Antibodies that specifically bind to GCC

Abstract

Provided herein are antibodies that specifically bind to guanylate cyclase C (GCC), and conjugates comprising the antibodies.

Description

[0001] Related Applications

[0002] This application claims the priority of International Application No. PCT / CN2022 / 133632, filed on November 23, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This application relates to antibodies that specifically bind to guanylate cyclase 2C (GCC). Also provided are methods for preparing the antibodies and uses of the antibodies. Background Art

[0004] Guanylate cyclase 2C, also known as guanylate cyclase C (GCC), intestinal guanylate cyclase, guanylate cyclase-C receptor, or heat-stable enterotoxin receptor (hSTAR), is an enzyme encoded by the GUCY2C gene in humans. It is expressed at mucosal cells (e.g., at the apical brush border membrane of the small intestine, large intestine, and rectum), where it can regulate intestinal homeostasis and has emerged as a tumor suppressor. In addition, it is ubiquitously expressed in advanced metastatic colorectal tumors and other cancer types arising through intestinal metaplasia, thus fulfilling many characteristics of an attractive target for treating malignancies. Summary of the Invention

[0005] In one aspect, provided herein is an antibody that specifically binds to guanylate cyclase 2C (GCC), comprising CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NOs. 1-10 and 118-123. In some embodiments, CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, CDR1, CDR2, and CDR3 are numbered according to Kabat.

[0006] In another aspect, the present disclosure provides an antibody that specifically binds to guanylate cyclase 2C (GCC), comprising: (1) CDR1, which comprises an amino acid sequence selected from: (i) SEQ ID NOs. 11-20 and 124-129, and (ii) an amino acid sequence having one or more amino acid alterations compared to SEQ ID NOs. 11-20 and 124-129; (2) CDR2, which comprises an amino acid sequence selected from: (i) SEQ ID NOs. 21-30 and 130-135, and (ii) an amino acid sequence having one or more amino acid alterations compared to SEQ ID NOs. 21-30 and 130-135; and (3) CDR3, which comprises an amino acid sequence selected from: (i) SEQ ID NOs. 31-40 and 136-141, and (ii) an amino acid sequence having one or more amino acid alterations compared to SEQ ID NOs. 31-40 and 136-141.

[0007] In some embodiments of the antibody of the present application, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 11 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 11, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 21 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 21, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 31 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 31.

[0008] In some embodiments of the antibody of the present application, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 12 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 12, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 22 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 22, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 32 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 32.

[0009] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 13 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 13, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 23 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 23, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 33 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 33.

[0010] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 14 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 14, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 24 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 24, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 34 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 34.

[0011] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 15 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 15, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 25 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 25, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 35 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 35.

[0012] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 16 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 16, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 26 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 26, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 36 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 36.

[0013] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 17 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 17, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 27 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 27, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 37 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 37.

[0014] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 18 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 18, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 28 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 28, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 38 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 38.

[0015] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 19 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 19, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 29 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 29, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 39 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 39.

[0016] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 20 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 20, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 30 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 30, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 40 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 40.

[0017] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 124 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 124, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 130 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 130, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 136 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 136.

[0018] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 125 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 125, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 131 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 131, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 137 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 137.

[0019] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 126 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 126, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 132 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 132, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 138 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 138.

[0020] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 127 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 127, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 133 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 133, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 139 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 139.

[0021] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 128 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 128, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 134 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 134, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 140 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 140.

[0022] In some embodiments of the antibodies of the present application, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 129 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 129, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 135 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 135, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 141 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 141.

[0023] In some embodiments of the antibodies of the present application, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity or at least 99% identity to any one selected from SEQ ID NOs. 1-10 and 82-123. In some embodiments of the antibodies of the present application, the antibody comprises an amino acid sequence selected from any one of SEQ ID NOs. 1-10 and 82-123.

[0024] In another aspect, provided herein are antibody constructs comprising an antibody as described herein. In some embodiments, the antibody construct further comprises an Fc fragment. In some embodiments, the Fc fragment is selected from IgG1, IgG2, IgG3 and IgG4. In some embodiments, the antibody construct is multispecific. In some embodiments, the antibody construct is bispecific.

[0025] In another aspect, the present disclosure provides nucleic acids encoding the antibodies or antibody constructs as described herein. In some embodiments, the nucleic acid comprises a polynucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to any one selected from SEQ ID NOs. 41 - 50 and 160 - 165. In some embodiments, the nucleic acid comprises a polynucleotide sequence selected from any one of SEQ ID NOs. 41 - 50 and 160 - 165.

[0026] In another aspect, the present disclosure provides a vector comprising the nucleic acid as described herein.

[0027] In another aspect, the present disclosure provides a host cell comprising the nucleic acid or vector as described herein.

[0028] In another aspect, the present disclosure provides a conjugate comprising: (1) an antibody or antibody construct as described herein, and (2) a drug. In some embodiments, the drug is selected from chemotherapeutic agents, toxins, cytokines, enzymes, immunomodulators, chelating agents, diagnostic agents, nanoparticles, and radioisotopes. In some embodiments, the conjugate further comprises a linker.

[0029] In another aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising the antibody construct as described herein. In another aspect, the present disclosure provides a T cell receptor (TCR) comprising the antibody construct as described herein.

[0030] In another aspect, the present disclosure provides a cell expressing the CAR or TCR as described herein.

[0031] In another aspect, the present disclosure provides a pharmaceutical composition comprising: (1) an antibody, antibody construct, nucleic acid, conjugate or cell as described herein, and (2) a pharmaceutically acceptable excipient.

[0032] In another aspect, the present disclosure provides a method for treating a disorder in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition as described herein. In another aspect, the present disclosure provides a method for treating a disorder associated with the presence of unwanted GCC+ cells, comprising administering to the subject an effective amount of the pharmaceutical composition as described herein. In some embodiments, the disorder is cancer. In another aspect, the present disclosure provides the use of the pharmaceutical composition as described herein in the manufacture of a medicament for treating a disorder in a subject in need thereof. In another aspect, the present disclosure provides the use of the antibody as described herein in detecting GCC in a sample from a subject.

[0033] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated herein by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained from the following detailed description that sets forth illustrative embodiments that utilize the principles of the invention, in the drawings in which:

[0035] Figure 1 The binding affinity of an exemplary anti-GCC antibody of the present application is illustrated by an SPR diagram.

[0036] Figure 2 The internalization of an exemplary anti-GCC antibody of the present application is illustrated. DETAILED DESCRIPTION

[0037] Before describing embodiments in detail, it is to be understood that the disclosure is not limited to the particular compositions or biological systems, which can of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0038] DEFINITIONS

[0039] As used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes combinations of two or more such molecules, and the like.

[0040] The term "antibody" is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.

[0041] Full-length antibodies contain two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. The variable regions in both chains typically contain three highly variable loops, called complementarity-determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CD2R, and LC-CDR3, and heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of their heavy chain constant regions. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the main antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0042] As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, diabodies, Fab, Fab’, F(ab’)2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv’), disulfide-stabilized diabodies (dsdiabody), single-chain Fv (scFv), scFv dimers (bivalent diabodies), multispecific antibodies formed from a portion of an antibody containing one or more CDRs, single-domain antibodies (e.g., camelized single-domain antibodies), nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds an antigen but does not contain the full antibody structure. An antigen-binding fragment is capable of binding the same antigen as the parent antibody or parent antibody fragment (e.g., parent scFv). In some embodiments, an antigen-binding fragment may contain one or more CDRs from a particular human antibody that are grafted into the framework regions from one or more different human antibodies.

[0043] As used herein, the term "single-domain antibody (sdAb, also known as nanobody)" refers to an antibody fragment consisting of a single monomeric variable antibody domain. Like a full antibody, it is capable of selectively binding to a specific antigen. The molecular weight of a single-domain antibody is only 12 - 15 kDa, much smaller than that of a conventional antibody composed of two protein heavy chains and two light chains (150 - 160 kDa), and even smaller than the Fab fragment (about 50 kDa, one light chain and half a heavy chain) and the single-chain variable fragment (about 25 kDa, two variable domains, one from the light chain and one from the heavy chain).

