last step-up dose or last treatment dose of the antibody. i. Clinically significant pulmonary compromise defined as requiring continuous supplemental oxygen (>2L / min by nasal cannula) j. Any episode of partial or complete bowel obstruction requiring hospitalization within 30 days prior to first dose of anti-GUCY2CxCD3 antibody k. diverting ileostomy, gastrojejunal stents, colorectal stents, or history of enterocutaneous fistula l. History of peptic ulcer disease in the past 3 months that required endoscopic intervention or blood transfusion m. Active hepatitis B or C. n. Human immunodeficiency virus unless viral load is undetectable and CD4 count is above 300 cells / mm3 on stable highly active antiretroviral therapy (HAART) therapy. o. Evidence of infection requiring parenteral treatment within 7 days before the first dose of anti-GUCY2CxCD3 antibody. p. History of solid organ or hematologic stem cell transplantation q. Active autoimmune disease within the 12 months prior to first dose of anti- GUCY2CxCD3 antibody that requires systemic immunosuppressive medications (eg, chronic corticosteroids, methotrexate, or tacrolimus). r. Any of the following within 6 months prior to first dose: i. Myocardial infarction ii. Severe or unstable angina iii. Coronary artery bypass grafting iv. Clinically significant arrhythmias (eg, ventricular arrhythmias, clinically significant atrial fibrillation with uncontrolled rate) v. New York Heart Association Class III or IV congestive heart failure vi. Myocarditis vii. Cerebrovascular accident or transient ischemic attack viii. Any grade arterial event s. Venous thromboembolic events (eg, pulmonary embolism or intracardiac thrombus) within 1 month prior to first dose of anti-GUCY2CxCD3 antibodyt. Any major surgery within 4 weeks before first dose of anti-GUCY2CxCD3 antibody or has not recovered adequately from surgery. u. Initiation or increase in dose of any guanylate cyclase agonists (eg, linaclotide or plecanatide) within 3 months prior to first dose of anti-GUCY2CxCD3 antibody. v. Known allergies, hypersensitivity, or intolerance to the excipients of GUCY2CxHumanCD3 bispecific antibody. w. Prolonged QT interval (QTc intervals corrected using Fredericia’s formula [QTcF]) >480 msec based on the average of triplicate assessments performed no more than 5 (±3) minutes apart x. Any serious underlying medical conditions or other issue that would impair the ability to receive or tolerate the planned treatment.

[0309] In some embodiments, the subject is a. a subject having histologically or cytologically confirmed diagnosis of colorectal adenocarcinoma (CRC) progressing after 2 or more prior lines of standard therapy in the metastatic / unresectable setting. b. a subject having evaluable or measurable disease per response evaluation criteria in solid tumors RECIST vl.l (or at least one measurable lesion) c. a subject having Eastern Cooperative Oncology Group (ECOG) performance status of 0-1 d. a subject having hematology laboratory parameters within the required range f. a subject having an estimated or measured glomerular filtration rate (GFR) >30 mL / min based on modification of diet in renal disease (MDRD) 4-variable formula.

[0310] In some embodiments, the subject is a. a subject having histologically or cytologically confirmed diagnosis of CRC progressing after 2 or more prior lines of standard therapy in the metastatic / unresectable setting. b. a subject having evaluable or measurable disease per RECIST vl .1 (or at least 1 measurable lesion) c. a subject having Eastern Cooperative Oncology Group (ECOG) performance status of 0-1.d. a subject having hematology laboratory parameters within the following range without prior growth factor support within 3 weeks or transfusion within 7 days before the day the laboratory test was performed: i. Absolute neutrophil count >1000* 109 / L ii. Hemoglobin >8.0 g / dL iii. Platelets >80* 109 / L e. a subject having clinical chemistry laboratory parameters within the following range during the screening period: i. aspartate aminotransferase (AST) and alanine aminotransferase (ALT) <3 x upper limit of normal (ULN) or < 5 x ULN if documented liver involvement by disease ii. Serum total bilirubin <1.5 x ULN; participants with congenital bilirubinemia, such as Gilbert’s syndrome, may enroll if conjugated bilirubin is < 1.5 x ULN f. a subject having an estimated or measured glomerular filtration rate (GFR) >30 mL / min based on modification of diet in renal disease (MDRD) 4-variable formula.

[0311] In some embodiments, the subject has histologically or cytologically confirmed diagnosis of CRC progressing after 2 or more prior lines of standard therapy in the metastatic / unresectable setting.

[0312] In some embodiments, the subject has evaluable or measurable disease per RECIST vl . l.

[0313] In some embodiments, the subject has Eastern Cooperative Oncology Group (ECOG) performance status of 0-1.

[0314] In some embodiments, the subject has hematology laboratory parameters within the following range: a. Absolute neutrophil count >1000* 109 / L; b. Hemoglobin >8.0 g / dL; or c. Platelets >80* 109 / L.

[0315] In some embodiments, the subject has clinical chemistry laboratory parameters within the following range:a. aspartate aminotransferase (AST) and alanine aminotransferase (ALT) <3 x upper limit of normal (ULN) or < 5 x ULN if documented liver involvement by disease; or b. Serum total bilirubin <1.5 x ULN; participants with congenital bilirubinemia, such as Gilbert’s syndrome, may enroll if conjugated bilirubin is < 1.5 x ULN.

[0316] In some embodiments, the subject has an estimated or measured glomerular filtration rate (GFR) >30 mL / min based on modification of diet in renal disease (MDRD) 4- variable formula.

[0317] In some embodiments, the method does not induce a non-hematologic treatment- related toxicity in the subject, wherein the non-hematologic toxicity is one or more of the following: a. Grade 2^3 CRS b. Grade 4 hypertension c. an increased Aspartate aminotransferase (AST) and Alanine Aminotransferase (ALT) within the following range: i. an increase >8x Upper Limit of Normal (ULN) for any duration, if the baseline is within normal limits; ii. an increase >5x ULN for >14 days, if the baseline is within normal limits; iii. ALT or AST value >3x ULN, total bilirubin >2x ULN, and ALP <2x ULN with no alternative etiology if Hy’s Law criteria are met and baseline is within normal limits; iv. an increase >5x baseline for any duration if the baseline AST / ALT is elevated above ULN; v. an increase >3x baseline for >14 days if the baseline AST / ALT is elevated above ULN; or vi. ALT or AST >3x baseline or AST or ALT >8x ULN, whichever is lower, combined with total bilirubin >2x baseline AND >2x ULN, with no alternative etiology, if modified Hy’s Law criteria baseline are met and baseline AST / ALT is elevated above ULN. d. any Grade >3 non-hematological toxicity with the following exceptions:i. First occurrence of Grade 3 systemic administration-related reaction (sARR) that improves to Grade <2 within 72 hours; ii. Grade 3 fatigue or asthenia lasting <7 days with best supportive care; iii. Grade 3 fever in the absence of neutropenia that resolves within 48 hours; iv. Grade 3 nausea, vomiting, constipation, or diarrhea / colitis lasting < 72 hours with best supportive care; v. Grade >3 hypertension that can be controlled by oral medical management and improves to Grade <2 within 72 hours; vi. Grade >3 lipase or amylase increase not associated with clinical or radiological evidence of pancreatitis; vii. Grade >3 pain due to tumor flare that does not improve to Grade <2 with best supportive care within 7 days; or viii. Grade >3 ALP, GGT, or electrolyte abnormalities that resolve spontaneously to Grade <2 or baseline within 7 days, or that respond to best supportive care.

[0318] In some embodiments, the method does not induce a hematologic treatment- related toxicity in the subject, wherein the hematologic toxicity is one or more of the following: a. Febrile neutropenia; b. Grade 4 neutropenia for >7 days despite supportive care according to institutional standards; and / or c. Grade > 3 thrombocytopenia with Grade 2 bleeding or any Grade 4 thrombocytopenia with duration >7 days.

[0319] In some embodiments, the method does not induce a. diarrhea or colitis in the subject; or b. a cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (iCANs) in the subject.

[0320] In some embodiments, the tumor is significantly reduced (or destroyed) in the subject.Compositions

[0321] In some embodiments, the method of the disclosure is practiced by administering a composition comprising a treatment dose of the anti-GUCY2CxCD3 bispecific antibody.

[0322] In the methods of the disclosure, the anti-GUCY2CxCD3 bispecific antibody may also be administered as a pharmaceutically composition that includes a treatment dose of the anti-GUCY2CxCD3 antibody and optionally a pharmaceutically acceptable carrier.

[0323] “Pharmaceutical composition” refers to a composition that results from combining an active ingredient and a pharmaceutically acceptable carrier.

[0324] “Pharmaceutically acceptable carrier” or “excipient” refers to an ingredient in a pharmaceutical composition, other than the active ingredient, which is nontoxic to a subject. Exemplary pharmaceutically acceptable carriers are a buffer, stabilizer or preservative.

[0325] The anti-GUCY2CxCD3 antibody disclosed herein can be administered in pharmaceutical compositions that include an effective amount of the antibody, and one or more pharmaceutically acceptable carriers or excipients, which can be formulated by methods known to those skilled in the art.

[0326] In some embodiments, the anti-GUCY2CxCD3 antibody useful for a method of the application can be packaged in one or more kits, which can optionally contain instructions for use.ITEMS DESCRIBED IN THE APPLICATION

[0327] The following list of items is intended to complement, rather than displace or supersede, the previous descriptions

[0328] Item 1. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein: a. the first antigen domain that binds GUCY2C comprises a first heavy chain variable region (VH1) of SEQ ID NO: 7 and a first light chain variable region (VL1) of SEQ ID NO: 8, andb. the second antigen domain that binds CD3 comprises a second heavy chain variable region (VH2) of SEQ ID NO: 17 and a second light chain variable region (VL2) of SEQ ID NO: 18.

[0329] Item 2. The method of item 1, wherein GUCY2C comprises human GUCY2C and wherein CD3 is or comprises, particularly is, human CD3.

[0330] Item 3. The method of item 1 or 2, wherein the VH1 and VL1 of the first antigen binding domain form a Fab or are comprised in a Fab.

[0331] Item 4. The method of any one of items 1-3, wherein the first antigen binding domain is (covalently) linked to a first Ig constant region or to a fragment of a first Ig constant region.

[0332] Item 5. The method of item 4, wherein the first Ig constant region or the fragment of the first Ig constant region comprises a. the mutations L234A, L235A, D265S (EU numbering)); and b. the mutations T366S, L368A, Y407V (EU numbering) or mutation T366W (EU numbering)).

[0333] Item 6. The method of item 5, wherein the first Ig constant region or fragment of first Ig constant region further comprises the mutations H435R and Y436F (EU numbering).

[0334] Item 7. The method of any one of items 4-6, wherein the first Ig constant region or fragment of first Ig constant region comprises the mutations L234A, L235A, D265S (EU numbering), the mutations T366S, L368A, Y407V (EU numbering) and the mutations H435R and Y436F (EU numbering).

[0335] Item 8. The method of any one of items 4-6, wherein the first Ig constant region or fragment of first Ig constant region comprises the mutations L234A, L235A, D265S (EU numbering) and the mutation T366W (EU numbering).

[0336] Item 9. The method of any one of items 4-8, wherein the Ig is of the Igl isotype.

[0337] Item 10. The method of any one of items 1-9, wherein the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9 and a first light chain (LC1) of SEQ ID NO: 10.

[0338] Item 11. The method of any one of items 1-10, wherein the VH2 and VL2 of the second antigen binding domain form a scFv or a spFv, particularly a spFv, or are comprised in a scFv or a spFv, particularly a spFv.

[0339] Item 12. The method of item 11, wherein the VH2 and VL2 of the second antigen binding domain are (covalently) linked via a linker, particularly a linker of SEQ ID NO: 31.

[0340] Item 13. The method of item 11 or 12, wherein the scFv or spFv, particularly the spFv, is in the VL-linker-VH orientation.

[0341] Item 14. The method of any one of items 1-13, wherein the second antigen binding domain is or comprises the amino acid sequence of SEQ ID NO: 19.

[0342] Item 15. The method of any one of items 1-14, wherein the second antigen binding domain is (covalently) linked to a second Ig constant region or a fragment of a second Ig constant region.

[0343] Item 16. The method of item 15, wherein the second Ig constant region or fragment of second Ig constant region comprises a. the mutations L234A, L235A, D265S (EU numbering); and b. the mutation T366W (EU numbering) or mutations T366S, L368A, Y407V (EU numbering).

[0344] Item 17. The method of item 16, wherein the second Ig constant region or fragment of second Ig constant region comprises the mutations L234A, L235A, D265S (EU numbering) and the mutation T366W (EU numbering).

[0345] Item 18. The method of item 16, wherein the second Ig constant region or fragment of second Ig constant region comprises the mutations L234A, L235A, D265S (EU numbering), and the mutations T366S, L368A, Y407V (EU numbering).

[0346] Item 19. The method of any one of items 15-18, wherein the second Ig constant region or fragment of second Ig constant region comprises the mutations H435R and Y436F (EU numbering).

[0347] Item 20. The method of any one of items 15-19, wherein the second Ig constant region or fragment of second Ig constant region comprises the mutations L234A, L235A, D265S (EU numbering), the mutations T366S, L368A, Y407V (EU numbering) and the mutations H435R and Y436F (EU numbering).

[0348] Item 21. The method of any one of items 15-20, wherein the second Ig constant region or fragment of second Ig constant region is of the Igl isotype.

[0349] Item 22. The method of any one of items 1-21, wherein the anti-GUCY2CxCD3 antibody comprises a second heavy chain (HC2) of SEQ ID NO: 20.

[0350] Item 23. The method of any one of items 1-22, wherein the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously or intravenously.

