Guanylate cyclase C (GCC) antigen-binding agent compositions and methods of use thereof

Abstract

Antigen-binding agents (e.g., single-domain antibodies) that bind to guanylate cyclase C (GCC) are disclosed. Nucleic acids, recombinant expression vectors, host cells, antigen-binding fragments, and pharmaceutical compositions comprising these antigen-binding agents and fragments thereof are also disclosed. The present invention also provides therapeutic methods utilizing the antibodies and antigen-binding molecules provided herein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the priority of U.S. Provisional Patent Application No. 63 / 123,333, filed on December 9, 2020, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII text file is hereby incorporated by reference in its entirety, was created on November 18, 2021, has the name "MIL - 011WO_SL", and is 33,391 bytes in size.

Background Art

[0003] Guanylate cyclase C (GCC) is a transmembrane cell - surface receptor that functions in the maintenance of intestinal fluid, electrolyte homeostasis, and cell proliferation. See, for example, Carrithers et al., Proc. Natl. Acad. Sci. USA 100:3018 - 3020 (2003). GCC is expressed in mucosal cells that line the inner surface of the small intestine, large intestine, and rectum (Carrithers et al., Dis Colon Rectum 39:171 - 181 (1996)). GCC expression is maintained during the neoplastic transformation of intestinal epithelial cells and is associated with expression in all primary and metastatic colorectal tumors (Carrithers et al., Dis Colon Rectum 39:171 - 181 (1996), Buc et al. Eur J Cancer 41:1618 - 1627 (2005), Carrithers et al., Gastroenterology 107:1653 - 1661 (1994)). There is a need for new and improved methods of targeting GCC.

Summary of the Invention

[0004] Disruption of the guanylate cyclase C (GCC) signaling pathway is associated with a number of gastrointestinal disorders, including colorectal cancer. The present invention provides, inter alia, novel anti-GCC antigen-binding molecules (e.g., single domain antibodies (sdAbs)).

[0005] In one aspect, the invention provides a guanylate cyclase C (GCC) binder comprising a heavy chain variable region (V H ) having the complementarity determining region (CDR) sequences of HYYWS (HCDR1) (SEQ ID NO: 8), RIYPSGSTSYNPSLKS (HCDR2) (SEQ ID NO: 11), and DRSTGWSEWNSDL (HCDR3) (SEQ ID NO: 16).

[0006] In one aspect, the invention provides a guanylate cyclase C (GCC) binder consisting of a heavy chain variable region (V H ) having the complementarity determining region (CDR) sequences of HYYWS (HCDR1) (SEQ ID NO: 8), RIYPSGSTSYNPSLKS (HCDR2) (SEQ ID NO: 11), and DRSTGWSEWNSDL (HCDR3) (SEQ ID NO: 16).

[0007] In one aspect, the invention provides a guanylate cyclase C (GCC) binder comprising a heavy chain variable region (V H ) having the complementarity determining region (CDR) sequences of RYWMS (HCDR1) (SEQ ID NO: 9), KIRHDGGEKYYVDSVKG (HCDR2) (SEQ ID NO: 12), and DYTRDV (HCDR3) (SEQ ID NO: 17).

[0008] In one aspect, the invention provides a guanylate cyclase C (GCC) binder consisting of a heavy chain variable region (V H ) having the complementarity determining region (CDR) sequences of RYWMS (HCDR1) (SEQ ID NO: 9), KIRHDGGEKYYVDSVKG (HCDR2) (SEQ ID NO: 12), and DYTRDV (HCDR3) (SEQ ID NO: 17).

[0009] In one aspect, the present invention provides a guanylate cyclase C (GCC) binder comprising a heavy chain variable region (V H ) having the complementarity determining region (CDR) sequences of RYWMT (HCDR1) (SEQ ID NO: 10), KIKYDGSEKYYADSVKG (HCDR2) (SEQ ID NO: 13), and DYNKDY (HCDR3) (SEQ ID NO: 18).