[0044] The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids; these are called VHH fragments. Cartilaginous fish also have heavy-chain antibodies (IgNAR, immunoglobulin new antigen receptor) from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimeric variable domains of common immunoglobulin G (IgG) from humans or mice into monomers. Although most current research on single-domain antibodies is based on heavy-chain variable domains, nanobodies derived from light chains have also shown specific binding to target epitopes.

[0045] Camelid nanobodies have been shown to be as specific as antibodies and in some cases they are more robust. They are easily isolated using the same phage display selection procedures used for antibodies, allowing them to be cultured at high concentrations in vitro. Their smaller size and single domain make these antibodies easier to transform into bacterial cells for mass production, making them ideal for research purposes.

[0046] As used herein, the term "CDR" or "complementary determining region" is intended to mean the non - contiguous antigen - binding sites found within the variable regions of heavy and light chain polypeptides. These specific regions have been described by Kabat et al., J. Biol. Chem. 252: 6609 - 6616 (1977); Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901 - 917 (1987); Al - Lazikani B. et al., J. Mol. Biol., 273:927 - 948 (1997); MacCallum et al., J. Mol. Biol. 262: 732 - 745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832 - 3839 (2008); Lefranc M. P. et al., Dev. Comp. Immunol., 27: 55 - 77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309: 657 - 670 (2001), where when compared to each other, these definitions include overlapping or subsets of amino acid residues. However, the application of any of these definitions to refer to the CDRs of an antibody or a graft - antibody or its variants is intended to fall within the scope of the terms defined and used herein. For comparison, the amino acid residues comprising the CDRs as defined by each of the references cited above are listed in Table 1 below. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832 - 3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301 - D307 (2010); and Adolf - Bryfogle J. et al., Nucleic Acids Res., 43: D432 - D438 (2015). The content of the references cited in this paragraph is hereby incorporated by reference in its entirety for this application and for one or more claims that may be included herein.

[0047] Table 1: CDR Definitions

[0048]

[0049] 1 The residue numbering follows the nomenclature of Kabat et al., ibid.

[0050] 2 The residue numbering follows the nomenclature of Chothia et al., ibid.

[0051] 3 The residue numbering follows the nomenclature of MacCallum et al., ibid.

[0052] 4 The residue numbering follows the nomenclature of Lefranc et al., ibid.

[0053] 5 The residue numbering follows the nomenclature of Honegger and Plückthun, ibid.

[0054] The expressions “residue numbering in the variable domain according to Kabat” or “amino acid position numbering in Kabat” and variations thereof refer to the numbering system for the heavy chain variable domain or light chain variable domain used in the antibody compilation in Kabat et al. (ibid.). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, which correspond to shortening or insertion in the FR or hypervariable region (HVR) of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and residues inserted after heavy chain FR residue 82 (such as residues 82a, 82b, and 82c, etc. according to Kabat). For a given antibody, the Kabat number of a residue can be determined by aligning the antibody sequence with the “standard” Kabat numbering sequence in the homologous region.

[0055] “Framework” or “FR” residues are those variable domain residues other than the CDR residues as defined herein.

[0056] The “percent amino acid sequence identity (%)” or “homology” for the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after aligning the sequences with any conservative substitutions being part of the sequence identity. The alignment for determining the percent amino acid sequence identity can be achieved in a variety of ways within the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) or MUSCLE software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes of this article, the percent amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R. C., Nucleic Acids Research 32 (5): 1792-1797, 2004; Edgar, R. C., BMCBioinformatics 5 (1): 113, 2004).

[0057] The term “Fc region” or “fragment crystallizable region” herein is used to define the C-terminal region of an immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as the segment from the amino acid residue at position Cys226, or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of an antibody, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies can include a population of antibodies in which all K447 residues have been removed, a population of antibodies in which the K447 residues have not been removed, and a population of antibodies that are a mixture of antibodies with and without the K447 residues. Suitable native sequence Fc regions for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0058] The term “epitope” as used herein refers to a specific atomic group or amino acid group on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they can bind to the same epitope within the antigen.

[0059] As used herein, the terms "specifically bind," "specifically recognize," and "is specific for" refer to a measurable and reproducible interaction, such as the binding between an antibody and its antigen, which is determinative of the presence of the target or antigen in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically recognizes an antigen is an antibody that binds that antigen with greater affinity, avidity, more readily, and / or for a longer duration compared to binding to other targets or antigens. In some embodiments, the degree of binding of an antibody to an unrelated target or antigen is less than about 10% of the degree of binding of the antibody to its antigen, as measured by radioimmunoassay (RIA). In some embodiments, the dissociation constant (K D ) of an antibody that specifically binds its antigen is ≤10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≤10 -12 M. In some embodiments, the specific binding may include, but does not require, exclusive binding. The binding specificity of an antibody can be determined experimentally by methods known in the art. Such methods include, but are not limited to, for example, Western blotting, ELISA testing, RIA testing, ECL testing, IRMA testing, EIA testing, BIACORE TM testing, and peptide scanning.

[0060] An "isolated" nucleic acid molecule encoding a polypeptide or cytokine as described herein is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in its native environment. Preferably, the isolated nucleic acid is not associated with all of the components associated with the native environment. In some embodiments, the isolated nucleic acid molecule encoding an antibody or antibody construct as described herein is in a form other than that found in nature or in its native environment.

[0061] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and vectors that are incorporated into the genome of the host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked thereto. Such vectors are referred to herein as "expression vectors."

[0062] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the original subject cell and its progeny.

[0063] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The nucleic acid content of the progeny may not be identical to that of the parental cell and may contain mutations. This article includes mutant progeny having the same function or biological activity as that screened or selected in the originally transformed cell.

[0064] As used herein, "treatment" or "treating" is a method for obtaining a beneficial or desired result, including a clinical result. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms caused by a disease, reduction in the severity of a disease, stabilization of a disease (e.g., preventing or delaying the worsening of a disease), prevention or delay of the spread of a disease (e.g., metastasis), prevention or delay of the recurrence of a disease, delay or slowing of the progression of a disease, improvement of the disease state, provision of a remission (partial or complete) of a disease, reduction in the dosage of one or more other drugs required to treat a disease, delay in the progression of a disease, improvement or enhancement of the quality of life, increase in body weight, and / or extension of survival. "Treatment" also encompasses reduction of the pathological consequences of cancer, such as, for example, tumor volume. The methods of this application contemplate any one or more of these aspects of treatment.

[0065] In the context of cancer, the term "treatment" includes any one or all of the following: inhibition of cancer cell growth, inhibition of cancer cell replication, reduction of the overall tumor burden, and improvement of one or more symptoms associated with the disease.

[0066] The terms "subject", "individual", and "patient" are used interchangeably herein and refer to a mammal, including but not limited to a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human.

[0067] It should be understood that the embodiments of the present application described herein include embodiments of "consisting of" and / or "consisting essentially of".

[0068] References herein to "about" a value or parameter include (and describe) variations that are directed to the value or parameter itself. For example, a description of "about X" includes a description of "X".

[0069] As used herein, a reference to "not" a value or parameter generally means and describes "excluding" a value or parameter. For example, the method is not for treating cancer type X means that the method is for treating other types of cancer than cancer type X.

[0070] The term "about X-Y" as used herein has the same meaning as "about X to about Y".

[0071] As used herein and in the appended claims, the singular forms "a" and / or "the" include plural referents unless the context clearly dictates otherwise.

[0072] Antibody binding affinity

[0073] The binding affinity or binding specificity of an antibody as described herein can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA assays, RIA assays, ECL assays, IRMA assays, EIA assays, BIACORETM assays, and peptide scanning.

[0074] In some embodiments, the K of the binding between the antibody (or antibody construct) and the target antigen (e.g., GCC) D is about 10 -7 M to about 10 -12 M, about 10 -7 M to about 10 -8 M, about 10 -8 M to about 10 -9 M, about 10 -9 M to about 10 -10 M, about 10 -10 M to about 10 -11 M, about 10 -11 M to about 10 -12 M, about 10 -7 M to about 10 -12 M, about 10 -8 M to about 10 -12 M, about 10 -9 M to about 10 -12 M, about 10 -10 M to about 10 -12 M, about 10 -7 M to about 10 -11 M, about 10 -8 M to about 10 -11 M, about 10 -9 M to about 10 -11 M, about 10 -7 M to about 10 -10 M, about 10 -8 M to about 10 -10 M or about 10 -7 M to about 10 -9 M. In some embodiments, the K of the binding between the antibody portion and the target antigen (e.g., GCC)D Less than about 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 Any of M.