[0351] Item 24. The method of any one of items 1-23, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose of less than about 1000 mg (per dose of administration).

[0352] Item 25. The method of any one of items 1-24, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose of less than about 500 mg (per dose of administration).

[0353] Item 26. The method of any one of items 1-25 wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose of less than about 100 mg (per dose of administration).

[0354] Item 27. The method of any one of items 1-26, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose ranging from about 1 mg to about 50 mg (per dose of administration).

[0355] Item 28. The method of any one of items 1-27, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose ranging from about 2 mg to about 50 mg (per dose of administration).

[0356] Item 29. The method of any one of the preceding items, wherein the anti- GUCY2CxCD3 antibody is administered at a treatment dose from about 0.5 to about 25 mg (per dose of administration), more particularly from about 0.5 to about 15 mg (per dose of administration), more particularly from about 0.5 to about 12 mg (per dose of administration), more particularly from about 0.5 to about 6.5 mg (per dose of administration).

[0357] Item 30. The method of any one of items 1-29, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose, which is a flat dose.

[0358] Item 31. The method of any one of items 1-30, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose, which is administered more than once.

[0359] Item 32. The method of any one of items 1-31, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose, which is administered several times at a regular frequency.

[0360] Item 33. The method of any one of items 1-32, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose, which is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0361] Item 34. The method of any one of items 1-33, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose, which is administered once every 2 weeks.

[0362] Item 35. The method of any one of items 1-34, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose, which is administered at a first frequency for a first period of time, and at a second frequency for a second period of time, wherein thesecond period of time immediately succeeds the first period of time, and wherein the second frequency is lower than the first frequency.

[0363] Item 36. The method of any one of items 1-35, wherein the first frequency is once every 2 weeks.

[0364] Item 37. The method of any one of items 1-36, wherein the second frequency is once every 4 weeks, once every 5 weeks or once every 6 weeks.

[0365] Item 38. The method of item 35, wherein the first period of time is of 4 to 8 weeks, for example 4, 6 or 6 weeks.

[0366] Item 39. The method of any one of items 1-38, wherein the administration of the anti-GUCY2CxCD3 antibody prevents disease progression in the subject.

[0367] Item 40. The method of any one of items 1-39 wherein the administering of the therapeutically effective amount of the anti-GUCY2CxCD3 antibody prevents disease progression in the subject.

[0368] Item 41. The method of any one of items 1-40 wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose that prevents disease progression in the subject.

[0369] Item 42. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO:9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose is lower than about 1000 mgs; and / ord. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0370] Item 43. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose is lower than about 500 mgs; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0371] Item 44. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose is lower than about 100 mgs; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0372] Item 45. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering tothe subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose ranges from about 2 mg to about 50 mg; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0373] Item 46. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose ranges from about 1 mg to about 50 mg; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0374] Item 47. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20;b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose ranges from about 0.5 to 25 mg (per dose of administration) more particularly from about 0.5 to 15 mg (per dose of administration), more particularly from about 0.5 to 12 mg (per dose of administration), more particularly from about 0.5 to 6.5 mg (per dose of administration); and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks, more particularly once every 2 weeks.

[0375] Item 48. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject intravenously; c. the treatment dose is lower than about 1000 mgs; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0376] Item 49. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject intravenously; c. the treatment dose is lower than about 500 mgs; and / ord. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0377] Item 50. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject intravenously; c. the treatment dose is lower than about 100 mgs; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0378] Item 51. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject intravenously; c. the treatment dose ranges from about 2 mg to about 50 mg; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0379] Item 52. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering tothe subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject intravenously; c. the treatment dose ranges from about 1 mg to about 50 mg; and / or d. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.Item 53. The method of any one of items 41-52, wherein the treatment dose is a flat dose.

[0380] Item 54. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose is lower than about 1000 mgs; d. the treatment dose is a flat dose; and / or e. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0381] Item 55. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigenbinding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose is lower than about 500 mgs; d. the treatment dose is a flat dose; and / or e. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0382] Item 56. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose is lower than about 100 mgs; d. the treatment dose is a flat dose; and / or e. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0383] Item 57. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, whereina. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose ranges from about 2 mg to about 50 mg; d. the treatment dose is a flat dose; and / or e. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0384] Item 58. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose ranges from about 1 mg to about 50 mg; d. the treatment dose is a flat dose; and / or e. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0385] Item 59. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, whereina. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously; c. the treatment dose ranges from about 0.5 to 25 mg (per dose of administration) more particularly from about 0.5 to 15 mg (per dose of administration), more particularly from about 0.5 to 12 mg (per dose of administration), more particularly from about 0.5 to 6.5 mg (per dose of administration); d. the treatment dose is a flat dose; and / or e. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks, more particularly once every 2 weeks.

[0386] Item 60. The method of any one of items 1-60, which comprises administering one or more step-up doses of the anti-GUCY2CxCD3 antibody prior to administering any treatment dose of the anti-GUCY2CxCD3 antibody, wherein the one or more step-up doses are lower than the treatment dose.

[0387] Item 61. The method of item 60, wherein the one or more step-up doses lower the risk of cytokine release syndrome (CRS) in the subject.

[0388] Item 62. The method of item 60 or 61, wherein the one or more step-up doses are administered 2-15 days, such as 1 week from each other and wherein the step-up dose that is administered as the last step-up dose before the first administration of the treatment dose, is administered 2-15 days, such as 1 week, prior to the first administration of the treatment dose.

[0389] Item 63. The method of any one of items 60-62, wherein each step-up dose ranges from about 1 mg to about 50 mg.

[0390] Item 64. The method of any one of items 60-63, wherein each step-up dose ranges from about 1 mg to about 50 mg and wherein the treatment dose ranges from about 1 mg to about 50 mg or from about 2 mg to about 50 mg.

[0391] Item 65. The method of any one of items 60-64, wherein each step-up dose ranges from about 0.5 mg to about 25 mg.

[0392] Item 66. The method of any one of items 60-65, wherein each step-up dose ranges from about 0.5 mg to about 12 mg.

[0393] Item 67. The method of any one of items 60-66, wherein each step-up dose ranges from about 0.5 mg to about 5 mg.

[0394] Item 68. The method of any one of items 60-67, wherein the one or more step-up doses are administered 2-15 days, such as 1 week or 2 weeks from each other.

[0395] Item 69. The method of any one of items 60-68, wherein the one or more step-up doses are administered 1 week from each other.

[0396] Item 70. The method of any one of items 60-69, wherein the one or more step-up doses are administered 2 weeks from each other.

[0397] Item 71. The method of any one of items 60-70, which comprises only one, or only two, or only three step-up doses.

[0398] Item 72. The method of any one of items 60-70, which comprises a first step-up dose and a second step-up dose, wherein the second step-up dose is higher than the first step- up dose.

[0399] Item 73. The method of item 72, wherein the first step-up dose is of about 0.5 mg to about 5 mg, and wherein the second step-up dose is of about 0.5 mg to about 12mg.

[0400] Item 74. The method of item 72 or 73, wherein the first and second step-up doses are administered 1 week from each other.

[0401] Item 75. The method of any one of items 72-74, wherein the first and second step- up doses are administered 2 weeks from each other.

[0402] Item 76. The method of any one of items 60-70, which comprises a first step-up dose and a second step-up dose and a third step-up dose, wherein the second step-up dose is higher than the first step-up dose, and wherein the third step-up dose is higher than the second step-up dose.

[0403] Item 77. The method of item 76, wherein the first step-up dose is of about 0.5 mg to about 5 mg, wherein the second step-up dose is of about 0.5 mg to about 12mg, and wherein the third step-up dose is of about 0.5 mg to about 25 mg.

[0404] Item 78. The method of item 76 or 77, wherein the first, second and third step-up doses are administered 1 week from each other.

[0405] Item 79. The method of any one of items 76 or 77, wherein the first, second and third step-up doses are administered 2 weeks from each other.

[0406] Item 80. The method of any one of items 60-79, wherein each step-up dose is a flat dose.

[0407] Item 81. The method of any one of items 60-70, wherein the method comprises administering one or two step-up doses of the anti-GUCY2CxCD3 antibody prior to administering any treatment dose of the anti-GUCY2CxCD3 antibody, wherein the one or two step-up doses are lower than the treatment dose.

[0408] Item 82. The method of item 81, wherein the method comprises administering a first step-up dose and a second step-up dose of the anti-GUCY2CxCD3 antibody, wherein the first step-dose is lower than the second step-up dose and is administered 2-15 days, such as 1 week, prior to the second step-up dose and wherein the second step-up dose is lower than the treatment dose and is administered 2-15 days, such as 1 week, prior to the first administration of the treatment dose.

[0409] Item 83. The method of item 82, wherein the method comprises administering only one step-up dose before the treatment dose, and the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0410] Item 84. The method of item 83, wherein the only one step-up dose is administered before the treatment dose, and the treatment dose is administered once every 2 weeks.

[0411] Item 85. The method of any one of items 81-84, wherein each step-up dose ranges from about 1 mg to about 50 mg.

[0412] Item 86. The method of any one of items 81-85, wherein each step-up dose ranges from about Img to about 50 mg and wherein the treatment dose ranges from about 1 mg to about 50 mg or from about 2 mg to about 50 mg.

[0413] Item 87. The method of item 83 or 84, wherein the only one step-up dose is administered, preferably 2-15 days, such as 1 week, prior to the treatment dose (or prior the first administration of the treatment dose).

[0414] Item 88. The method of item 87, wherein the step-up dose ranges from about 1 mg to about 50 mg.

[0415] Item 89. The method of item 87 or 88, wherein the step-up dose ranges from about 1 mg to about 50 mg and wherein the treatment dose ranges from about 1 mg to about 50 mg or from about 2 mg to about 50 mg.

[0416] Item 90. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, whereina. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10, and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and the treatment dose are administered subcutaneously; c. the one or more step-up doses and the treatment dose are lower than about 1000 mgs; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step-up doses are administered 2-15 days, such as 1 week from each other; and the last step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0417] Item 91. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10, and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and the treatment dose are administered subcutaneously; c. the one or more step-up doses and the treatment dose are lower than about 500 mgs; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step-up doses are administered 2-15 days, such as 1 week from each other; and the last step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0418] Item 92. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10, and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and the treatment dose are administered subcutaneously; c. the one or more step-up doses and the treatment dose are lower than about 100 mgs; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step-up doses are administered 2-15 days, such as 1 week from each other; and the last step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0419] Item 93. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10, and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and the treatment dose are administered subcutaneously; c. the one or more step-up doses and the treatment dose range from about 2 mg to about 50 mg; d. the one or more step-up doses are lower than the treatment dose;e. the one or more step-up doses are administered 2-15 days, such as 1 week from each other; and the last step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0420] Item 94. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10, and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and the treatment dose are administered subcutaneously; c. the one or more step-up doses and the treatment dose range from about 1 mg to about 50 mg; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step-up doses are administered 2-15 days, such as 1 week from each other; and the last step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0421] Item 95. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, whereina. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10, and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and the treatment dose are administered intravenously; c. the one or more step-up doses and the treatment dose are lower than about 1000 mgs; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step-up doses are administered 2-15 days, such as 1 week from each other; and the last step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0422] Item 96. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10, and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and the treatment dose are administered intravenously; c. the one or more step-up doses and the treatment dose are lower than about 500 mgs; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step-up doses are administered 2-15 days, such as 1 week from each other; and the last step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0423] Item 97. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10, and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and the treatment dose are administered intravenously; c. the one or more step-up doses and the treatment dose are lower than about 100 mgs; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step-up doses are administered 2-15 days, such as 1 week from each other; and the last step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0424] Item 98. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10, and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and the treatment dose are administered intravenously; c. the one or more step-up doses and the treatment dose range from about 2 mg to about 50 mg; d. the one or more step-up doses are lower than the treatment dose;e. the one or more step-up doses are administered 2-15 days, such as 1 week from each other; and the last step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0425] Item 99. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject one or more step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10, and a second heavy chain (HC2) of SEQ ID NO: 20; b. the one or more step-up doses and the treatment dose are administered intravenously; c. the one or more step-up doses and the treatment dose range from about 1 mg to about 50 mg; d. the one or more step-up doses are lower than the treatment dose; e. the one or more step-up doses are administered 2-15 days, such as 1 week from each other; and the last step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or f. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0426] Item 100. A method of treating cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, whereina. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the first step-up dose, the second step-up dose and the treatment dose are administered subcutaneously; c. the first step-up dose, the second step-up doses and the treatment dose are lower than about 1000 mgs; d. the first step-up dose is lower than the second step-up dose and the second step-up dose is lower than the treatment dose; e. the first step-up dose and the second step-up dose are each administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the first step-up dose is administered 2-15 days, such as 1 week before the second step-up dose; the second step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0427] Item 101. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the first step-up dose, the second step-up dose and the treatment dose are administered subcutaneously; c. the first step-up dose, the second step-up doses and the treatment dose are lower than about 500 mgs; d. the first step-up dose is lower than the second step-up dose and the second step-up dose is lower than the treatment dose; e. the first step-up dose and the second step-up dose are each administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times);f. the first step-up dose is administered 2-15 days, such as 1 week before the second step-up dose; the second step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0428] Item 102. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the first step-up dose, the second step-up dose and the treatment dose are administered subcutaneously; c. the first step-up dose, the second step-up doses and the treatment dose are lower than about 100 mgs; d. the first step-up dose is lower than the second step-up dose and the second step-up dose is lower than the treatment dose; e. the first step-up dose and the second step-up dose are each administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the first step-up dose is administered 2-15 days, such as 1 week before the second step-up dose; the second step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0429] Item 103. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibodycomprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the first step-up dose, the second step-up dose and the treatment dose are administered subcutaneously; c. the first step-up dose, the second step-up doses and the treatment dose range from about 2 mg to about 50 mg; d. the first step-up dose is lower than the second step-up dose and the second step-up dose is lower than the treatment dose; e. the first step-up dose and the second step-up dose are each administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the first step-up dose is administered 2-15 days, such as 1 week before the second step-up dose; the second step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0430] Item 104. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the first step-up dose, the second step-up dose and the treatment dose are administered subcutaneously; c. the first step-up dose, the second step-up doses and the treatment dose range from about 1 mg to about 50 mg; d. the first step-up dose is lower than the second step-up dose and the second step-up dose is lower than the treatment dose;e. the first step-up dose and the second step-up dose are each administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the first step-up dose is administered 2-15 days, such as 1 week before the second step-up dose; the second step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0431] Item 105. A method of treating cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the first step-up dose, the second step-up dose and the treatment dose are administered intravenously; c. the first step-up dose, the second step-up doses and the treatment dose are lower than about 1000 mgs; d. the first step-up dose is lower than the second step-up dose and the second step-up dose is lower than the treatment dose; e. the first step-up dose and the second step-up dose are each administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the first step-up dose is administered 2-15 days, such as 1 week before the second step-up dose; the second step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0432] Item 106. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering tothe subject two step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the first step-up dose, the second step-up dose and the treatment dose are administered intravenously; c. the first step-up dose, the second step-up doses and the treatment dose are lower than about 500 mgs; d. the first step-up dose is lower than the second step-up dose and the second step-up dose is lower than the treatment dose; e. the first step-up dose and the second step-up dose are each administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the first step-up dose is administered 2-15 days, such as 1 week before the second step-up dose; the second step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0433] Item 107. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the first step-up dose, the second step-up dose and the treatment dose are administered intravenously; c. the first step-up dose, the second step-up doses and the treatment dose are lower than about 100 mgs;d. the first step-up dose is lower than the second step-up dose and the second step-up dose is lower than the treatment dose; e. the first step-up dose and the second step-up dose are each administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the first step-up dose is administered 2-15 days, such as 1 week before the second step-up dose; the second step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0434] Item 108. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the first step-up dose, the second step-up dose and the treatment dose are administered intravenously; c. the first step-up dose, the second step-up doses and the treatment dose range from about 2 mg to about 50 mg; d. the first step-up dose is lower than the second step-up dose and the second step-up dose is lower than the treatment dose; e. the first step-up dose and the second step-up dose are each administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the first step-up dose is administered 2-15 days, such as 1 week before the second step-up dose; the second step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0435] Item 109. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject two step-up doses and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the first step-up dose, the second step-up dose and the treatment dose are administered intravenously; c. the first step-up dose, the second step-up doses and the treatment dose range from about 1 mg to about 50 mg; d. the first step-up dose is lower than the second step-up dose and the second step-up dose is lower than the treatment dose; e. the first step-up dose and the second step-up dose are each administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the first step-up dose is administered 2-15 days, such as 1 week before the second step-up dose; the second step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0436] Item 110. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose are lower than about 1000 mgs; d. the only one step-up is lower than the treatment dose;e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0437] Item 111. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose are lower than about 500 mgs; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0438] Item 112. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, whereina. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose are lower than about 100 mgs; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0439] Item 113. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose range from about 2 mg to about 50 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0440] Item 114. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose range from about 1 mg to about 50 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0441] Item 115. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose are lower than about 25 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times);f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0442] Item 116. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose are lower than about 15 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0443] Item 117. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously;c. the only one step-up dose and the treatment dose are lower than about 12 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0444] Item 118. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ IDNO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered subcutaneously; c. the only one step-up dose and the treatment dose are lower than about 6.5 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0445] Item 119. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a first step-up dose, a second step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ IDNO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the first step-up dose, the second step-up dose and the treatment dose are flat doses and are administered subcutaneously; c. the first step-up dose is equal to or lower than the second step-up dose, and the second step-up dose is lower than the treatment dose; d. the first step-up dose is administered only once and the second step-up dose is administered only once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); e. the first step-up dose is about 0.5 to about 5 mg, the second step-up dose is of about 0.5 mg to about 12 mg, and the treatment dose is lower than about 100 mg; f. the first step-up dose is administered 2-15 days, such as 1 week or 2 weeks before the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week or 2 weeks, before the treatment dose; and g. the treatment dose is administered once every 1-9 weeks, for example, once every2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0446] Item 120. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject a first step-up dose, a second step-up dose, and a treatment dose of an anti- GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the first step-up dose, the second step-up dose and the treatment dose are flat doses and are administered subcutaneously; c. the first step-up dose is equal to or lower than the second step-up dose, and the second step-up dose is lower than the treatment dose;d. the first step-up dose is administered only once and the second step-up dose is administered only once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); e. the first step-up dose is about 0.5 to about 5 mg, the second step-up dose is of about 0.5 mg to about 12 mg, and the treatment dose is lower than about 100 mg; f. the first step-up dose is administered 2-15 days, such as 1 week or 2 weeks before the second step-up dose, and the second step-up dose is administered 2-15 days, such as 1 week or 2 weeks, before the treatment dose; and g. the treatment dose is administered at a first frequency for a first period of time, and at a second frequency for a second period of time, wherein the second period of time immediately succeeds the first period of time, and wherein the second frequency is lower than the first frequency.

[0447] Item 121. The method of item 120, wherein the treatment dose is administered once every 2 weeks for a first period of time, and once every 4 weeks or once every 6 weeks for a second period of time, wherein the first period of time is of 4-6 weeks.

[0448] Item 122. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered intravenously; c. the only one step-up dose and the treatment dose are lower than about 1000 mgs; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / org. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0449] Item 123. A method of treating cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered intravenously; c. the only one step-up dose and the treatment dose are lower than about 500 mgs; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0450] Item 124. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered intravenously; c. the only one step-up dose and the treatment dose are lower than about 100 mgs; d. the only one step-up is lower than the treatment dose;e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0451] Item 125. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein a. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered intravenously; c. the only one step-up dose and the treatment dose range from about 2 mg to about 50 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0452] Item 126. A method of treating a cancer of the digestive system, preferably of the gastrointestinal system, in a human subject, wherein the method comprises administering to the subject only one step-up dose and a treatment dose of an anti-GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, whereina. the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10 and a second heavy chain (HC2) of SEQ ID NO: 20; b. the only one step-up dose and the treatment dose are administered intravenously; c. the only one step-up dose and the treatment dose range from about 1 mg to about 50 mg; d. the only one step-up is lower than the treatment dose; e. the only one step-up dose is administered once, and the treatment dose is administered at least twice (e.g., 2, 3, 4 or more times); f. the only one step-up dose is administered 2-15 days, such as 1 week before the treatment dose; and / or g. the treatment dose is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

[0453] Item 127. The method of any one of items 1-126, wherein the cancer of the digestive system, preferably of the gastrointestinal system, is a colorectal cancer.

[0454] Item 128. The method of any one of items 1-127, wherein the cancer of the digestive system, preferably of the gastrointestinal system, is a metastatic colorectal cancer.

[0455] Item 129. The method of any one of the foregoing items, wherein the human subject does not have one or more of the following a. Active (new or progressive) brain metastases, leptomeningeal disease, or untreated spinal cord compression. b. Toxicity from prior anticancer therapy that has not resolved to Grade <1 (except alopecia, vitiligo, Grade <= 2 peripheral neuropathy, or endocrinopathies that are stable on hormone replacement) c. Prior or concurrent second malignancy (other than mCRC) unless natural history or treatment is unlikely to interfere with any study endpoints of safety or the efficacy of the study treatment. c. Received glucocorticoids (doses >10 mg / day prednisone or equivalent) within 7 days prior to the first dose of the anti-GUCY2CxCD3 antibody.e. Received or plans to receive any live, attenuated vaccine within 4 weeks before the dose of the antibody or within 4 weeks after the last step-up dose or last treatment dose of the antibody.

[0456] Item 130. The method of any one of the foregoing items, wherein the human subject does not have one or more of the following a. Active (new or progressive) brain metastases, leptomeningeal disease, or untreated spinal cord compression. b. Toxicity from prior anticancer therapy that has not resolved to Grade <1 (except alopecia, vitiligo, Grade <= 2 peripheral neuropathy, or endocrinopathies that are stable on hormone replacement) c. A history of Grade >2 immune-related adverse event (AE) on prior immunotherapy. d. Prior or concurrent second malignancy (other than mCRC) unless natural history or treatment is unlikely to interfere with any study endpoints of safety or the efficacy of the study treatment. e. Prior treatment with any other GUCY2C targeted anti-cancer therapy. f. Prior anti-cancer treatments discontinued for less than 3 weeks or 5 half-lives (whichever is shorter) prior to the first dose of the antibody. i. Prior immune checkpoint inhibitors and / or T-cell redirecting bispecific antibodies (eg, CD3 targeting or other bispecific antibodies) discontinued for less than 6 weeks before the first dose of the antibody . ii. Prior Car-T therapies less than 12 weeks prior to first dose of the antibody iii. Palliative radiation less than 14 days before prior to first dose of the antibody g. Received glucocorticoids (doses >10 mg / day prednisone or equivalent) within 7 days prior to the first dose of the anti-GUCY2CxCD3 antibody. h. Received or plans to receive any live, attenuated vaccine within 4 weeks before the dose of the antibody or within 4 weeks after the last step-up dose or last treatment dose of the antibody. i. Clinically significant pulmonary compromise defined as requiring continuous supplemental oxygen (>2L / min by nasal cannula) j. Any episode of partial or complete bowel obstruction requiring hospitalization within 30 days prior to first dose of the anti-GUCY2CxCD3 antibody.k. diverting ileostomy, gastrojejunal stents, colorectal stents, or history of enterocutaneous fistula l. History of peptic ulcer disease in the past 3 months that required endoscopic intervention or blood transfusion m. Active hepatitis B or C. n. Human immunodeficiency virus unless viral load is undetectable and CD4 count is above 300 cells / mm3 on stable highly active antiretroviral therapy (HAART) therapy. o. Evidence of infection requiring parenteral treatment within 7 days before the first dose of the anti-GUCY2CxCD3 antibody. p. History of solid organ or hematologic stem cell transplantation q. Active autoimmune disease within the 12 months prior to first dose of the anti- GUCY2CxCD3 antibody that requires systemic immunosuppressive medications (eg, chronic corticosteroids, methotrexate, or tacrolimus). r. Any of the following within 6 months prior to first dose: i. Myocardial infarction ii. Severe or unstable angina iii. Coronary artery bypass grafting iv. Clinically significant arrhythmias (eg, ventricular arrhythmias, clinically significant atrial fibrillation with uncontrolled rate) v. New York Heart Association Class III or IV congestive heart failure vi. Myocarditis vii. Cerebrovascular accident or transient ischemic attack viii. Any grade arterial event s. Venous thromboembolic events (eg, pulmonary embolism or intracardiac thrombus) within 1 month prior to first dose of the anti-GUCY2CxCD3 antibody t. Any major surgery within 4 weeks before first dose of the anti-GUCY2CxCD3 antibody or has not recovered adequately from surgery. u. Initiation or increase in dose of any guanylate cyclase agonists (eg, linaclotide or plecanatide) within 3 months prior to first dose of the anti-GUCY2CxCD3 antibody. v. Known allergies, hypersensitivity, or intolerance to the excipients of GUCY2CxHumanCD3 bispecific antibody.w. Prolonged QT interval (QTc intervals corrected using Fredericia’s formula [QTcF]) >480 msec based on the average of triplicate assessments performed no more than 5 (±3) minutes apart. x. Any serious underlying medical conditions or other issue that would impair the ability to receive or tolerate the planned treatment. y. Grade >2 diarrhea and / or colitis.

[0457] Item 131. The method of item 129 or 130, wherein the human subject does not have active (new or progressive) brain metastases, leptomeningeal disease, or untreated spinal cord compression.

[0458] Item 132. The method of any one of items 129-131, wherein the human subject does not have toxicity from prior anticancer therapy that has not resolved to Grade <1.

[0459] Item 133. The method of any of the foregoing items, wherein the subject is a. a subject having histologically or cytologically confirmed diagnosis of colorectal adenocarcinoma (CRC) progressing after 2 or more prior lines of standard therapy in the metastatic / unresectable setting. b. a subject having evaluable or measurable disease per response evaluation criteria in solid tumors RECIST vl. l (or at least one measurable lesion) c. a subject having Eastern Cooperative Oncology Group (ECOG) performance status of 0-1 d. a subject having hematology laboratory parameters within the required range f. a subject having an estimated or measured glomerular filtration rate (GFR) >30 mL / min based on modification of diet in renal disease (MDRD) 4-variable formula.

[0460] Item 134. The method of any one of the foregoing items wherein the subject is a. a subject having histologically or cytologically confirmed diagnosis of colorectal adenocarcinoma (CRC) progressing after 2 or more prior lines of standard therapy in the metastatic / unresectable setting. b. a subject having evaluable or measurable disease per RECIST vl .1 (or at least 1 measurable lesion) c. a subject having Eastern Cooperative Oncology Group (ECOG) performance status of 0-1.d. a subject having hematology laboratory parameters within the following range without prior growth factor support within 3 weeks or transfusion within 7 days before the day the laboratory test was performed: i. Absolute neutrophil count >1000>< 109 / L; ii. Hemoglobin >8.0 g / dL; or iii. Platelets >80x 109 / L e. a subject having clinical chemistry laboratory parameters within the following range during the screening period: i. aspartate aminotransferase (AST) and alanine aminotransferase (ALT) <3 x upper limit of normal (ULN) or < 5 x ULN if documented liver involvement by disease, or ii. Serum total bilirubin <1.5 x ULN; participants with congenital bilirubinemia, such as Gilbert’s syndrome, may enroll if conjugated bilirubin is < 1.5 x ULN; f. a subject having an estimated or measured glomerular filtration rate (GFR) >30 mL / min based on modification of diet in renal disease (MDRD) 4-variable formula.