[0075] In some embodiments, the K of the binding between the antibody (or antibody construct) and the target antigen (e.g., GCC) on Is about 10 3 M -1 s -1 To about 10 8 M -1 s -1 , about 10 3 M-1s-1 to about 10 4 M -1 s -1 , about 10 4 M -1 s -1 To about 10 5 M -1 s -1 , about 10 5 M -1 s -1 To about 10 6 M -1 s -1 , about 10 6 M -1 s -1 To about 10 7 M -1 s -1 Or about 10 7 M -1 s -1 To about 10 8 M -1 s -1 . In some embodiments, the Kon of the binding between the antibody portion and the target antigen (e.g., GCC) is about 10 3 M -1 s -1 To about 10 5 M -1 s -1 , about 10 4 M -1 s -1 To about 10 6 M -1 s -1 , about 10 5 M -1 s -1from about 0 to about 10 7 M -1 s -1 from about 10 6 M -1 s -1 to about 10 8 M -1 s -1 from about 10 4 M - 1 s -1 to about 10 7 M -1 s -1 or about 10 5 M -1 s -1 to about 10 8 M -1 s -1 。In some embodiments, the K of the binding between the antibody portion and the target antigen (e.g., GCC) on is not more than about 10 3 M -1 s -1 、10 4 M -1 s -1 、10 5 M -1 s -1 、10 6 M -1 s -1 、10 7 M -1 s -1 or 10 8 M -1 s -1 any of which.

[0076] In some embodiments, the K of the binding between the antibody (or antibody construct) and the target antigen (e.g., GCC) off is from about 1 s -1 to about 10 -6 s -1 from about 1 s -1 to about 10 -2 s -1 from about 10 -2 s -1 to about 10 -3 s -1 from about 10 -3 s -1 to about 10 -4 s -1 from about 10 -4 s -1 to about 10-5 s -1 , about 10 -5 s -1 to about 10 -6 s -1 , about 1 s -1 to about 10 -5 s -1 , about 10 -2 s -1 to about 10 -6 s -1 , about 10 -3 s -1 to about 10 -6 s -1 , about 10 -4 s -1 to about 10 -6 s -1 , about 10 -2 s -1 to about 10 -5 s -1 or about 10 -3 s -1 to about 10 -5 s -1 . In some embodiments, the K off of the binding between the antibody portion and the target antigen (e.g., GCC) is at least about 1 s -1 , 10 -2 s -1 , 10 -3 s -1 , 10 -4 s -1 , 10 -5 s -1 or 10 -6 s -1 Any one of them.

[0077] Chimeric or humanized antibodies

[0078] In some embodiments, one or more of the antibodies of the present application are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some embodiments, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse) and a human constant region. In some embodiments, the chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from that of the parental antibody. Chimeric antibodies include their antigen-binding fragments.

[0079] In some embodiments, the chimeric antibody is a humanized antibody. Generally, non-human antibodies are humanized to reduce their immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. In general, a humanized antibody comprises one or more variable domains in which the HVRs (e.g., CDRs) (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. A humanized antibody optionally further comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the HVRs are derived), for example to restore or improve the specificity or affinity of the antibody.

[0080] Humanized antibodies and methods for their preparation are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat’l Acad. Sci. USA 86: 10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36: 25-34 (2005) (describing SDR (α-CDR) grafting); Padlan, Mol. Immunol. 28: 489-498 (1991) (describing “surface resurfacing”); Dall’Acqua et al., Methods 36: 43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36: 61-68 (2005) and Klimka et al., Br. J.Cancer, 83:252-260 (2000) (describing “guided selection” methods of FR shuffling).

[0081] Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the "best fit" method (see, e.g., Sims et al., J. Immunol. 151: 2296 (1993)); framework regions derived from the consensus sequences of human antibodies of specific subgroups of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992) and Presta et al., J. Immunol., 151 :2623 (1993)); human mature (somatic mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684(1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

[0082] Substitution, insertion and deletion variants

[0083] In some embodiments, antibody variants comprising one or more amino acid substitutions are included in the antibodies described herein. Sites of interest for substitution mutagenesis include HVR (or CDR) and FR. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 2. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 2 and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened to obtain the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity or improved ADCC or CDC.

[0084] Table 2. Amino acid substitutions

[0085]

[0086] Amino acids can be grouped according to common side chain characteristics: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0087] Cysteine-engineered antibody variants

[0088] In some embodiments, it may be desirable to create cysteine-engineered antibody moieties, such as "thioMAbs", in which one or more residues of one or more antibody moieties in the multispecific constructs herein are replaced with cysteine residues. In certain embodiments, the residues being replaced occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates, as further described herein. Cysteine-engineered antibody moieties can be generated as described, for example, in U.S. Patent No. 7,521,541.

[0089] Anti-GCC single domain antibody

[0090] Guanylate cyclase C (GCC) (also known as STAR, ST receptor, GUC2C, and GUCY2C) is a transmembrane cell surface receptor that functions in maintaining intestinal fluid, electrolyte homeostasis, and cell proliferation. It is expressed at mucosal cells (e.g., at the apical brush border membrane of the inner walls of the small intestine, large intestine, and rectum), and remains expressed upon neoplastic transformation of intestinal epithelial cells and is expressed in all primary and metastatic colorectal tumors. Tumor cells from the stomach, esophagus, and gastroesophageal junction also express GCC, which is considered a potential therapeutic target for receptor-binding proteins such as antibodies or ligands. The GCC protein includes a signal sequence, an extracellular domain for a ligand (e.g., guanylin or heat-stable enterotoxin (ST)), a transmembrane domain, a kinase homology domain, and a guanylate cyclase catalytic domain. In some embodiments, the GCC protein as described herein is wild-type GCC. In some embodiments, the GCC protein is a native variant GCC. In some embodiments, the GCC protein is a mutant GCC. In some embodiments, the GCC protein is human GCC, such as comprising the amino acid sequence shown in SEQ ID NO. 81.

[0091]

[0092] In one aspect, the present disclosure provides antibodies that specifically bind guanylate cyclase C (GCC) and comprise CDR1, CDR2, and CDR3 of a single domain antibody selected from any one of SEQ ID NOs. 1-10 and 118-123. In some embodiments, CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, CDR1, CDR2, and CDR3 are numbered according to Chothia. In some embodiments, CDR1, CDR2, and CDR3 are numbered according to Abm. In some embodiments, CDR1, CDR2, and CDR3 are numbered according to Contact.

[0093] In another aspect of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any of SEQ ID NOs. 1-10 and 118-123, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1, which comprises an amino acid sequence selected from: (i) SEQ ID NOs. 11-20 and 124-129, and (ii) an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NOs. 11-20 and 124-129; (2) CDR2, which comprises an amino acid sequence selected from: (i) SEQ ID NOs. 21-30 and 130-135, and (ii) an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NOs. 21-30 and 130-135; and (3) CDR3, which comprises an amino acid sequence selected from: (i) SEQ ID NOs. 31-40 and 136-141, and (ii) an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NOs. 31-40 and 136-141.

[0094] In some embodiments of the present application, antibodies that specifically bind to guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 1. In some embodiments of the present application, antibodies that specifically bind to guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 1, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 11 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 11, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 21 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 21, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 31 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 31. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 11, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 21, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 31. In some embodiments of the present application, antibodies that specifically bind to guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 1, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 51 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 51, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 61 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 61, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 71 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 71. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 51, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 61, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 71.In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO. 1. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 1.

[0095] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 2. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 2, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 12 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 12, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 22 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 22, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 32 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 32. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 12, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 22, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 32. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 2, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 52 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 52, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 62 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 62, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 72 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 72. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 52, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 62, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 72.In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to SEQ ID NO. 2. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 2.

[0096] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 3. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 3, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 13 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 13, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 23 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 23, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 33 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 33. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 13, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 23, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 33. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 3, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 53 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 53, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 63 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 63, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 73 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 73. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 53, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 63, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 73.In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO. 3. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 3.

[0097] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 4. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 4, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 14 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 14, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 24 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 24, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 34 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 34. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 14, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 24, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 34. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 4, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 54 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 54, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 64 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 64, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 74 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 74. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 54, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 64, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 74.In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to SEQ ID NO. 4. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 4.

[0098] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 5. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 5, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 15 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 15, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 25 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 25, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 35 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 35. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 15, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 25, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 35. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 5, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 55 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 55, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 65 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 65, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 75 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 75. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 55, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 65, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 75.In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO. 5. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 5.

[0099] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 6. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 6, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 16 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 16, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 26 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 26, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 36 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 36. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 16, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 26, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 36. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 6, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 56 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 56, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 66 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 66, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 76 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 76. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 56, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 66, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 76.In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to SEQ ID NO. 6. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 6.

[0100] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 7. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 7, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 17 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 17, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 27 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 27, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 37 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 37. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 17, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 27, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 37. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 7, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 57 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 57, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 67 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 67, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 77 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 77. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 57, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 67, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 77.In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to SEQ ID NO. 7. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 7.

[0101] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 8. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 8, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 18 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 18, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 28 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 28, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 38 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 38. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 18, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 28, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 38. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 8, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 58 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 58, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 68 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 68, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 78 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 78. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 58, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 68, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 78.In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to SEQ ID NO. 8. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 8.

[0102] In some embodiments of the present application, antibodies that specifically bind to guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any of SEQ ID NO. 9. In some embodiments of the present application, antibodies that specifically bind to guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any of SEQ ID NO. 9, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 19 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 19, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 29 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 29, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 39 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 39. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 19, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 29, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 39. In some embodiments of the present application, antibodies that specifically bind to guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 9, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 59 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 59, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 69 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 69, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 79 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 79. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 59, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 69, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 79.In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to SEQ ID NO. 9. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 9.

[0103] In some embodiments of the present application, antibodies that specifically bind to guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 10. In some embodiments of the present application, antibodies that specifically bind to guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 10, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 20 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NO. 20, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 30 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NO. 30, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 40 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NO. 40. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 20, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 30, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 40. In some embodiments of the present application, antibodies that specifically bind to guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 10, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 60 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NO. 60, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 70 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NO. 70, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 80 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NO. 80. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 60, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 70, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 80.In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO. 10. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 10.

[0104] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any of SEQ ID NO. 118. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any of SEQ ID NO. 118, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 124 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 124, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 130 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 130, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 136 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 136. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 124, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 130, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 136. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 118, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 142 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 142, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 148 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 148, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 154 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 154.In some embodiments, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 142, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 148, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 154. In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to SEQ ID NO. 118. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 118.

[0105] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 119. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 119, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 125 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 125, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 131 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 131, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 137 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 137. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 125, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 131, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 137. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody of SEQ ID NO. 119, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 142 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 142, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 148 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 148, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 154 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 154.In some embodiments, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 142, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 148, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 154. In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to SEQ ID NO. 119. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 119.

[0106] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 120. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 120, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 126 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NO. 126, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 132 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NO. 132, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 138 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NO. 138. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 126, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 132, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 138. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody of SEQ ID NO. 120, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 144 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NO. 144, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 150 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NO. 150, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 156 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) as compared to SEQ ID NO. 156.In some embodiments, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 144, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 150, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 156. In some embodiments, the antibody comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO. 120. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 120.

[0107] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 121. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 121, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 127 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 127, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 133 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 133, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 139 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 139. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 127, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 133, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 139. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 121, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 145 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 145, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 151 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 151, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 157 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 157.In some embodiments, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 145, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 151, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 157. In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to SEQ ID NO. 121. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 121.

[0108] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any of SEQ ID NO. 122. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any of SEQ ID NO. 122, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 128 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 128, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 134 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 134, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 140 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 140. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 128, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 134, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 140. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody of SEQ ID NO. 122, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 146 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 146, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 152 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 152, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 158 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 158.In some embodiments, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 146, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 152, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 158. In some embodiments, the antibody comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO. 122. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 122.

[0109] In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 123. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NO. 123, and CDR1, CDR2, and CDR3 are numbered according to IMGT. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 129 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 129, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 135 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 135, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 141 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 141. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 129, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 135, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 141. In some embodiments of the present application, antibodies that specifically bind guanylate cyclase C (GCC) are provided herein, which comprise CDR1, CDR2, and CDR3 of the single-domain antibody of SEQ ID NO. 123, and CDR1, CDR2, and CDR3 are numbered according to Kabat. In some embodiments, the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 147 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 147, (2) CDR2 comprising the amino acid sequence of SEQ ID NO. 153 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 153, and (3) CDR3 comprising the amino acid sequence of SEQ ID NO. 159 or an amino acid sequence having one or more amino acid alterations (e.g., substitutions, deletions, or insertions) compared to SEQ ID NO. 159.In some embodiments, the antibody comprises: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO. 147, (2) a CDR2 comprising the amino acid sequence of SEQ ID NO. 153, and (3) a CDR3 comprising the amino acid sequence of SEQ ID NO. 159. In some embodiments, the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO. 123. In some embodiments, the antibody comprises the amino acid sequence of SEQ ID NO. 123.

[0110] In some embodiments of the antibodies of the present application, the antibody is a humanized antibody. In some embodiments, the humanized antibody is derived from any one selected from SEQ ID NOs. 1-10 and 118-123.

[0111] In some embodiments, the humanized antibody is derived from SEQ ID NO. 1 and comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one selected from SEQ ID NOs. 82-86. In some embodiments, the humanized antibody is derived from SEQ ID NO. 1 and comprises the amino acid sequence of any one selected from SEQ ID NOs. 82-86.

[0112] In some embodiments, the humanized antibody is derived from SEQ ID NO. 2 and comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one selected from SEQ ID NOs. 87-90. In some embodiments, the humanized antibody is derived from SEQ ID NO. 2 and comprises the amino acid sequence of any one selected from SEQ ID NOs. 87-90.

[0113] In some embodiments, the humanized antibody is derived from SEQ ID NO. 3 and comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one selected from SEQ ID NOs. 91-94. In some embodiments, the humanized antibody is derived from SEQ ID NO. 3 and comprises an amino acid sequence selected from any one of SEQ ID NOs. 91-94.

[0114] In some embodiments, the humanized antibody is derived from SEQ ID NO. 4 and comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one selected from SEQ ID NOs. 95-98. In some embodiments, the humanized antibody is derived from SEQ ID NO. 4 and comprises an amino acid sequence selected from any one of SEQ ID NOs. 95-98.

[0115] In some embodiments, the humanized antibody is derived from SEQ ID NO. 5 and comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one selected from SEQ ID NOs. 99-101. In some embodiments, the humanized antibody is derived from SEQ ID NO. 5 and comprises an amino acid sequence selected from any one of SEQ ID NOs. 99-101.

[0116] In some embodiments, the humanized antibody is derived from SEQ ID NO. 6 and comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one selected from SEQ ID NOs. 102-105. In some embodiments, the humanized antibody is derived from SEQ ID NO. 6 and comprises an amino acid sequence selected from any one of SEQ ID NOs. 102-105.

[0117] In some embodiments, the humanized antibody is derived from SEQ ID NO. 7 and comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to any one selected from SEQ ID NOs. 106 - 110. In some embodiments, the humanized antibody is derived from SEQ ID NO. 7 and comprises an amino acid sequence selected from any one of SEQ ID NOs. 106 - 110.

[0118] In some embodiments, the humanized antibody is derived from SEQ ID NO. 8 and comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to any one selected from SEQ ID NOs. 111 - 114. In some embodiments, the humanized antibody is derived from SEQ ID NO. 8 and comprises an amino acid sequence selected from any one of SEQ ID NOs. 111 - 114.

[0119] In some embodiments, the humanized antibody is derived from SEQ ID NO. 9 and comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to any one selected from SEQ ID NOs. 115 - 117. In some embodiments, the humanized antibody is derived from SEQ ID NO. 9 and comprises an amino acid sequence selected from any one of SEQ ID NOs. 115 - 117.

[0120] In some embodiments of the antibodies of the present application, the antibody is a single - domain antibody.

[0121] In another aspect, the present disclosure provides an antibody construct comprising an antibody as described herein. In some embodiments, the antibody construct further comprises one or more additional antibody moieties. In some embodiments, the antibody construct is a multi - specific antibody capable of specifically binding GCC and one or more additional targets. In some embodiments, the antibody construct is a bispecific antibody capable of specifically binding GCC and a second target.