[0461] Item 135. The method of item 133 or 134 wherein the subject has histologically or cytologically confirmed diagnosis of CRC progressing after 2 or more prior lines of standard therapy in the metastatic / unresectable setting.

[0462] Item 136. The method of any one of items 133-135 wherein the subject has evaluable or measurable disease per RECIST vl .1.

[0463] Item 137. The method of any one of items 133-136 wherein the subject has Eastern Cooperative Oncology Group (ECOG) performance status of 0-1.

[0464] Item 138. The method of any one of items 133-137 wherein the subject has hematology laboratory parameters within the following range: a. Absolute neutrophil count >1000x l09 / L; b. Hemoglobin >8.0 g / dL; or c. Platelets >80 x109 / L.

[0465] Item 139. The method of any one of items 133-138 wherein the subject has clinical chemistry laboratory parameters within the following range during the screening period: a. aspartate aminotransferase (AST) and alanine aminotransferase (ALT) <3 x upper limit of normal (ULN) or < 5 x ULN if documented liver involvement by disease; or b. Serum total bilirubin <1.5 x ULN; participants with congenital bilirubinemia, such as Gilbert’s syndrome, may enroll if conjugated bilirubin is < 1.5 x ULN.

[0466] Item 140. The method of any one of items 133-139 wherein the subject has an estimated or measured glomerular filtration rate (GFR) >30 mL / min based on modification of diet in renal disease (MDRD) 4-variable formula.

[0467] Item 141. The method of any one of the foregoing items, wherein the method does not induce a non-hematologic treatment-related toxicity in the subject, wherein the non- hematologic toxicity is one or more of the following: a. Grade 2^3 CRS b. Grade 4 hypertension c. an increased Aspartate aminotransferase (AST) and Alanine Aminotransferase (ALT) within the following range: i. an increase >8x Upper Limit of Normal (ULN) for any duration, if the baseline is within normal limits; ii. an increase >5x ULN for >14 days, if the baseline is within normal limits; iii. ALT or AST value >3x ULN, total bilirubin >2x ULN, and ALP <2x ULN with no alternative etiology if Hy’s Law criteria are met and baseline is within normal limits; iv.an increase >5x baseline for any duration if the baseline AST / ALT is elevated above ULN; v. an increase >3x baseline for >14 days if the baseline AST / ALT is elevated above ULN; or vi. ALT or AST >3x baseline or AST or ALT >8x ULN, whichever is lower, combined with total bilirubin >2x baseline AND >2x ULN, with no alternative etiology, if modified Hy’s Law criteria baseline are met and baseline AST / ALT is elevated above ULN.d. any Grade >3 non -hematological toxicity with the following exceptions: i. First occurrence of Grade 3 systemic administration-related reaction (sARR) that improves to Grade <2 within 72 hours; ii. Grade 3 fatigue or asthenia lasting <7 days with best supportive care; iii. Grade 3 fever in the absence of neutropenia that resolves within 48 hours; iv. Grade 3 nausea, vomiting, constipation, or diarrhea / colitis lasting < 72 hours with best supportive care; v. Grade>3 hypertension that can be controlled by oral medical management and improves to Grade <2 within 72 hours; vi. Grade >3 lipase or amylase increase not associated with clinical or radiological evidence of pancreatitis;Grade >3 pain due to tumor flare that improves to Grade <2 with best supportive care within 5 days; or vii. Grade >3 ALP, GGT, or electrolyte abnormalities that resolve spontaneously to Grade <2 or baseline within 5 days, or that respond to best supportive care.

[0468] Item 142. The method of any one of the foregoing items, wherein the method does not induce a hematologic treatment-related toxicity in the subject, wherein the hematologic toxicity is one or more of the following: a. Febrile neutropenia; b. Grade 4 neutropenia for >7 days despite supportive care according to institutional standards; and / or c. Grade > 3 thrombocytopenia with Grade 2 bleeding or any Grade 4 thrombocytopenia with duration >7 days.

[0469] Item 143. The method of any one of the foregoing items, wherein the method does not induce a. diarrhea or colitis in the subject; or b. a cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (iCANs) in the subject.

[0470] Item 144. The method of any one of the foregoing items, wherein a. less than 28% of subjects experience Dose Limiting Toxicity (DLT), orb. the probability of the rate of subjects experiencing DLT exceeding 28%, is lower than 95%.

[0471] Item 145. The method of item 144 wherein the DLT comprises one or more events of hematologic toxicity; or one or more events of non-hematological toxicity.

[0472] Item 146. The method of item 141, wherein the one or more events of hematological toxicity are one or more from among a. Febrile neutropenia; b. Grade 4 neutropenia for >7 days despite supportive care according to institutional standards; or c. Grade > 3 thrombocytopenia with Grade 2 bleeding or any Grade 4 thrombocytopenia with duration >7 days.

[0473] Item 147. The method of item 141, wherein the one or more events of non- hematologic toxicity are one or more of the following: a. an increased Aspartate aminotransferase (AST) and Alanine Aminotransferase (ALT) within the following range: i. an increase >8x Upper Limit of Normal (ULN) for any duration if the baseline is within normal limits; ii. an increase >5x ULN for >14 days if the baseline is within normal limits; iii. an ALT or AST value >3x ULN, total bilirubin >2x ULN, and ALP <2x ULN with no alternative etiology if Hy’s Law criteria are met and the baseline is within normal limits; iv. an increase >5x baseline for any duration if baseline AST / ALT elevated above ULN; v. an increase >3x baseline for >14 days if baseline AST / ALT elevated above ULN; or vi. an ALT or AST >3x baseline or AST or ALT >8x ULN, whichever is lower, combined with total bilirubin >2x baseline AND >2x ULN, with no alternative etiology, if modified Hy’s Law criteria are met defined and baseline AST / ALT elevated above ULN b. any Grade >3 non-hematological toxicity with the following exceptions:i. First occurrence of Grade 3 CRS or systemic administration-related reaction (sARR) that improves to Grade <2 within 72 hours; ii. Grade 3 fatigue or asthenia lasting <7 days with best supportive care; iii. Grade 3 fever in the absence of neutropenia that resolves within 48 hours; iv. Grade 3 nausea, vomiting, constipation, or diarrhea / colitis lasting <5 days with best supportive care; v. Grade >3 hypertension that can be controlled by oral medical management and improves to Grade <2 within 72 hours; vi. Grade >3 lipase or amylase increase not associated with clinical or radiological evidence of pancreatitis; vii. Grade >3 pain due to tumor flare that does not improve to Grade <2 with best supportive care within 7 days; or vii. Grade >3 ALP, GGT, or electrolyte abnormalities that resolve spontaneously to Grade <2 or baseline within 7 days, or that respond to best supportive care.

[0474] Item 148. The method of any one of the foregoing items, wherein the tumor is significantly reduced (or destroyed) in the subject.

[0475] Item 149. The method of any one of the preceding items, wherein the human subject has received a pre-medication prior to the administration the GUCY2CxHumanCD3 antibody, wherein the premedication is a. a glucocorticoid: i. 16 mgs of Dexamethasone or equivalent prior to step-up doses; ii. 8 mgs of Dexamethasone or equivalent prior to second step-up dose if 2 step-up doses are administered within 1 week; iii. 16 mgs of Dexamethasone or equivalent prior to first treatment dose iv. 16 mgs of Dexamethasone or equivalent prior to step-up dose or treatment dose if CRS or sARR occurs in previous dose; v. 8 mgs of Dexamethasone or equivalent prior to step-up dose or treatment dose if no CRS or sARR occurs in previous dose; b. an antihistamine i. 50 mgs of Diphenhydramine or equivalent prior to step-up doses and first treatment dose;ii. 50 mgs of Diphenhydramine or equivalent prior to next two step-up doses or treatment doses if Grade >2 CRS or sARR occurred in the previous dose; c. an antipyretic i. 650 mgs to 1,000 mg of Acetaminophen or equivalent prior to step-up doses and first treatment; ii. 650 mgs to 1,000 mg of Acetaminophen or equivalent prior to next two step-up doses and treatment doses if Grade >2 CRS or sARR occurred in previous; d. optionally a H2-antagonist, (50 mgs of Ranitidine or equivalent); and / or e. optionally an antiemetic (16 mgs of Ondansetron or equivalent).

[0476] Item 150. The method of item 149, wherein the pre-medication is administered about 1 to 3 hours prior to the administration the GUCY2CxHumanCD3 antibody (or 30 minutes prior to the administration the GUCY2CxHumanCD3 if the premedication is given IV).

[0477] Item 151. The method of any one of the preceding items, wherein the human subject receives a treatment, more particularly a prophylactic treatment after the administration of the GUCY2CxHumanCD3 antibody, wherein the treatment, or prophylactic treatment is or comprises a cortico-steroid .

[0478] Item 152. The method of any one of the preceding items, which comprises administering to the subject only one step-up dose and a treatment dose, and which further comprises administering to the human subject receives a treatment, more particularly a prophylactic treatment, after the administration of the step-up dose and after the first administration of the first treatment dose, wherein the treatment, or prophylactic treatment is or comprises a cortico-steroid

[0479] Item 153. The method of any one of the foregoing items, wherein the colorectal cancer is unresectable metastatic colorectal cancer.

[0480] Item 154. The method of any one of items 1-126, wherein the cancer of the digestive system, preferably of the gastrointestinal system, is a gastric cancer.

[0481] Item 155. The method of any one of items 1-126 and item 154, wherein the cancer of the digestive system, preferably of the gastrointestinal system, is a primary or metastatic gastric cancer.

[0482] Item 156. The anti-GUCY2CxCD3 antibody as defined in any one of the foregoing items for use in the method of any one of the foregoing items.

[0483] Item 157. Use of the anti-GUCY2CxCD3 antibody as defined in any one of items 1-155 for the manufacture of a medicament for the treatment of a cancer of the digestive system, preferably of the gastrointestinal system.EXAMPLESEXAMPLE 1. GENERATION OF ANTLGUCY2C ANTIBODIESImmunization of Ablexis Kappa mice

[0484] Ablexis Kappa mice were immunized with alternating boosts of 10 pg of cynomolgus monkey GUCY2C extracellular domain (ECD) (day 0, 14, 28) or human GUCY2C ECD (day 7, 21). Zinc chitosan nanoparticles were used in conjunction in some instances. The mice received 5 weekly boosts and a final boost at day 44 with human GUCY2C ECD (with zinc chitosan nanoparticles in some instances) and 50 pg of antimouse CD40 (R&D Systems MAB440).

[0485] Immune sera samples were collected once during the immunization (Day 28) and tested for binding via flow cytometry to T84 (GUCY2C+) and T84 GUCY2C KO (GUCY2C-) cells. Protein serology was done on human and cynomolgus monkey GUCY2C ECD proteins by MSD. Quality of Immune Response analysis was done by SPR on human and cynomolgus monkey GUCY2C ECD coated chips.Single B-cell recovery

[0486] On Day 51 of the immunization schedule, spleens and inguinal lymph nodes were harvested from each mouse of group 4. All lymph nodes were pooled and homogenized into a single-cell suspension, sorted on double positive AF647-labeled cynomolgus monkeyGUCY2C and biotinylated human GUCY2C (with streptavidin-PE detection). Cells were then plated and cultured for 7 days.

[0487] Supernatants from the sorted B cells were screened by protein MSD on biotinylated human and cynomolgus monkey GUCY2C. Primary hits were sent for V gene recovery. Selected recovered hits were expressed as human IgGl .Expi293 Small Scale Transfection and Purification

[0488] Antibodies identified from the immunization campaign were cloned and expressed as human IgGl at 2 ml scale and purified. Expi293 cells were cultured in Expi293 Expression Medium at 37°C, 7% CO2. Cells were sub-cultured when density reached the log phase growth at 2.5-3 x 10A6 viable cells / mL with a 98-99% viability. On the day of transfection, the viable cell density and percent viability was determined. Cells were transfected at a density of 2.5-3 x 10A6 viable cells / mL following manufacturer’s Transfection protocol (ThermoFisher ExpiCHO Expression System Protocols for 24 and 96 deep well blocks and mini bioreactor tubes). Culture supernatants were harvested on Day 5 post-transfection by centrifugation at 2000 RPM for 15 minutes prior to purification.Antibodies were purified from the clarified supernatants using Capture Select CHI -XL resin slurry (Thermo Cat 2943452050). Antibodies were eluted with 0.1 M Na-Acetate, pH 3.5 and each elution fraction was neutralized with 2.5 M Tris HC1, pH 7.5 before dialyzes into DPBS. Protein concentrations were determined by measurement of absorbance at 280 nm on the filtrate using a DropSense Instrument (Trinean NV / SA).Recombinant antibody screening

[0489] The antibodies were screened for binding on T84 cells, CHO-huGUCY2C and EL4-huGUCY2C and counter screened on untransfected EL4 in dose response titrations. The best binders were then rescreened in dose response titrations on CHO-huGUCY2C, CHO-cynoGUCY2C, untransfected CHO and EL-4 huGUCY2C. They were also tested for competition against index GUCY2C binders and a known GUCY2C binder (reference binder) for binding to CHO-huGUCY2C cells. The variable regions of selected antibodies were sequenced. A mAb called GUCYC2_mAb was selected for further studies.Sequences of GUCY2C_mAb

[0490] Sequences of representative GUCY2C_mAb are provided in Tables 1-7. Table 1 shows the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences ofGUCY2C_mAb under the Kabat delineation. Table 2 shows the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of GUCY2C_mAb under the Chothia delineation. Table 3 shows the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of GUCY2C_mAb under the ABM delineation. Table 4 shows the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of GUCY2C_mAb under the Contact delineation. Table 5 shows the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of GUCY2C_mAb under the IMGT delineation.Table 1. HCDRs and LCDRs of GUYC2C_mAb under the Kabat delineation.Table 2. HCDRs and LCDRs of GUYC2C_mAb under the Chothia delineation.Table 3. HCDRs and LCDRs of GUYC2C_mAb under the AbM delineation.Table 4. HCDRs and LCDRs of GUYC2C_mAb under the Contact delineation.Table 5. HCDRs and LCDRs of GUYC2C_mAb under the IMGT delineation.

[0491] Table 6 shows the VH1 and VL1 amino acid sequences of GUCY2C_mAb. Table 7 shows the VH1 and VL1 nucleic acid sequences of GUCY2C_mAb.Table 6. Amino acid sequences of the VH1 and VL1 of GUCY2C_mAbTable 7. Nucleic acid sequences of the VH1 and VL1 of GUCY2C_mAbEXAMPLE 2. CHARACTERIZATION OF GUCY2C_mAb

[0492] Three (3) cell lines, Chinese hamster ovary (CHO) cell line, CHO transfected with cynomolgus monkey GUCY2C, and CHO transfected with human GUCY2C, were incubated with a titrating dose of GUCY2C_mAb for 60 min at 37°C, washed twice, then incubated with AF647 labeled anti human secondary for 30 minutes at 4°C. Cells were washed twice again, resuspended and acquired in a flow cytometer. Specific antibody binding was assessed in the RL1H channel for each cell line. Data presented as signal / background where background is secondary only. As shown in FIG. 1, GUCY2C_mAb as generated herein binds to both human and cynomolgus monkey GUCY2C, with higher binding affinity to human CUCY2C.

[0493] EC50 values for binding of GUCY2C_mAb to CHO cells transfected with human or cynomolgus monkey GUCY2C is shown in Table 8.Table 8.EXAMPLE 3. GENERATION OF GUCY2CxCD3 BISPECIFIC ANTIBODIESEngineering of GUCY2CxCD3 bispecific antibodies

[0494] In this example, two (2) GUCY2CxCD3 bispecific antibodies were generated (GUCY2CxCD3_biAb-l and GUCY2CxCD3_biAb-2). GUCY2CxCD3_biAb-l and GUCY2CxCD3_biAb-2 comprise the same GUCY2C binding arm, which corresponds to the GUCY2C_mAb described above and a medium and high affinity CD3 binding arm, respectively.Engineering of GUCY2CxCD3_biAb-l

[0495] Briefly, the GUCY2C binding arm of GUCY2CxCD3_biAb-l comprises an anti- GUCY2C Fab (GUCY2C_Fab) derived from GUCY2C_mAb describe in Example 1. Toprepare the bispecific antibody, the VH1 and VL1 of GUCY2C_mAb were engineered in VHl-CHl-hinge-CH2-CH3 (Heavy Chain 1, HC1) and VL1-CL (Light Chain 1, LC1) formats respectively and expressed as IgGl. The Fc silencing mutation L234A / L235A / D265S were introduced in the Fc region of HC1. Mutations designed to promote selective heterodimerization (“hole” mutation T366S, L368A and Y407V) were also engineered in the Fc domain of HC1.

[0496] The CD3 binding arm of GUCY2CxCD3_biAb-l comprises a stabilized (or stapled) scFv domain herein described as spFv-1. The CD3 arm of GUCY2CxCD3_biAb-l is a low-medium affinity CD3 binding arm which will herein be referred to as CD3_spFv-l. The CD3_spFv-l is derived from an anti-CD3 scFv fragment described in W02022 / 201053, which is incorporated herein by reference in its entirety.

[0497] The anti-CD3 scFv as disclosed in W02022 / 201053 was engineered into a stabilized scFv (herein referred to “stapled scFv” or spFv) by restraining the scFv structure, without negatively impacting, the relative movements between the VH2 and the VL2. The stabilized scFv was generated by engineering disulfide bonds between the VH2 and the linker and between the VL2 and the linker. Two structurally conserved surface exposed framework positions (anchor points) that are not involved in antigen binding, were identified, one on the VH2 at position Hl 05 and one on the VL2 at position L42, and mutated into cysteine (Cys) residues to generate the VH2 of SEQ ID NO: 17 and VL2 of SEQ ID NO: 18. A flexible linker of sequence GGGSGGSGGCPPCGGSGG (SEQ ID NO: 31) comprising two Cys residues was used to conjugate the VL2 and the VH2 in the VL- linker-VH (LH) format. The distance and location of the Cys residues of the linker and the Cys residues of the VH2 and the VL2 is critical for the formation of the disulfide bonds between the Cys residues of the Linker and each anchor point of the VH2 and the VL2.

[0498] The stapled scFv in the VL-Linker-VH was further engineered into a Heavy Chain 2 (HC2) and expressed as IgGl. Additionally, the Fc silencing mutation L234A / L235A / D265S and the heterodimerization (“knob” mutation T366W) were engineered in the Fc domain of HC2.

[0499] HC2 comprising CD3_spFv-l was then paired with HC1 and LC1 comprising the GUCY2C_Fab using the knob-in-hole heterodimerization technology to generate the stapled GUCY2CxCD3_biAb-l.

[0500] The affinity of CD3_spFv-l to T cells was measured by flow cytometry based Intellicyt assay. Briefly, pan T cells were isolated from PBMC from healthy donors. Cells were incubated with titrating amounts test molecules for 1 hour, then washed. Boundantibodies were detected with Alexa Fluor 647 F(ab')2 fragment goat anti-human IgG. Cells were then washed and resuspended in 0.1% pluronic acid containing Sytox Green, then immediately analyzed in a flow cytometer. Median fluorescent intensity (MFI) was recorded (on RL1-A). The loglO of the molecule concentration was plotted against the MFI. Best fit curves were evaluated using a 4PL algorithm (log(agonist) vs. response — Variable slope) in GraphPad Prism 9.0. 50% effective concentrations (EC50) values were derived from the best-fit curves. The EC50 value of CD3_spFv-l for human CD3 is 108-112 nM.

[0501] CDR sequences, VH2, VL2 and spFv sequence of CD3_spFv-l are shown in Table 9-11.Table 9. HCDRs and LCDRs of CD3_spFv-l under the Kabat delineation.Table 10. Amino Acid sequences of VH2, VL2 and stapled VL-Linker-VH of CD3_spFv-lTable 11. Nucleotide sequences of VH2, VL2 and stapled VL-Linker-VH of CD3_spFv-l

[0502] The full-length amino acid sequences of GUCY2C-CD3_biAb-l are shown in Table 12-13. Table 12 shows the full-length amino acid sequences of the GUCY2C arm (HC1 and LC1) and of the CD3 arm (HC2). Table 13 shows the nucleic acid sequences of the GUCY2C arm (HC1 and LC1) and of the CD3 arm (HC2).Table 12. Full length amino acid sequence of GUCY2CxCD3 bisp-AblTable 13. Full length nucleic acid sequence of GUCY2CxCD3 bisp-AblEngineering of GUCY2CxCD3 biAb-2

[0503] The GUCY2C binding arm of GUCY2CxCD3_biAb-2 is the same as for GUCY2CxCD3_biAb-l described above.

[0504] The CD3 binding arm of GUCY2CxCD3_biAb-2 is a high affinity CD3 binding arm in a spFv format, and will herein be referred to as anti-CD3 spFv-2 (CD3_spFv-2). The affinity of CD3_spFv-2 for human CD3 was measured by flow cytometry based Intellicyt assay as described above. The calculated EC50 of CD3_spFv-2 for human CD3 is 5-19 nM.

[0505] The stapled scFv in the VL-Linker-VH was further engineered into a Heavy Chain 2 (HC2) and expressed as IgGl comprising the Fc silencing mutation L234A / L235A / D265S and the heterodimerization (“knob” mutation T366W) in the Fc domain. HC2 comprising CD3_spFv-2 was then paired with the HC1 and the LC1 comprising the GUCY2C_Fab using the knob-in-hole heterodimerization technology to generate the stapled GUCY2CxCD3_biAb-2.Expression of GUCY2CxCD3 antibodies

[0506] The GUCY2CxCD3 bispecific antibodies were expressed in ExpiCHO-S cells (ThermoFisher; Cat # A29127) by transient transfection with purified plasmid DNAs encoding LC1, HC1, and HC2 using the ExpiFectamine CHO transfection kit (ThermoFisher; Cat # A29131). ExpiCHO-S cells were maintained in suspension inExpiCHO Expression Medium (ThermoFisher; Cat # A2910001) in an orbital shaking incubator set to 37 °C, 8% CO2 and 125 rpm. The cells were passaged into 4 sterile, vented, non-baffled Erlenmeyer flasks (Coming 431255) with 400mL starting culture volume per flask.

[0507] On the day of transfection (Culture Day 0), the flask cultures were between 5.15 - 8.99 x 10A6 viable cells / mL with a minimum viability of 98.2%. For the transfection, 200 pg of plasmid DNA encoding HC2, 200 pg plasmid DNA encoding HC1, and 400 pg plasmid DNA encoding LC1 (HC2:HC1 :LC1 chain ratio at 1 : 1 :2) were diluted in 64 mL of OptiPRO medium (ThermoFisher Cat # 12309019). 5.12 mL of ExpiFectamine CHO transfection reagent was diluted in another 64 mL of OptiPRO medium. The plasmid DNA plus OptiPRO medium mixture and the ExpiFectamine CHO reagent plus OptiPRO medium mixture were combined and incubated at room temperature for one minute. This was then equivalently distributed across the 4 flasks of cells, ~33 mL / flask. The transfected cells were returned to the orbital shaking incubator.

[0508] After overnight incubation (Culture Day 1), 240 mL of ExpiCHO Feed and 4.8 mL ExpiFectamine CHO Enhancer were combined and then distributed equivalently across the 4 flasks. Cells were further incubated until harvest (Culture Day 7). The culture supernatant from the transiently transfected cells (6.06 * 10A66 viable cells / mL, 84.7% viability) was clarified by centrifugation (15 min, 5000 rpm (5316 RCF)) followed by 0.2 pm filtration.Purification of GUCY2CxCD3 antibodies

[0509] The GUCY2CxCD3 bispecific antibodies were purified as follows. The filtered cell culture supernatant as prepared above was loaded onto a pre-equilibrated (l x DPBS, pH 7.2) custom 60 mL MabSelect PrismA column at 20 mL / min using an AKTA Purel50. After loading, the column was washed with 1 x DPBS, pH 7.2 until UV stabilized near baseline. The protein was eluted with 0.1 M sodium acetate, pH 3.45, and neutralized inline by the addition of 2.5 M Tris HC1, pH 7.5 to 10% (v / v) final volume, filtered (0.2 pm), and dialyzed into 20 mM MES, pH 5.5. The dialyzed eluate was further purified by cation exchange chromatography (CEX) using a custom Capto S ImpAct column (GE Healthcare, 2.6 cm x 36 cm, CV = 193 mL) pre-equilibrated into buffer A (20 mM MES, pH 6.5). The protein was loaded at 10 mL / min and then eluted from the column with a gradient of buffer B (20 mM MES, pH 6.5, I M NaCl: 0 - 5% buffer B over 10 min, 5 - 35% buffer B over135 min). The peak fractions containing only monomeric protein were pooled, dialyzed into l x dPBS, PH 7.2 and filtered (0.2 pm).EXAMPLE 4. CHARACTERIZATION OF GUCY2CxCD3 ANTIBODIESTumor cell binding specificity and T-cell binding specificity

[0510] One hour cell binding studies by flow cytometry were carried out at physiologically relevant temperature (37°C) on GUCY2CxCD3 bispecific antibodies (GUCY2CxCD3_biAb-l and GUCY2CxCD3_biAb-2), known reference GUCY2CxCD3 bispecific antibody (biAb), and NullxCD3 control molecules. The goal was to characterize and quantify specific, cell binding for GUCY2CxCD3_biAb-l and 2. As shown in FIG. 2A, GUCY2CxCD3_biAb-l and 2 displayed, strong, specific binding to the GUCY2C endogenously expressing cell line T84. Maximum binding of GUCY2CxCD3_biAb-l and 2 was higher than reference biAb. The NullxCD3 control did not bind to T84.

[0511] GUCY2CxCD3_biAb-l bound to primary T cells to a similar extent as the NullxCD3, suggesting no impact of the GUCY2C Fab binding arm on T-cell engagement. GUCY2CxCD3 biAb-2, bearing a higher affinity CD3 binder, bound primary T cells with higher potency than GUCY2CxCD3_biAb-l or NullxCD3 (FIG. 2B). Reference biAb also bound T cells as expected.