[0122] As used herein, the term "bispecific" refers to an antibody in which one portion of the antibody binds to one epitope on an antigen, while a second portion binds to a different epitope on the same antigen or a different antigen. The different epitopes are typically on different antigens. In some embodiments, a bispecific antibody is an antibody that comprises portions of two different antibodies and thus binds to two different types of antigens. One arm of the bispecific antibody typically contains the variable domain of one antibody, while the other arm contains the variable domain of the other antibody. In some embodiments, one arm of the bispecific antibody typically contains the variable domain that targets GCC, while the other arm contains the variable domain of another antibody that targets a second target. In some embodiments, one arm of the bispecific antibody typically contains a single-domain antibody that specifically binds GCC as described herein, while the other arm contains the variable domain of another antibody that targets a second target.

[0123] In some embodiments, the second target is a tumor antigen. In some embodiments, the tumor antigen can be selected from CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GCC, GD2, O-acetyl-GD2, O-acetyl-GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, NY-ESO-1, PSMA, RANK, RORl, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-I-4, APRIL, CS1, MAGE3, Claudin18.2, folate receptor α, folate receptor β, GPC2, CD70, BAFF-R, and TROP-2.

[0124] In some embodiments, the second target is an immune cell-specific antigen. In some embodiments, the immune cell antigen can be selected from CD2, CD3, CD4, CD5, CD7, CD8, CD16a, CD16b, CD25, CD27, CD28, CD30, CD38, CD45, CD48, CD50, CD52, CD56, CD57, CD62L, CD69, CD94, CD100, CD102, CD122, CD127, CD132, CD137, CD160, CD161, CD178, CD218, CD226, CD244, CD159a (NKG2A), CD159c (NKG2C), NKG2E, CD279, CD314 (NKG2D), CD305, CD335 (NKP46), CD337, CD319 (CS1), TCRα, TCRβ, and SLAMF7.

[0125] In some embodiments, the antibody construct further comprises an Fc fragment. In some embodiments, the Fc fragment is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the Fc fragment further comprises one or more mutations to alter the function of the Fc fragment, e.g., to decrease or enhance the ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement-dependent cytotoxicity), and / or ADCP (antibody-dependent cell phagocytosis) functions of the Fc fragment.

[0126] Conjugate

[0127] In another aspect, the present disclosure provides a conjugate comprising: (1) an antibody or antibody construct that specifically binds to GCC as described herein, and (2) a drug. In some embodiments, the drug is selected from chemotherapeutic agents, toxins, hormones, enzymes, immunomodulators, chelating agents, imaging agents, nanoparticles, and radioisotopes.

[0128] In some embodiments, the drug is a chemotherapeutic agent useful for treating cancer. Examples of chemotherapeutic agents include Erlotinib (TARCEVA®, Genentech / OSI Pharm.), Bortezomib (VELCADE®, Millennium Pharm.), Fulvestrant (FASLODEX®, Astrazeneca), Sunitinib malate (Sutent) (SUl 1248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), Oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (RAPAMUNE®, Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs.), and Gefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as Thiotepa and CYTOXANE® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimine and methylamelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; polyacetylenes; camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin, and bizelesin);Cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I; dynemicin, including dynemicin A); bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoproteid enediyne antibiotic chromophores), aclacinomysin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino doxorubicin, cyano morpholino doxorubicin, 2-pyrrolino doxorubicin, and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin; mitomycin, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin;Sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2’’-trichloroethylamine; trichothecene (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoid, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), CREMOPHOR-free ABRAXANETM, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and a pharmaceutically acceptable salt, acid, or derivative of any of the foregoing.

[0129] In some embodiments, the chemotherapeutic agent further includes, but is not limited to: (i) antihormonal agents for modulating or inhibiting the action of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON-toremifene; (ii) aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazole, aminoglutethimide, MEGASE® medroxyprogesterone acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and troxacitabine (1,3-dioxolane nucleoside cytosine analogue); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those that inhibit gene expression in signal pathways associated with abnormal cell proliferation, such as, for example, PKC-α, Ralf, and H-Ras; (viii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME® ribozyme) and HER2 expression inhibitors; (ix) vaccines, such as gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; (x) antiangiogenic agents, such as bevacizumab (AVASTIN®, Genentech); and (xi) pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0130] In some embodiments, the drug is a cytokine. The term "cytokine" is a general term for proteins released by one cell population that act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, and conventional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-a and tumor necrosis factor-β; mullerian-inhibiting substance; murine gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-β; platelet-derived growth factor; transforming growth factors (TGF) such as TGF-a and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); osteogenic factor; interferons such as interferon-a, interferon-β, and interferon-γ; colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; tumor necrosis factors such as TNF-a or TNF-β; and other polypeptide factors including LIF and kit ligand (KL).

[0131] In some embodiments of the conjugates of the present application, the conjugate further comprises a linker. The term "linker" as used herein refers to a chemical moiety comprising a covalent bond or chain of atoms that covalently links an antibody as described herein and / or a drug as described herein.

[0132] Nucleic acids, vectors, host cells, and methods for producing GCC antibodies

[0133] Nucleic acid molecules encoding antibodies or antibody constructs that specifically bind GCC as described herein are also contemplated. In some embodiments, nucleic acids encoding antibodies as described herein are provided. In some embodiments, the nucleic acid comprises a polynucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to any one selected from SEQ ID NOs. 41 - 50 and 160 - 165. In some embodiments, the nucleic acid comprises a polynucleotide sequence selected from any one of SEQ ID NOs. 41 - 50 and 160 - 165. The nucleic acid sequences of SEQ ID NOs: 41 - 50 and 160 - 165 are provided in the sequence summary table below the examples.

[0134] Vectors incorporating the nucleic acids described herein are also provided. In some embodiments, expression of an antibody that specifically binds GCC as described herein can be achieved by inserting the nucleic acid as described herein into a suitable expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, including for example a promoter (e.g., constitutive, regulatable tissue - specific promoter) and a 3' untranslated region (UTR). The vector can be suitable for replication and integration in a eukaryotic host cell. Typical cloning and expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence.

[0135] The nucleic acid can be cloned into a variety of types of vectors. For example, the nucleic acid can be cloned into vectors including but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, probe - generating vectors, and sequencing vectors.

[0136] In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include but are not limited to retroviruses, adenoviruses, adeno - associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0137] A variety of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells in vivo or in vitro. A variety of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. A variety of adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene into daughter cells and its propagation. Lentiviral vectors have additional advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus because they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0138] Additional promoter elements (e.g., enhancers) regulate the frequency of transcriptional initiation. Typically, they are located in the region from 30 to 110 base pairs (bp) upstream of the start site, although many promoters have recently been found to also contain functional elements downstream of the start site. The spacing between promoter elements is generally flexible, so promoter function is still retained when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline.

[0139] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked thereto when expression is desired, or turn off the expression when expression is not desired. Examples of inducible promoters include but are not limited to metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0140] In some embodiments, the expression of a nucleic acid encoding an antibody that specifically binds GCC as described herein is inducible. In some embodiments, the nucleic acid encoding an antibody that specifically binds GCC as described herein is operably linked to an inducible promoter, including any inducible promoter known in the art. In some embodiments, the nucleic acid encoding an antibody that specifically binds GCC as described herein has been engineered to encode an epitope tag, for example to facilitate purification or detection of the polypeptide. Exemplary epitope tags include, but are not limited to, for example, 6x His (also referred to as His-tag or hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, such as enhanced green fluorescent protein or EGFP), GST (glutathione-S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein).

[0141] The antibodies that specifically bind GCC of the present application can be produced by any method known in the art. Exemplary techniques for polypeptide production are described below; however, these exemplary techniques are provided for illustrative purposes only and are not intended to be limiting.

[0142] The antibodies that specifically bind GCC of the present application can be produced by recombinant methods. To recombinantly produce the antibodies that specifically bind GCC of the present application, the nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. The DNA encoding the antibody can be readily isolated and sequenced using conventional procedures (such as by using oligonucleotide probes that specifically bind to the gene encoding the antibody). There are many vectors available. Vector components generally include, but are not limited to, one or more of the following: signal sequence, origin of replication, one or more marker genes, enhancer elements, promoter, and transcription termination sequence.