[0512] EC50 values for binding of GUCY2CxCD3_biAb-l and 2 to T84 cells and T cells are summarized in Table 14.Table 14.Epitope comparison

[0513] To determine whether the GUCY2C binder in GUCY2CxCD3_biAb-l and 2 share the same epitope as reference biAb, competition flow cytometry experiments were performed using parental GUCY2C mAbs from which GUCY2CxCD3_biAb-l,GUCY2CxCD3_biAb-2 and reference Ab were derived. To that end, human GUCY2C transfected CHO cells were stained with AF647-labeled reference mAb in conjunction with a titration of unlabeled GUCY2C mAb from which GUCY2CxCD3 biAb-1 and 2 are derived. Plates were then washed and bound AF647-labeled reference mAb was then detected in a flow cytometer. As shown in FIG. 3, unlabeled GUCY2C mAb did not compete with AF647-labeled reference mAb for binding, while unlabeled reference biAb did. This indicates that the epitope of the GUCY2C binder in GUCY2CxCD3_biAb-l and 2 is distinct from the epitope of the reference mAb.In vitro killing and T-cell activation

[0514] Impedance-based cytotoxicity assays performed on the RTCA xCELLigence (Agilent) platform were the primary means used to characterize the in vitro potency of GUCY2CxCD3 bispecific antibodies (GUCY2CxCD3_biAb-l and GUCY2CxCD3_biAb- 2), reference GUCY2CxCD3 bispecific antibody (biAb), and Null*CD3 control molecules. Experiments conducted on the IncuCyte® S3 (Sartorius) real-time, live cell imaging system were investigated as an orthogonal readout. CRC cell lines HT55, LS1034, and T84 were tested. They express GUCY2C at a range of 15000-40000 receptors / cell, characterized as a medium level of GUCY2C. To best approximate physiological relevance in the tumor microenvironment (TME), the lowest E:T ratios where maximal cytotoxicity was reached were used for lead identification and characterization.

[0515] As shown in FIG. 4A- FIG. 4F, GUCY2CxCD3_biAb-l induced T-cell mediated cytotoxicity and T-cell activation at an E:T ratio of 1 : 1 for T84 and a E:T ratio of 3 : 1 for both HT55 and LS1034 cells. GUCY2CxCD3_biAb-2 also induced T-cell mediated cytotoxicity and T-cell activation against these cell lines, but showed a hook effect at high concentrations. The reference biAb showed weaker T-cell mediated cytotoxicity and T-cell activation than both GUCY2CxCD3_biAb-l and 2. The control nullxCD3 spFv did not show significant T-cell mediated cytotoxicity or T-cell activation against these cell lines.

[0516] EC50 values are shown in Table 15.Table 15.Abbreviations: n / a = not applicable because curve fit was ambiguous and did not allow for accurate calculation of EC 50 valuesConformational Stability

[0517] The thermal stability of the GUCY2CxCD3_biAb-l was characterized by capillary VP-DSC microcalorimeter (Malvern Panalytical Malvern, UK). Temperature scans were performed from 25 to 100 °C in duplicate. A buffer reference scan was subtracted from protein scan and the concentration of protein was normalized prior to thermodynamic analysis. The data were plotted and analyzed using MicroCai PEAQ software. The DSC curve was fitted using non-two-state model to obtain the enthalpy and apparent transition temperature (Tm) values. GUCY2CxCD3_biAb-l showed good thermal stability (Table 16).Table 16.Human Protein Screening Array

[0518] GUCY2CxHumanCD3 bispecific antibody (evaluated as a monoclonal antibody to test the GUCY2C binder) was screened for binding against fixed HEK293 cells individually expressing a library of 6,108 full-length human plasma membrane, secreted, and cell-surface-tethered secreted proteins and of 397 human heterodimers.GUCY2CxHumanCD3 bispecific antibody was determined to be specific to its primary targets GUCY2C and CD3s. GUCY2CxHumanCD3 bispecific antibody bound to heterodimers of CD3y + CD3s and CD35 + CD3s. No off-target interactions were identified, demonstrating the target specificity of GUCY2CxHumanCD3 bispecific antibody.EXAMPLE 5. ORGANOID EXPERIMENT

[0519] T cell-mediated cytotoxic activity of GUCY2CxCD3_biAb-l was evaluated in tumor and WT intestinal human organoids. Organoids are in vitro 3D cultures derived fromnormal and diseased tissues. They faithfully mimic in vivo structure, functionality, and genetic features of original tissues. Because organoids retain the polarity of the original tissues, organoids could be utilized to probe the therapeutic hypothesis that GUCY2CxCD3_biAb-l would differentially affect tumors, which are depolarized, compared to normal tissue where GUCY2C maintains polarity.

[0520] To evaluate T cell-mediated cytotoxic activity of GUCY2CxCD3_biAb-l on WT and CRC tumor organoids, T cells and antibodies were incubated under normal culture condition (ie, with basal side facing outward) for 5 days. To measure T cell-mediated cytotoxic activity, an apoptosis signal (ie, NucView488-Caspase3 / 7 dye) was detected within prestained organoids (ie, CellTracer-Yellow) using IncuCyte live imaging. T-cell activation against organoids were measured using flow cytometry. GUCY2CxCD3_biAb-l induced dose-dependent apoptosis in CRC organoids and showed a clear selectivity window of cytotoxic activity between CRC and WT colon organoids, while a control EpCAMxCD3 biAb antibody (targeting EpCAM, a non-apically polarized tumor target) induced comparable robust cytotoxicity in both CRC and WT organoids (FIG. 5). Consistent with the high-content imaging result, evaluation of CD69, an early activation marker, on CD8+ T cells indicated a significant increase of CD69+ CD8+ cells in CRC organoids as compared to WT organoids in the presence of GUCY2CxCD3_biAb-l (data not shown).EXAMPLE 6. IN VIVO STUDIES IN MICE

[0521] The antitumor efficacy of GUCY2CxCD3_biAb-l was evaluated in the SC human colorectal HT55 and LS1034 cell line-derived xenograft (CDX) GUCY2C+ models in immune-compromised NSG (non-obese [NOD] diabetic SCID gamma) mice implanted with human CD3+ pan-T-cells. Twice weekly intraperitoneal (IP) treatments of GUCY2CxCD3_biAb-l were initiated after SC tumors were established.Anti -tumor efficacy in a T-cell humanized HT55 CDX model

[0522] The antitumor efficacy and pharmacodynamic effect of GUCY2CxCD3_biAb-l were evaluated in female NSG mice bearing SC HT55 human colorectal cell line derived tumors. HT55 tumor cells were implanted on Day 0, T-cells were implanted on Day 7, and treatment with GUCY2CxCD3_biAb-l occurred on Days 8, 12, 15, 19, 23, 27, 30, and 33 (dosing period indicated by the bar below the X-axis, FIG. 6). Mice were dosed twice weekly IP with GUCY2CxCD3_biAb-l at 0.005, 0.05, 0.5 and 1 mg / kg or DPBS for a totalof 8 doses (n=10 / group). Significant antitumor efficacy was observed with GUCY2CxCD3_biAb-l treatment at 0.05, 0.5, and 1 mg / kg with 111% delta tumor growth inhibition (ATGI) at all 3 dose levels (p<0.05, FIG. 6), as compared to the DPBS-treated control mice on Day 33 post tumor implantation. GUCY2CxCD3_biAb-l treatment at 0.05, 0.5, and 1 mg / kg resulted in 100% tumor regression (TR) and 10 of 10 complete tumor responses (CRs) in each treatment group by the end of the study. Treatment with GUCY2CxCD3_biAb-l at 0.005 mg / kg did not demonstrate tumor growth inhibition.

[0523] Additional SC HT55 tumor-bearing mice were dosed IP with GUCY2CxCD3_biAb-l twice weekly at 0.05 and 0.5 mg / kg, or DPBS for 3 doses for pharmacodynamic evaluation. Tumors were collected from 3 animals per treatment group on Day 12 (48 hours post first dose), Day 14 (24 hours post second dose), and Day 18 (24 hours post third dose) for H4C analysis. Representative micrographs of CD8, GUCY2C, and pan- cytokeratin immune-stained tumor samples are shown in FIG. 7. GUCY2CxCD3_biAb-l treatment demonstrated a time- and dose-dependent increase in CD8+T-cell tumor infiltrate, with T-cell tumor infiltrate detected beginning on Day 12, 48 hours after the first treatment. Similar results were observed with CD4+staining (data not shown). As compared with HT55 tumors treated with DPBS, GUCY2CxCD3_biAb-l treatment resulted in a time- and dosedependent reduction of human GUCY2C+ tumor cells. Similar results with the same expression pattern were observed with the human pan-cytokeratin staining, confirming that viable tumor cells still expressed target. After 3 doses of GUCY2CxCD3_biAb-l at either 0.05 or 0.5 mg / kg, tumor cells were no longer detected by GUCY2C or human pan- cytokeratin immunostaining.

[0524] Anti-tumor efficacy in female NSG mice bearing SC LS1034 human colorectal CDX tumors

[0525] The efficacy of GUCY2CxCD3_biAb-l was further evaluated in female NSG mice bearing SC LSI 034 human colorectal CDX tumors. LSI 034 tumor cells were implanted on Day 0, T-cells were implanted on Day 12, and treatment occurred on Days 13, 17, 20, 24, 27, 31, 34, and 38 (dosing period indicated by the bar below the X-axis, FIG. 8). Mice were dosed twice weekly IP with GUCY2CxCD3_biAb-l at 0.005, 0.01, 0.05, 0.5 and 1 mg / kg or DPBS for a total of 8 doses (n=10 / group). Significant antitumor efficacy was observed with GUCY2CxCD3_biAb-l antibody treatment at 0.05, 0.5, and 1 mg / kg with 73%, 106%, and 106% ATGI respectively, as compared to the DPBS-treated control mice on Day 38 post tumor implantation (p<0.05, FIG. 8). Treatment with GUCY2CxCD3_biAb-l at 0.5 and 1 mg / kg resulted in 100% TR and 10 of 10 CRs for each group by the end of thestudy. Treatment with GUCY2CxCD3_biAb-l at 0.05 mg / kg resulted in 2 of 10 partial tumor responses (PRs) by the end of the study. GUCY2CxCD3_biAb-l treatment at 0.005 and 0.01 mg / kg did not elicit biologically significant tumor growth inhibition (defined as >60% TGI, with 0% and 8% TGI respectively, as compared to the DPBS-treated control mice on Day 38 post tumor implantation.

[0526] Collectively, these data demonstrated the potent in vivo antitumor efficacy of GUCY2CxCD3_biAb-l across multiple in vivo tumor models expressing GUCY2C, which correlated to intratumoral T-cell infiltration.EXAMPLE 7. AFFINITY AND FUNCTIONAL ACTIVITY OF GUCY2CXCD3 BISPECIFIC ANTIBODY WITH CYNOMOLGUS MONKEY PBMCSAffinity of GUCY2CxHumanCD3 bispecific antibody (GUCY2CxCD3 biAb-1) and GUCY2CxCynoCD3 bispecific antibody (GUCY2CxCD3_biAb-3)

[0527] The GUCY2C-binding arm of GUCY2CxCD3_biAb-l of Tables 12-13 (GUCY2CxHumanCD3) is cynomolgus monkey cross-reactive (cell binding EC50 = 21.31 nM) but its CD3-binding arm is not cynomolgus monkey cross-reactive (cf. Table 20 below).

[0528] When pharmacological activity is absent with a large molecule, a toxicology study will have limited value for risk assessment to support early clinical development and is generally discouraged per International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). Therefore, an anti-GUCY2CxCD3 bispecific antibody was generated containing the same GUCY2C-binding arm as GUCY2CxCD3_biAb-l (GUCY2CxHumanCD3 bispecific antibody) but including a cynomolgus monkey cross-reactive CD3 -binding arm (CynoCD3 arm). This CD3 cynomolgus monkey cross reactive bispecific antibody will be referred to as GUCY2CxCynoCD3 bispecific antibody. The CDR sequences of the CynoCD3 arm are listed in Table 17.Table 17. HCDRs and LCDRs of CynoCD3 arm of GUCY2CxCynoCD3 under the Kabat delineationTable 18. VH, VL and VH-Linker-VL of the CynoCD3 arm of GUCY2CxCynoCD3Table 19. Full length amino acid sequence of the CynoCD3 arm of GUCY2CxCynoCD3

[0529] The binding affinity to cynomolgus monkey CD3 of the cynomolgus monkey cross-reactive CD3 arm (CynoCD3 arm) of GUCY2CxCynoCD3 was 2 to 20 times higher than the binding affinity to human CD3 of the human CD3 -specific arm of the GUCY2CxHumanCD3 bispecific antibody (Table 20). In one monkey T cell donor, the binding affinity of the CD3 arm of GUCY2CxCynoCD3 for monkey CD3 was 6 times higher than the binding affinity of the CD3 arm of GUCY2CxhumanCD3 for human CD3 in a human T cell donor. This higher CD3 binding affinity of GUCY2CxCynoCD3 bispecific antibody may result in higher levels of cytokine release. On the other hand, tumor-associated antigen levels will be higher in cancer patients with active disease, which may cause an underestimation of the cytokine release triggered by GUCY2CxCynoCD3 bispecific antibody in the cynomolgus monkey. Therefore, the repeat-dose toxicology studies conducted with GUCY2CxCynoCD3 bispecific antibody are considered hazard identification (ID) studies that inform on potential safety concerns associated with GUCY2CxHumanCD3 bispecific antibody, but not the clinical dose or exposure at which these findings may manifest. GUCY2CxCynoCD3 bispecific antibody remains however an appropriate surrogate molecule in cynomolgus monkey to model the pharmacology and any associated potential toxicity resulting from GUCY2CxHumanCD3 bispecific antibody administration in humans.