[0143] Both expression vectors and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells, for example, to enable the vector to replicate independently of the host chromosomal DNA. Such sequences can include an origin of replication or an autonomous replication sequence. Such sequences are well known in a variety of bacteria, yeasts, and viruses. Generally, mammalian expression vectors do not require an origin of replication component (the SV40 origin can be used as it contains an early promoter).

[0144] Expression and cloning vectors can contain a selectable gene or selectable marker. Typical selectable genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), (b) complement auxotrophic deficiencies, or (c) supply critical nutrients unavailable from complex media. Examples of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin. Another example of suitable selectable markers for mammalian cells are those that identify cells capable of incorporating nucleic acids encoding antibodies, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and metallothionein-II, preferably the primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, etc. For example, Chinese hamster ovary (CHO) cell lines lacking endogenous DHFR activity transformed with the DHFR gene can be identified by culturing the transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR).

[0145] Alternatively, host cells (especially wild-type hosts containing endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR gene, and the selectable marker can be selected by culturing the cells in a medium containing a selection agent (such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418) for another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH).

[0146] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and operably linked to the nucleic acid encoding the antibody. Promoters suitable for prokaryotic hosts include the phoA promoter, the beta-lactamase and lactose promoter systems, the alkaline phosphatase promoter, the tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoter sequences are known for eukaryotes. Yeast promoters are well known in the art and can include inducible promoters / enhancers that are regulated by growth conditions. Almost all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Examples include but are not limited to the promoters of 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Transcription of the antibody from the vector in mammalian host cells can be controlled, for example, by a promoter obtained from a viral genome. The early and late promoters of the SV40 virus can be conveniently obtained as SV40 restriction fragments that also contain the SV40 virus origin of replication. The immediate early promoter of the human cytomegalovirus can be conveniently obtained as a HindIII restriction fragment. Alternatively, the long terminal repeat of the Rous sarcoma virus can be used as a promoter.

[0147] Transcription of DNA encoding an antibody as described herein in higher eukaryotes is often increased by inserting enhancer sequences into the vector. Many enhancer sequences are currently known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from eukaryotic cell viruses are often used.

[0148] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for termination of transcription and stabilization of the mRNA.

[0149] Suitable host cells for cloning or expressing the DNA in the vectors herein are the above-described prokaryotes, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, the Enterobacteriaceae, such as the genus Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), and Shigella, etc. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable as cloning or expression hosts for vectors encoding antibodies that specifically bind GCC as described herein. Saccharomyces cerevisiae, also known as common baker's yeast, is the most commonly used yeast among lower eukaryotic host microorganisms. Certain fungal and yeast strains can be selected in which the glycosylation pathway has been "humanized" to produce antibodies with a partially or fully human glycosylation pattern. See, for example, Li et al., Nat. Biotech. 24: 210-215 (2006).

[0150] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Leninaceae), alfalfa (Medicago truncatula), and tobacco can also be used as hosts.

[0151] Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A variety of baculovirus strains and variants and the corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori have been identified.

[0152] Vertebrate cells can be used as hosts, and the propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); the human embryonic kidney line (293 or subclones of 293 cells adapted for growth in suspension culture, Graham et al., J. GenVirol. 36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and the human hepatoma cell line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines such as NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N. J., 2003), pp. 255-268.

[0153] The host cells of the present disclosure can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are all suitable for culturing host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44 (1979); Barnes et al., Anal. Biochem. 102: 255 (1980); U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Reissue Patent 30,985 can be used as the medium for host cells. Any of these media can be supplemented with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM drug), trace elements (defined as inorganic compounds that are usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Appropriate concentrations of any other necessary supplements known to those skilled in the art can also be included. The culture conditions (such as temperature, pH, etc.) are those previously used for the selected host cells for expression and are apparent to those skilled in the art.

[0154] When recombinant techniques are used, the antibody can be produced intracellularly, in the periplasmic space, or secreted directly into the medium. If the polypeptide is produced intracellularly, in the first step, particulate debris (host cells or lysed fragments) is removed by methods such as centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describe procedures for isolating antibodies secreted into the periplasmic space of E. coli.

[0155] Polypeptide compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being one of the commonly preferred purification steps. In some embodiments, the antibodies as described herein comprise an epitope tag (e.g., a tag attached via a cleavable linker to an antibody that specifically binds GCC as described herein) to facilitate purification. Exemplary epitope tags include, but are not limited to, for example, 6xHis (also referred to as His-tag or hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, such as enhanced green fluorescent protein or EGFP), GST (glutathione-S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein).

[0156] Chimeric antigen receptors and T cell receptors

[0157] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to transplant one or more antigen specificities onto an immune effector cell (such as a T cell). Some CARs are also referred to as "artificial T cell receptors", "chimeric T cell receptors", or "chimeric immune receptors". In some embodiments, a CAR comprises an extracellular antigen-binding domain that is specific for one or more antigens (such as GCC), a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptor. "CAR-T" refers to a T cell that expresses a CAR.

[0158] In another aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising an antibody construct as described herein. In some embodiments, the CAR comprises an antibody that specifically binds GCC as described herein, a transmembrane domain, and an intracellular signaling domain.

[0159] As used herein, the term "T cell receptor" or "TCR" refers to the molecule on the surface of a T cell or T lymphocyte that is responsible for antigen recognition. The TCR is a heterodimer composed of two different protein chains. In some embodiments, the TCR of the present disclosure is composed of an α chain and a β chain and is referred to as an αβ TCR. The αβ TCR recognizes antigenic peptides degraded from proteins that are bound to major histocompatibility complex molecules (MHC) on the cell surface. In some embodiments, the TCR of the present disclosure is composed of a γ chain and a δ chain and is referred to as a γδ TCR.

[0160] In another aspect, provided herein are T cell receptors (TCRs) comprising antibody constructs as described herein. In some embodiments, the T cell receptor (TCR) comprises an antibody that specifically binds GCC as described herein, wherein the antibody that specifically binds GCC as described herein is linked to: (i) at least one TCR chain selected from the α, β, γ, and δ chains of the T cell receptor; (ii) the ε, δ, and / or γ chains of cluster of differentiation 3 (CD3); or (iii) the CD3 ζ chain.

[0161] In some embodiments, provided herein are also cells expressing a CAR or TCR as described herein. In some embodiments, the cell is an immune cell. In some embodiments, the cell can be selected from T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells.

[0162] Pharmaceutical composition

[0163] In another aspect, provided herein is a pharmaceutical composition comprising an antibody, nucleic acid, conjugate, and / or cell as described herein, and a pharmaceutically acceptable excipient.

[0164] Any suitable excipient can be used in the pharmaceutical compositions of the present application. In some embodiments, the excipients include, but are not limited to, diluents; carriers; excipients; stabilizers; buffers such as phosphate, citrate, and other organic acid buffers; antioxidants, including ascorbic acid and methionine; preservatives such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl esters of p-hydroxybenzoic acid (such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0165] Method

[0166] The present disclosure also provides a method of treating a disorder in a subject in need thereof, which comprises administering to the subject an effective amount of a pharmaceutical composition as described herein. In some embodiments, the method comprises administering to the subject an antibody as described herein, a nucleic acid as described herein, a vector as described herein, a cell as described herein, and / or a pharmaceutical composition as described herein. The present disclosure also provides a method for treating a disorder associated with the presence of unwanted GCC+ cells, which comprises administering to the subject an antibody as described herein, a nucleic acid as described herein, a vector as described herein, a cell as described herein, and / or a pharmaceutical composition as described herein.

[0167] In some embodiments, the disorder is a proliferative disorder. In some embodiments, the disorder is cancer.

[0168] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present invention. The present invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the present invention, but should not be construed in any way as limiting its scope.

[0169] Examples

[0170] The following examples are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the present invention, but the examples are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments that have been conducted. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. The following examples are only intended to illustrate the present application and should not be regarded as limiting the present application in any way. The following examples and detailed description are provided by way of illustration and not limitation.

[0171] Example 1: Generation of a single-domain antibody (sdAb) that specifically binds GCC

[0172] 1.1 Animal immunization and phage library construction

[0173] Two alpacas were subcutaneously injected with human GCCx10*His, 6 doses, 100 to 200 μg of antigen per dose, every 1 - 2 weeks. Blood samples were collected at different time points, and the induction of an antigen-specific humoral immune response was verified by ELISA using serum prepared from the blood samples.

[0174] Seven days after the last immunization, 50 mL of blood samples were collected from each animal, and approximately 1×10 8Peripheral blood mononuclear cells (PBMCs). Total RNA was extracted from PBMCs according to the manufacturer's recommendations and transcribed into cDNA using oligo-dT primers and the SuperScript III First-Strand Synthesis SuperMix system (Invitrogen, Carlsbad, CA, USA).

[0175] The purified cDNA was then used as a template to amplify the Ig heavy chain-encoding gene segment library by using signal peptide domain-specific primers and CH2 domain-specific primers. Fragments of approximately 700 bp (representing heavy chain IgG lacking the CH1 domain) were isolated from the agarose gel and purified by the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). The purified fragments were further used as templates to amplify the VHH library by using framework 1 (FR1) and framework 4 (FR4) specific primers.

[0176] After amplification, the purified VHH genes were cloned into the phagemid vector pFL249 and electrotransformed into Escherichia coli TG1. After transformation, the TG1 cells were cultured in SOC medium at 37 °C for 1 h, then inoculated onto plates containing solid 2YT medium supplemented with 100 μg / mL Carb and 1% (w / v) glucose and cultured overnight at 37 °C. The next day, the colonies were scraped into liquid 2YT medium supplemented with 1 / 3 (v / v) 80% glycerol and stored at -80 °C. The library size was approximately 1×10 8 , the VHH insertion rate was higher than 95%, and the phage display rate was higher than 85%.

[0177] 1.2 Antibody panning and screening

[0178] The constructed library was subjected to four rounds of panning, and then the binding ability was screened via ELISA. Thirty selected clones were further ranked by SPR, and 10 clones were selected for sequencing. The sequences are shown in Table 3.

[0179] Table 3 Sequences of exemplary anti-GCC single-domain antibodies

[0180]

[0181] Example 2. Characterization of the exemplary anti-GCC single-domain antibodies of the present application

[0182] 2.1 Detection of the binding affinity of anti-GCC sdAb via surface plasmon resonance (SPR)

[0183] Dilute the anti-GCC sdAb with 1xHBS-EP buffer. Capture human GUCY2C on an anti-human Fc capture Ab immobilized sCM5 chip. Then inject the anti-GCC sdAb sample at a flow rate of 10 μl / min for 180 seconds, followed by dissociation for 800 seconds. Use the 1:1 Langmuir binding model and locally fit the binding curve using the Biacore® T200 analysis software (BIAevaluation). The results are shown in Table 4 and Figure 1 as follows.

[0184] Table 4 Binding affinities of exemplary anti-GCC sdAbs of the present application

[0185]

[0186] 2.2 Binding affinities of exemplary anti-GCC sdAbs of the present application to T84 cells

[0187] Use T84 cells expressing GCC to evaluate the binding affinity of the anti-GCC sdAb. Briefly, incubate 50 μL of T84 cells at a density of 3×10 6 cells / mL with 50 μL of the anti-GCC sdAb at 4 °C for 2 hours. After incubation, wash the cells three times with 400 μL of FACS buffer, resuspend with the PE-anti-human Fc secondary antibody (eBioscience 12-4998-82, diluted 1:1000), and incubate at 4 °C for 60 min. Then collect the cells and perform FACS analysis. The results are shown in Table 5A and Table 5B below.

[0188] Table 5A. Geometric mean MFI (300 nm) of exemplary anti-GCC sdAbs of the present application

[0189]

[0190] Table 5B. Geometric mean MFI (300 nm) of anti-GCC sdAbs of the present application

[0191]

[0192] Example 3. Internalization of anti-GCC single domain antibodies of the present application

[0193] Label the anti-His antibody with the pHrodo iFLRed STP ester amine-reactive dye according to the manufacturer's instructions and purify it through a zeba TM spin desalting column. Seed 293F cells overexpressing GCC expressing GCC at 1.5×10 4Cells were seeded at a density of Figure 2 per well in a 96-well plate containing complete medium with 10% FBS and cultured overnight at 37 °C and 5% CO2. Different concentrations of the anti-GCC of the present application were mixed with the pHrodo iFL Red / anti-His antibody conjugate at a ratio of 1:2 (unit: mol / L). Then, the GCC-overexpressing 293F cells were incubated with the mixture at 4 °C for 6 hours. After incubation, the cells were treated with 100 μL of Hoechst 33342 (4 μg / ml) diluted in DPBS at 4 °C for 15 min. The fluorescence intensity of the GCC-overexpressing 293F cells was analyzed by the Perkin Elmer Operetta CLS high-content analysis system. The internalization results are shown in

[0194] Example 4. Antibody binning

[0195] The anti-GCC sdAb was diluted with 1xHBS-EP buffer. Human GUCY2C was captured on an anti-human Fc capture Ab immobilized sCM5 chip. Then, the anti-GCC sdAb sample was injected at a flow rate of 30 μl / min for 120 seconds, and then dissociated for 120 seconds. Then, the competitive anti-GCC sdAb sample was injected at a flow rate of 30 μl / min for 120 seconds, and then dissociated for 120 seconds. The results were locally fitted using the Biacore® T200 analysis software (BIAevaluation), as shown in Table 6. It can be seen from Table 6 that the anti-GCC sdAbs with SEQ ID NO. 1-4 basically bind to the same epitope in the GCC antigen; the anti-GCC sdAbs with SEQ ID NO. 5 and 6 basically bind to the same epitope in the GCC antigen; and the anti-GCC sdAbs with SEQ ID NO. 7-9 basically bind to the same epitope in the GCC antigen.

[0196] Table 6 Antibody binning

[0197]

[0198] Example 5. Humanization of the exemplary anti-GCC single-domain antibodies of the present application

[0199] The selected anti-GCC sdAb was further humanized. Briefly, the amino acid sequences of the candidate antibodies (hits) in Table 3 were compared with the available human Ig gene sequence database to obtain the best-matched human germline Ig gene sequences. Then, the CDR1, CDR2, and CDR3 coding sequences of each selected sdAb were transplanted onto their best-matched human germline Ig gene frameworks to obtain humanized sdAbs, as shown in Table 7 below.

[0200] Table 7 Humanized anti-GCC sdAb

[0201]

[0202]

[0203]

[0204] Example 6. Characterization of the Humanized Anti-GCC Single Domain Antibody of the Present Application

[0205] The exemplary humanized anti-GCC sdAb of the present application was diluted with 1xHBS-EP buffer. Human GUCY2C was captured on an anti-human Fc capture Ab immobilized sCM5 chip. Then, the humanized anti-GCC sdAb sample was injected at a flow rate of 10 μl / min for 180 seconds and then dissociated for 800 seconds. Using a 1:1 Langmuir binding model, the binding curve was locally fitted using Biacore® T200 analysis software (BIAevaluation). The results are shown in Table 8.

[0206] Table 8. Binding Affinity of the Exemplary Humanized Anti-GCC sdAb of the Present Application

[0207]

[0208]

[0209] The scope of the present disclosure is not limited by the specific embodiments, which are intended to be a single illustration of various aspects of the present disclosure, and any compositions or methods that are functionally equivalent are within the scope of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Accordingly, the present disclosure is intended to cover modifications and variations of the present disclosure as long as they come within the scope of the appended claims and their equivalents.

[0210] All publications and patent applications mentioned in this specification are incorporated herein by reference to the extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0211] The present invention has been described in terms of specific embodiments found or proposed by the inventors to include preferred modes of practicing the invention. Those skilled in the art will appreciate that various modifications and changes can be made to the specific embodiments illustrated without departing from the intended scope of the invention. For example, due to codon redundancy, the underlying DNA sequence can be altered without affecting the protein sequence. In addition, for considerations of biological functional equivalence, the protein structure can be changed without affecting the type or amount of biological activity. All such modifications are intended to be included within the scope of the appended claims.

[0212] Sequence Listing

[0213]

[0214]

[0215]

[0216]

Claims

1. An antibody that specifically binds to guanylate cyclase 2C (GCC), comprising CDR1, CDR2, and CDR3 of a single-domain antibody selected from any one of SEQ ID NOs. 1-10 and 118-123.