[0530] Table 20 contains a summary of the molecular characteristics of GUCY2CxHumanCD3 bispecific antibody and GUCY2CxCynoCD3 bispecific antibody.Table 20. Summary of molecular characteristics of GUCY2CxHumanCD3 bispecific antibody (GUCY2CxCD3_biAb-l) and GUCY2CxCynoCD3 bispecific antibody.Identity Method Human NHPGUCY2C arm GUCY2C binder of GUCY2C EC504.385 to EC5014.57 toGUCY2CxHumanCD3 cell-based 7.092 nM 37.49 nM bispecific antibody and binding ofGUCY2CxCynoCD3 bispecific antibodyCD3 arm of CD3 binder of SPR (CD3) 110±48 nM Not cross-GUCY2CxHumanCD3 GUCY2CxHumanCD3 (SPR) reactive bispecific antibody bispecific antibody T-cell 94.28 to binding 126.3 nMCD3 arm of CD3 binder of SPR (CD3) 15.7±8.31 nM 11.7±0.47 nMGUCY2CxCynoCD3 GUCY2CxCynoCD3 T-cell 4.5 nM 5 nM bispecific antibody bispecific antibody bindingNHP, nonhuman primate; SPR, surface plasmon resonance.Functional activity of GUCY2CxHumanCD3 bispecific antibody and GUCY2CxCynoCD3 bispecific antibody

[0531] An assessment of the in vitro functional activity of GUCY2CxCynoCD3 bispecific antibody was conducted. T-cell activation induced by GUCY2CxCynoCD3 bispecific antibody, and a known GUCY2CxCD3 bispecific antibody comparator (herein referred to as Reference GUCY2CxCD3 bisp), in the presence of cynomolgus monkey T cells was measured by flow cytometry after a 48hr co-culture with CHO-K1 cells overexpressing cynomolgus monkey GUCY2C (FIG.9A-9C and Table 21) and compared to the activity of GUCY2CxCynoCD3 bispecific antibody or the reference GUCY2CxCD3 bispecific, in the presence of human T cells after a co-culture with CHO-K1 cells overexpressing human GUCY2C (FIG. 10). A commercially available anti-GUCY2C mAb that cross-reacts with cynomolgus monkey GUCY2C was used to confirm the cell surface expression of cynomolgus monkey or human GUCY2C on the CHO-K1 target cell lines. Parental untransduced CHO-K1 target cell lines, as well as a NullxCD3 compound, were used as negative controls for both human and cynomolgus monkey T cell activity. These data demonstrated that the functional impact of GUCY2CxCynoCD3 bispecific antibody on cynomolgus monkey T cell activation is pharmacologically similar to that mediated by GUCY2CxHumanCD3 bispecific antibody on human T cell activation (Table 21). Further supporting the translational relevance of GUCY2CxCynoCD3 bispecific antibody, these data also demonstrate similar relative potency between GUCY2CxCynoCD3 bispecific antibody relative to reference GUCY2C bisp on cynomolgus monkey T cells and GUCY2CxCynoCD3 bispecific antibody relative to the reference GUCY2CxCD3 bispecific antibody on human T cells. These data support that GUCY2CxCynoCD3 bispecific antibody is an appropriate surrogate molecule in cynomolgus monkey to evaluate the pharmacology and any associated potential toxicity that may result from GUCY2CxHumanCD3 bispecific antibody administration in humans.Table 21. Estimated ECso and maximum cynomolgus monkey T-cell CD25 activation after 48 hours co-culture with recombinant GUCY2C-expressing CHO-K1 target cell lines, and human T-cell CD25 activation for comparison.Cynomolgus Monkey CD25Human CD25CD, cluster of differentiation; ECso, 50% effective concentrationEXAMPLE 8. IN VIVO TOXICOLOGY OF GUCY2CxCynoCD3 BISPECIFIC ANTIBODY IN CYNOMOLGOUS MONKEY2-Week Subcutaneous Repeat-Dose Toxicity Study of GUCY2CxCynoCD3 bispecific antibody in Cynomolgus Monkeys

[0532] A dose-range finding study (TOX15268) was conducted in female cynomolgus monkeys (n = 2 females / dose) using GUCY2CxCynoCD3 antibody to assess relevant target- related toxicity and TK, and to enable dose selection for the GLP compliant 1 -month hazard ID study in male and female cynomolgus monkeys. GUCY2CxCynoCD3 antibody was administered SC as a solution in Dulbecco’s PBS and 0.05% PS80 (pH 7.2) once weekly for two weeks at 0.03, 0.3 and 1.2 mg / kg, respectively. Criteria for evaluation included: mortality, clinical observations, body weights, qualitative food consumption, veterinary physical and vital signs examinations, body temperature, clinical pathology parameters(hematology, coagulation, clinical chemistry, and urinalysis), toxicokinetic parameters, immunophenotyping, cytokine analysis, organ weights, and macroscopic and microscopic examinations. Scheduled necropsies were conducted on Day 15.

[0533] TK parameters are illustrated in Table 22. Average Cmax and AUC0-7d increased in an approximate dose-proportionate manner. The accumulation ratios forGUCY2CxCynoCD3 antibody in the systemic circulation were 1.57, 1.59 and 1.88 for the 0.03, 0.3 and 1.2 mg / kg dose groups respectively, indicating a trend toward a slight accumulation on Day 8.Table 22. Mean serum GUCY2CxCynoCD3 bispecific antibody TK parameter estimates following weekly SC dose of GUCY2CxCynoCD3 bispecific antibody in female cynomolgus monkeys.

[0534] There were no unscheduled deaths at any dose level tested throughout the study. At all doses, GUCY2CxCynoCD3 antibody -related findings included minimal to moderate increases in C-reactive protein (CRP; 19 to 132-fold above baseline) and minimally elevated fibrinogen levels (1 to 2-fold) (acute phase response), minimally to mildly decreasedcholesterol concentrations (0.6 to 0.8-fold), and dose-independent increases in plasma monocyte chemoattractant protein-1 (MCP-1), ZFNy and IL-10 levels in most animals, peaking at 6 hours post-dose. At >0.3 mg / kg, bilirubin levels were minimally elevated, but not associated with hepatobiliary findings, and diffuse interstitial lymphoid cell infiltrates were noted in exocrine and endocrine pancreas of one female at 0.3 mg / kg (minimal) and one female at 1.2 mg / kg (slight), which were considered to be likely related to the CD3 targeting arm and were consistent with the modality of CD3 redirectors. At 1.2 mg / kg, one animal was noted with vomitus and brown particles in the cage two hours post-dose, and slight to moderate liquid particles were present in the cage of two animals on Day 1. These findings were absent after Day 1. A decreased body weight was noted for one animal (5.7% decrease by Day 7 versus Day -1, which remained stable afterwards until the end of the study), which did not correlate with clinical signs or reduced food consumption. No GI tissue toxicities were observed at necropsy by histopathology assessment.

[0535] All findings described above were considered non-adverse due to their low magnitudes of change, the generally minimal severity, and / or lack of correlative findings. The highest tolerated dose was considered to be 1.2 mg / kg, the highest dose administered in this study, corresponding with mean Cmax and AUCo-7d values of 7.36 pg / mL and 38.29 pg.day / mL respectively, after Day 8 of treatment.1 -Month Repeat-Dose Toxicity Study of GUCY2CxCynoCD3 antibody in Cynomolgus Monkeys

[0536] The GLP compliant repeated dose 1 -month hazard ID study was performed in male and female cynomolgus monkeys (n = 3 / sex / dose) with GUCY2CxCynoCD3 antibody. GUCY2CxCynoCD3 antibody was administered in formulation buffer once weekly by SC injection at doses of 0 mg / kg or 0.3 mg / kg on Days 1, 8, 15, 22 and 29, or a step-up dose of 1 mg / kg on Day 1 followed by a dose of 3 mg / kg on subsequent dosing days. Criteria for evaluation included: mortality, clinical observations, body weights, food consumption, injection site observations, veterinary physical examinations, ophthalmology, qualitative and quantitative electrocardiology, body temperature, neurologic examination, blood pressure and heart rate, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), anti-drug antibody analysis, bioanalysis and toxicokinetics, immunophenotyping, cytokines, organ weights, and macroscopic and microscopic examinations. Scheduled necropsies were conducted at the end of the dosing period.

[0537] TK data are summarized in Table 23. At all dose levels, mean exposure of GUCY2CxCynoCD3 antibody was comparable between males and females, except AUCo-7d on Day 22 at 0.3 mg / kg likely due to ADA impact. Exposure increased approximately dose- proportionally from 0.3 to 1 mg / kg on Day 1, ie, a 3.1-fold increase in Cmax and approximate 4.1-fold increase in AUCo-7d, and from 0.3 to 3.0 mg / kg on Day 22, a 6.6-fold and 8.7-fold increase in mean Cmax and AUCo-7d values, respectively. Systemic exposure generally increased following repeated SC administration of GUCY2CxCynoCD3 antibody with a mean accumulation AUCo-7d ratio of 3.48 on Day 22.Table 23. Mean Serum TK Parameters of GUCY2CxCynoCD3 bispecific antibody Following 1 -Month Repeated SC Dosing to Cynomolgus Monkeys.DayaAnimals in Group 3 received a single administration of 1 mg / kg GUCY2CxCynoCD3 bispecific antibody on Day 1 followed by weekly administration of 3 mg / kg GUCY2CxCynoCD3 bispecific antibody from Day 8 onwards. b RAUC: Mean of individual accumulation ratios based on AUC

[0538] The presence of AD As was evaluated for all treated animals on Day 1, 8, 22 and29 predose. Anti-GUCY2CxCynoCD3 bispecific antibodies were detected in serum of all animals, except one animal dosed at 1 then 3 mg / kg (no. 3003), and initially appeared onDay 22 at pre-dose. GUCY2CxCynoCD3 serum concentration-time profiles and AUCo ?d values (post dosing on Day 22) were reduced for all ADA positive animals, except for one female at 0.3 mg / kg (no. 2503) and one male at 1 then 3 mg / kg (no. 3501) (FIG. 11 A and FIG. 1 IB).

[0539] There were no unscheduled deaths in this study. In addition, no GUCY2CxCynoCD3 antibody -related findings were observed on clinical observations, food consumption, body weights, injection site observations, physical examinations, ophthalmology, clinical pathology parameters, immunophenotyping, organ weights, macroscopy or microscopic histopathology.

[0540] At >0.3 mg / kg, GUCY2CxCynoCD3 antibody administration triggered a subtle acute phase response, which included minimal transient increases in CRP in some animals (up to 14-fold above baseline on Day 15 and / or 30) and dose-dependent increases in plasma MCP-1 concentrations (3 to 25-fold changes at 0.3 mg / kg, and 9 to 41-fold changes for the step-dose of 1 mg / kg followed by 3 mg / kg) on Day 1 : 6 hours post-dose in all animals at >0.3 mg / kg. These increases in MCP-1 levels generally peaked at Day 1 : 6 hours post-dose and trended toward baseline by Day 1 : 24 hours post-dose, with other transient increases of a lesser magnitude observed in some animals following dosing on Days 8, 22, and 29.

[0541] Slight abdominal distension was noted in three animals from the high dose group on veterinary examinations at Day 24 / 25, and vomitus was observed in one animal on Day 29. However, there were no signs of abdominal distension at necropsy and no GI tissue toxicities were observed by histopathology assessment. Since these findings were transient, not associated with GI tissue toxicity, and common background observations in cynomolgus monkey, these findings were considered incidental and not related to the administration of GUCY2CxCynoCD3 antibody.

[0542] Based on these data, the no-observed-adverse-effect level (NOAEL) is considered to be 1 mg / kg (Day 1) followed by 3 mg / kg (Day 8 onwards), the highest dose tested, corresponding with a Cmax of 28.3 pg / mL and AUC0-7d of 175 day.pg / mL on Day 22 (males / females combined).EXAMPLE 9. SINGLE-DOSE PHARMACOKINETICS OF GUCY2CxHumanCD3 BISPECIFIC ANTIBODY

[0543] The PK properties of the GUCY2CxHumanCD3 bispecific antibody were characterized in female cynomolgus monkeys (n = 3) following a single bolus IV injection at0.5 mg / kg. PK parameters are summarized in Table 10. Mean Cmax and AUCinf following a single IV dose of 0.5 mg / kg GUCY2CxHumanCD3 bispecific antibody were 12.1 pg / mL and 41.8 pg.day / mL, respectively. The mean clearance (Cl) in cynomolgus monkeys was 12.1 mL / day / kg, volume of distribution (Vz) 102 mL / kg, and estimated half-life (T1 / 2) approximately 6 days. Table 24 shows mean PK parameters of GUCY2CxHumanCD3 bispecific antibody Following a Single IV Dose of 0.5 mg / kg in Cynomolgus MonkeyTable 24.AUCinf=area under the serum drug concentration-time curve from time 0 to infinity;AUCiast=area under the serum concentration-time curve from time zero to the time of the last quantifiable concentration; CL=systemic clearance; Cmax=maximum observed concentration; IV=intravenous; PK=pharmacokinetics; SD=standard. deviation; tl / 2=terminal half-life; Vz=volume of distribution in the terminal phase.EXAMPLE 10. A PHASE 1 STUDY OF GUCY2CxCD3 BISPECIFIC ANTIBODY FOR ADVANCED STAGE COLORECTAL CANCER AND OTHER SOLID TUMORS

[0544] This is a first-in-human (FIH), Phase 1, open-label, multi-center study to determine the safety, PK, PD, and preliminary antitumor activity of GUCY2CxHumanCD3 bispecific antibody in participants with gastrointestinal cancer, specifically colorectal cancer, specifically metastatic colorectal cancer (mCRC), specifically refractory mCRC, or unresectable metastatic colorectal cancer.

[0545] The primary objectives of this study are to determine the RP2Ds of GUCY2CxHumanCD3 bispecific antibody and to determine the safety and tolerability of GUCY2CxHumanCD3 bispecific antibody at the RP2D(s). Key secondary objectives are to assess PK, PD, immunogenicity, and preliminary antitumor activity of GUCY2CxHumanCD3 bispecific antibody.