2. The antibody according to claim 1, wherein the CDR1, CDR2, and CDR3 are numbered according to IMGT.

3. The antibody according to claim 1, wherein the CDR1, CDR2, and CDR3 are numbered according to Kabat.

4. An antibody that specifically binds to guanylate cyclase 2C (GCC), comprising: (1) CDR1, which comprises an amino acid sequence selected from: (i) SEQ ID NOs. 11-20 and 124-129, and (ii) an amino acid sequence having one or more amino acid alterations compared to SEQ ID NOs. 11-20 and 124-129; (2) CDR2, which comprises an amino acid sequence selected from: (i) SEQ ID NOs. 21-30 and 130-135, and (ii) an amino acid sequence having one or more amino acid alterations compared to SEQ ID NOs. 21-30 and 130-135; and (3) CDR3, which comprises an amino acid sequence selected from: (i) SEQ ID NOs. 31-40 and 136-141, and (ii) an amino acid sequence having one or more amino acid alterations compared to SEQ ID NOs. 31-40 and 136-141.

5. The antibody according to claim 4, wherein the antibody comprises: (1) CDR1 comprising the amino acid sequence of SEQ ID NO. 11 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 11, CDR2 comprising the amino acid sequence of SEQ ID NO. 21 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 21, and CDR3 comprising the amino acid sequence of SEQ ID NO. 31 or an amino acid sequence having one or more amino acid alterations compared to SEQ ID NO. 31; (2) CDR1 comprising the amino acid sequence of SEQ ID NO. 12 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 12, CDR2 comprising the amino acid sequence of SEQ ID NO. 22 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 22, and CDR3 comprising the amino acid sequence of SEQ ID NO. 32 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 32; (3) CDR1 comprising the amino acid sequence of SEQ ID NO. 13 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 13, CDR2 comprising the amino acid sequence of SEQ ID NO. 23 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 23, and CDR3 comprising the amino acid sequence of SEQ ID NO. 33 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 33; (4) CDR1 comprising the amino acid sequence of SEQ ID NO. 14 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 14, CDR2 comprising the amino acid sequence of SEQ ID NO. 24 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 24, and CDR3 comprising the amino acid sequence of SEQ ID NO. 34 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 34; (5) CDR1 comprising the amino acid sequence of SEQ ID NO. 15 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 15, CDR2 comprising the amino acid sequence of SEQ ID NO. 25 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 25, and CDR3 comprising the amino acid sequence of SEQ ID NO. 35 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 35; (6) CDR1 comprising the amino acid sequence of SEQ ID NO. 16 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 16, CDR2 comprising the amino acid sequence of SEQ ID NO. 26 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 26, and CDR3 comprising the amino acid sequence of SEQ ID NO. 36 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 36; (7) A CDR1 comprising the amino acid sequence of SEQ ID NO. 17 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 17, a CDR2 comprising the amino acid sequence of SEQ ID NO. 27 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO. 37 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 37; (8) A CDR1 comprising the amino acid sequence of SEQ ID NO. 18 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 18, a CDR2 comprising the amino acid sequence of SEQ ID NO. 28 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 28, and a CDR3 comprising the amino acid sequence of SEQ ID NO. 38 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 38; (9) A CDR1 comprising the amino acid sequence of SEQ ID NO. 19 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 19, a CDR2 comprising the amino acid sequence of SEQ ID NO. 29 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 29, and a CDR3 comprising the amino acid sequence of SEQ ID NO. 39 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 39; (10) A CDR1 comprising the amino acid sequence of SEQ ID NO. 20 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 20, a CDR2 comprising the amino acid sequence of SEQ ID NO. 30 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 30, and a CDR3 comprising the amino acid sequence of SEQ ID NO. 40 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 40; (11) A CDR1 comprising the amino acid sequence of SEQ ID NO. 124 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 124, a CDR2 comprising the amino acid sequence of SEQ ID NO. 130 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 130, and a CDR3 comprising the amino acid sequence of SEQ ID NO. 136 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 136; (12) A CDR1 comprising the amino acid sequence of SEQ ID NO. 125 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 125, a CDR2 comprising the amino acid sequence of SEQ ID NO. 131 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 131, and a CDR3 comprising the amino acid sequence of SEQ ID NO. 137 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 137; (13) A CDR1 comprising the amino acid sequence of SEQ ID NO. 126 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 126, a CDR2 comprising the amino acid sequence of SEQ ID NO. 132 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 132, and a CDR3 comprising the amino acid sequence of SEQ ID NO. 138 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 138; (14) A CDR1 comprising the amino acid sequence of SEQ ID NO. 127 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 127, a CDR2 comprising the amino acid sequence of SEQ ID NO. 133 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 133, and a CDR3 comprising the amino acid sequence of SEQ ID NO. 139 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 139; (15) CDR1 comprising the amino acid sequence of SEQ ID NO. 128 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 128, CDR2 comprising the amino acid sequence of SEQ ID NO. 134 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 134, and CDR3 comprising the amino acid sequence of SEQ ID NO. 140 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 140; or (16) CDR1 comprising the amino acid sequence of SEQ ID NO. 129 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 129, CDR2 comprising the amino acid sequence of SEQ ID NO. 135 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO. 135, and CDR3 comprising the amino acid sequence of SEQ ID NO. 141 or an amino acid sequence having one or more amino acid alterations as compared to SEQ ID NO.

141.

6. The antibody according to any one of claims 1-5, wherein the antibody comprises an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity or at least 99% identity to any one selected from SEQ ID NOs. 1-10 and 82-123.

7. The antibody according to claim 6, wherein the antibody comprises an amino acid sequence selected from any one of SEQ ID NOs. 1-10 and 82-123.

8. An antibody construct comprising the antibody according to any one of claims 1-7.

9. The antibody construct according to claim 8, which further comprises an Fc fragment.

10. The antibody construct according to claim 9, wherein the Fc fragment is selected from IgG1, IgG2, IgG3 and IgG4.

11. The antibody construct according to claim 9 or 10, wherein the antibody construct is multispecific.

12. The antibody construct according to claim 11, wherein the antibody construct is bispecific.

13. A nucleic acid encoding the antibody according to any one of claims 1-7 or the antibody construct according to any one of claims 8-12.

14. The nucleic acid according to claim 13, which comprises a polynucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to any one selected from SEQ ID NOs. 41 - 50 and 160 - 165.

15. The nucleic acid according to claim 14, which comprises a polynucleotide sequence selected from any one of SEQ ID NOs. 41 - 50 and 160 - 165.

16. A vector, which comprises the nucleic acid according to claim 15.

17. A host cell, which comprises the nucleic acid according to any one of claims 13 - 15 or the vector according to claim 16.

18. A conjugate, which comprises: (1) An antibody according to any one of claims 1 - 7 or an antibody construct according to any one of claims 8 - 12, and (2) A drug.

19. The conjugate according to claim 18, wherein the drug is selected from chemotherapeutic agents, toxins, cytokines, enzymes, immunomodulators, chelating agents, diagnostic agents, nanoparticles and radioisotopes.

20. The conjugate according to claim 18 or 19, which further comprises a linker.

21. A chimeric antigen receptor (CAR), which comprises an antibody construct according to any one of claims 8 - 12.

22. A T cell receptor (TCR), which comprises an antibody construct according to any one of claims 8 - 12.

23. A cell, which expresses the CAR according to claim 21 or the TCR according to claim 22.

24. A pharmaceutical composition, which comprises: (1) An antibody according to any one of claims 1 - 7, an antibody construct according to any one of claims 8 - 12, a nucleic acid according to any one of claims 13 - 15, a conjugate according to claims 18 - 20 or a cell according to claim 23, and (2) A pharmaceutically acceptable excipient.

25. A method for treating a disorder in a subject in need thereof, which comprises administering to the subject an effective amount of the pharmaceutical composition according to claim 24.

26. A method for treating a disorder associated with the presence of unwanted GCC+ cells, which comprises administering to a subject an effective amount of the pharmaceutical composition according to claim 24.

27. The method according to claim 25 or 26, wherein the disorder is cancer.

28. Use of the pharmaceutical composition according to claim 24 in the preparation of a medicament for treating a disorder in a subject in need thereof.

29. Use of an antibody according to any one of claims 1-7 for detecting GCC in a sample from a subject.

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