[0546] The study will be conducted in 2 parts: Dose Escalation (Part 1) and Dose Expansion (Part 2). During the study, all available data will be monitored at each dose escalation step and at regular intervals during expansion.

[0547] Approximately 120 participants (60 participants in Part 1 and 60 participants in Part 2) are estimated to be enrolled in this study.Part 1 - Dose escalation

[0548] Part 1 is designed to identify the RP2D(s) of GUCY2CxHumanCD3 bispecific antibody in participants with mCRC who have progressed on at least 2 or more lines of standard therapy. Participants must have received or are not eligible to receive prior fluoropyrimidine (such as 5 -fluorouracil or capecitabine), oxaliplatin, irinotecan, and bevacizumab.

[0549] A MABEL approach has been used to determine the FIH starting dose.Subsequent dose levels will be determined based on review of the totality of the data at each dose level. Multiple dose regimens may be enrolled simultaneously, provided each new dose level and schedule is supported by the PK analysis and BOIN escalation rules. To better understand the safety, tolerability, PK, PD, and preliminary antitumor activity, additional participants may be enrolled at one or more PK / PD cohorts at or below doses or schedules considered to be safe. These participants will be required to provide additional blood samples and mandatory pre- and on-treatment tumor biopsies.Part 2 - Dose expansion

[0550] In Part 2, GUCY2CxHumanCD3 bispecific antibody will be administered at or a dose level below the RP2D as determined in Part 1 to participants with mCRC.Justification of Dose

[0551] The proposed FIH starting dose is less than 1 mg Q2W via SC administration. This starting dose was projected using a MABEL approach matching the median EC50 estimates for T-cell activation marker (CD3+ / CD69+).

[0552] The human GUCY2C-expressing cell line, T84, and PBMCs from 6 healthy donors were used to derive MABEL concentrations. The ability of GUCY2CxHumanCD3 bispecific antibody to induce T-cell activation, cytotoxicity and cytokine release was evaluated in T84 (human colorectal) cells co-cultured with human PBMCs from healthy donors (n=6) at E:T-cell ratio of 1 : 1, the lowest tested E:T ratio where maximal cytotoxicitywas reached. GUCY2CxHumanCD3 bispecific antibody induced concentration-dependent T-cell activation assessed by CD69+ T-cells at 96 hours. GUCY2CxHumanCD3 bispecific antibody induced concentration-dependent and T-cell dependent cytotoxicity of T84 cells at 144, 168 and 192 hours. Cytokines were tested at 96 hours and included IFN-y, IL-ip, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, TNF-a and IL-12p70. Concentration-dependent increases in IFN-y, IL-2, IL-6, IL-8, IL-10, IL-13 and TNF-a were observed.

[0553] The EC values for T'-cell activation were consistently lower than that of cytotoxicity as well as cytokine release. The most sensitive read out, T-cell activation (CD3+ / CD69+), was used to determine the MABEL concentration.

[0554] The PK of GUCY2CxHumanCD3 bispecific antibody was studied in cynomolgus monkeys. A two-compartment model was built for the calculation of human PK. In this model, the CL and apparent volume of distribution based on the terminal phase (Vz) were allometrically scaled (with an exponent of 0.85 for CL and 1.00 for Vz). The human PK was calculated based on an average body weight of 75 kg.Study treatment administration

[0555] The study treatment administration is summarized in Table 25.Table 25.Objectives and End PointsPrimary endpoints and secondary endpoints

[0556] The primary endpoints are the occurrence of DLTs and occurrence of and severity of AEs assessed as the frequency of DLTs and the incidence and severity of AEs for Part 1 and Part 2, respectively.

[0557] The secondary endpoints are:(1) Serum concentration-time profiles and PK parameters, including but not limited to Cmax, Cmin, Tmax, and AUC(2) Presence of GUCY2CxHumanCD3 antibody antibodies(3) Using the Response Evaluation Criteria in Solid Tumors (RECIST) vl .1 :(4) Overall response (OR) is defined as a best response of PR or better(5) Complete response is defined as a best response of complete response (CR).(6) Time to Response (TTR): is defined for participants who achieved an overall response as the time from the first dose of study treatment to the first response of partial response (PR) or better. TTR is not defined for participants who did not achieve a response of partial response or better.(7) Duration of response (DOR) is defined for participants who achieved an overall response as the time between the date of initial documentation of first response of PR or better to the date of first documented evidence of progressive disease or death due to any cause, whichever occurs first. DOR is not defined for participants who did not achieve a response of PR or better.

[0558] The Objectives and Endpoints of Trial are summarized in Table 26.Table 26.Study Population

[0559] The inclusion and exclusion criteria for enrolling participants in this study are described below.Inclusion criteria

[0560] Each potential participant must satisfy all of the following criteria to be enrolled in the study.(1) Be >18 years of age (or the legal age of consent in the jurisdiction in which the study is taking place) at the time of informed consent.(2) Have histologically or cytologically confirmed diagnosis of CRC progressing after 2 or more prior lines of standard therapy in the metastatic / unresectable setting. Participants must have previously received fluoropyrimidine (such as 5 -fluorouracil, capecitabine, or SI), oxaliplatin, irinotecan, and bevacizumab. Participants with microsatellite unstable (MSI) / deficient mismatch repair (dMMR) CRC must additionally have received prior immune checkpoint inhibitor (such as nivolumab + / - ipilimumab, pembrolizumab, or dostarlimab).(3) Have evaluable or measurable disease per RECIST vl. l (or at least 1 measurable lesion) a. Part 1 : Must have either measurable or evaluable disease b. Part 2: Must have at least 1 measurable lesion(4) Eastern Cooperative Oncology Group (ECOG) performance status of 0-1.(5) Can have a prior or concurrent second malignancy (other than the disease under study) whose natural history or treatment is unlikely to interfere with any study endpoints of safety or the efficacy of the study treatment(s).(6) Hematology laboratory parameters within the following range without prior growth factor support within 3 weeks or transfusion within 7 days before the day the laboratory test was performed: a. Absolute neutrophil count >1000* 109 / L b. Hemoglobin >8.0 g / dL c. Platelets >80x 109 / L(7) Clinical chemistry laboratory parameters within the following range during the screening period:a. aspartate aminotransferase (AST) and alanine ami...

Claims

CLAIMS1. A method of treating a cancer of the digestive system, in a human subject, wherein the method comprises administering to the subject a therapeutically effective amount of an anti- GUCY2CxCD3 antibody comprising a first antigen binding domain that binds specifically to GUCY2C, and a second antigen binding domain that binds specifically to CD3, wherein: a. the first antigen domain that binds GUCY2C comprises a first heavy chain variable region (VH1) of SEQ ID NO: 7 and a first light chain variable region (VL1) of SEQ ID NO: 8, and b. the second antigen domain that binds CD3 comprises a second heavy chain variable region (VH2) of SEQ ID NO: 17 and a second light chain variable region (VL2) of SEQ ID NO: 18.

2. The method of claim 1, wherein the anti-GUCY2CxCD3 antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9 and a first light chain (LC1) of SEQ ID NO: 10.

3. The method of claim 1 or 2, wherein the anti-GUCY2CxCD3 antibody comprises a second heavy chain (HC2) of SEQ ID NO: 20.

4. The method of any one of claims 1-3, wherein the anti-GUCY2CxCD3 antibody is administered to the subject subcutaneously or intravenously.

5. The method of any one of claims 1-4, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose of less than about 1000 mg per dose of administration.

6. The method of any one of claims 1-5, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose ranging from about 1 mg to about 50 mg (per dose of administration).

7. The method of any one of claims 1-6, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose ranging from about 2 mg to about 50 mg (per dose of administration).

8. The method of any one of the preceding claims, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose from about 0.5 to about 25 mg (per dose of administration), more particularly from about 0.5 to about 15 mg (per dose of administration), more particularly from about 0.5 to about 12 mg (per dose of administration), more particularly from about 0.5 to about 6.5 mg (per dose of administration).

9. The method of any one of claims 1-8, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose, which is a flat dose.

10. The method of any one of claims 1-9, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose, which is administered once every 1-9 weeks, for example, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 6 weeks, once every 8 weeks or once every 9 weeks.

11. The method of any one of claims 1-10, wherein the administering of the therapeutically effective amount of the anti-GUCY2CxCD3 antibody prevents disease progression in the subject.

12. The method of any one of claims 1-11, wherein the anti-GUCY2CxCD3 antibody is administered at a treatment dose that prevents disease progression in the subject.

13. The method of any one of claims 1-12, which comprises administering one or more step- up doses of the anti-GUCY2CxCD3 antibody prior to administering any treatment dose of the anti-GUCY2CxCD3 antibody, wherein the one or more step-up doses are lower than the treatment dose.

14. The method of claim 13, wherein the one or more step-up doses are administered 2-15 days, such as 1 week from each other and wherein the step-up dose that is administered as the last step-up dose before the first administration of the treatment dose, is administered 2- 15 days, such as 1 week, prior to the first administration of the treatment dose.

15. The method of claim 13 or 14, wherein each step-up dose ranges from about 1 mg to about 50 mg and wherein the treatment dose ranges from about 1 mg to about 50 mg or from about 2 mg to about 50 mg.

16. The method of claim 13 or 14, wherein each step-up dose ranges from about 0.5 mg to about 25 mg.

17. The method of claim 16, wherein each step-up dose ranges from about 0.5 mg to about 12 mg.

18. The method of claim 17, wherein each step-up dose ranges from about 0.5 mg to about 5 mg.

19. The method of any one of the foregoing claims, wherein the method does not induce a non-hematologic treatment-related toxicity in the subject, wherein the non -hematologic toxicity is one or more of the following: a. Grade >3 CRS b. Grade 4 hypertension c. an increased Aspartate aminotransferase (AST) and Alanine Aminotransferase (ALT) within the following range: i. an increase >8x Upper Limit of Normal (ULN) for any duration, if the baseline is within normal limits; ii. an increase >5x ULN for >14 days, if the baseline is within normal limits; iii. ALT or AST value >3x ULN, total bilirubin >2x ULN, and ALP <2x ULN with no alternative etiology if Hy’s Law criteria are met and baseline is within normal limits; iv.an increase >5x baseline for any duration if the baseline AST / ALT is elevated above ULN; v. an increase >3x baseline for >14 days if the baseline AST / ALT is elevated above ULN; or vi. ALT or AST >3x baseline or AST or ALT >8x ULN, whichever is lower, combined with total bilirubin >2x baseline AND >2x ULN, with no alternativeetiology, if modified Hy’s Law criteria baseline are met and baseline AST / ALT is elevated above ULN; and d. any Grade >3 non -hematological toxicity with the following exceptions: i. First occurrence of Grade 3 systemic administration-related reaction (sARR) that improves to Grade <2 within 72 hours; ii. Grade 3 fatigue or asthenia lasting <7 days with best supportive care; iii. Grade 3 fever in the absence of neutropenia that resolves within 48 hours; iv. Grade 3 nausea, vomiting, constipation, or diarrhea / colitis lasting < 72 hours with best supportive care; v. Grade>3 hypertension that can be controlled by oral medical management and improves to Grade <2 within 72 hours; vi. Grade >3 lipase or amylase increase not associated with clinical or radiological evidence of pancreatitis; vii. Grade >3 pain due to tumor flare that improves to Grade <2 with best supportive care within 5 days; or viii. Grade >3 ALP, GGT, or electrolyte abnormalities that resolve spontaneously to Grade <2 or baseline within 5 days, or that respond to best supportive care.

20. The method of any one of the foregoing claims wherein the subject is a. a subject having histologically or cytologically confirmed diagnosis of colorectal adenocarcinoma (CRC) progressing after 2 or more prior lines of standard therapy in the metastatic / unresectable setting. b. a subject having evaluable or measurable disease per RECIST vl .1 (or at least 1 measurable lesion) c. a subject having Eastern Cooperative Oncology Group (ECOG) performance status of 0-1. d. a subject having hematology laboratory parameters within the following range without prior growth factor support within 3 weeks or transfusion within 7 days before the day the laboratory test was performed: i. Absolute neutrophil count >1000>< 109 / L; ii. Hemoglobin >8.0 g / dL; or iii. Platelets >80x 109 / Le. a subject having clinical chemistry laboratory parameters within the following range during the screening period: i. aspartate aminotransferase (AST) and alanine aminotransferase (ALT) <3 x upper limit of normal (ULN) or < 5 x ULN if documented liver involvement by disease, or ii. Serum total bilirubin <1.5 x ULN; participants with congenital bilirubinemia, such as Gilbert’s syndrome, may enroll if conjugated bilirubin is < 1.5 x ULN; and f. a subject having an estimated or measured glomerular filtration rate (GFR) >30 mL / min based on modification of diet in renal disease (MDRD) 4-variable formula.

21. The method of any one of claims 1-20, wherein the cancer of the digestive system is a cancer of the gastrointestinal system.

22. The method of any one of claims 1-21, wherein the cancer of the digestive system is a colorectal cancer.

23. The method of claim 22, wherein the colorectal cancer is a metastatic colorectal cancer.

24. The method of claim 23, wherein the metastatic colorectal cancer is unresectable metastatic colorectal cancer.

25. The method of any one of claims 1-24, wherein the cancer of the digestive system is a gastric cancer.

26. The method of claim 25, wherein the gastric cancer is a primary or metastatic gastric cancer.

27. The anti-GUCY2CxCD3 antibody as defined in any one of the foregoing claims for use in the method of any one of the foregoing claims.

Images