[0010] In one aspect, the present invention provides a guanylate cyclase C (GCC) binder consisting of a heavy chain variable region (V H ) having the complementarity determining region (CDR) sequences of RYWMT (HCDR1) (SEQ ID NO: 10), KIKYDGSEKYYADSVKG (HCDR2) (SEQ ID NO: 13), and DYNKDY (HCDR3) (SEQ ID NO: 18).

[0011] In one aspect, the present invention provides a guanylate cyclase C (GCC) binder comprising a heavy chain variable region (V H ) having the complementarity determining region (CDR) sequences of RYWMT (HCDR1) (SEQ ID NO: 10), KIRHDGGEKYYPDSVKG (HCDR2) (SEQ ID NO: 14), and DYNKDL (HCDR3) (SEQ ID NO: 19).

[0012] In one aspect, the present invention provides a guanylate cyclase C (GCC) binder consisting of a heavy chain variable region (V H ) having the complementarity determining region (CDR) sequences of RYWMT (HCDR1) (SEQ ID NO: 10), KIRHDGGEKYYPDSVKG (HCDR2) (SEQ ID NO: 14), and DYNKDL (HCDR3) (SEQ ID NO: 19).

[0013] In one aspect, the present invention provides a guanylate cyclase C (GCC) binder comprising a heavy chain variable region (V H ) having the complementarity determining region (CDR) sequences of RYWMT (HCDR1) (SEQ ID NO: 10), KIRHDGGEKYYADSVKG (HCDR2) (SEQ ID NO: 15), and DYNKDY (HCDR3) (SEQ ID NO: 18).

[0014] In one embodiment, the present invention relates to a heavy chain variable region (V) having complementarity determination region (CDR) sequences of RYWMT(HCDR1)(SEQ ID NO: 10), KIRHDGGEKYYADSVKG(HCDR2)(SEQ ID NO: 15), and DYNKDY(HCDR3)(SEQ ID NO: 18). H The present invention provides a guanylate cyclase C (GCC) binder consisting of the following components.

[0015] In some embodiments, the GCC binder includes an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 20.

[0016] In some embodiments, the GCC binder comprises an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 20.

[0017] In some embodiments, the GCC binder includes an immunoglobulin heavy chain variable (VH) region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 21.

[0018] In some embodiments, the GCC binder comprises an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 21.

[0019] In some embodiments, the GCC binder includes an immunoglobulin heavy chain variable (VH) region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 26.

[0020] In some embodiments, the GCC binder comprises an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 26.

[0021] In some embodiments, the GCC binder includes an immunoglobulin heavy chain variable (VH) region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 27.

[0022] In some embodiments, the GCC binder comprises an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 27.

[0023] In some embodiments, the GCC binder includes an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 28.

[0024] In some embodiments, the GCC binder comprises an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 28.

[0025] In one embodiment, the present invention provides a guanylate cyclase C (GCC) binder comprising an immunoglobulin heavy chain variable (VH) region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 20.

[0026] In one embodiment, the present invention provides a guanylate cyclase C (GCC) binder comprising an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 20.

[0027] In one embodiment, the present invention provides a guanylate cyclase C (GCC) binder comprising an immunoglobulin heavy chain variable (VH) region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 21.

[0028] In one embodiment, the present invention provides a guanylate cyclase C (GCC) binder comprising an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 21.

[0029] In one embodiment, the present invention provides a guanylate cyclase C (GCC) binder comprising an immunoglobulin heavy chain variable (VH) region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 26.

[0030] In one embodiment, the present invention provides a guanylate cyclase C (GCC) binder comprising an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 26.

[0031] In one embodiment, the present invention provides a guanylate cyclase C (GCC) binder comprising an immunoglobulin heavy chain variable (VH) region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 27.

[0032] In one embodiment, the present invention provides a guanylate cyclase C (GCC) binder comprising an immunoglobulin heavy chain variable (VH) region containing an amino acid sequence that is at least 90% identical to SEQ ID NO: 27.

[0033] In one embodiment, the present invention provides a guanylate cyclase C (GCC) binder comprising an immunoglobulin heavy chain variable (VH) region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 28.

[0034] In one embodiment, the present invention provides a guanylate cyclase C (GCC) binder comprising or consisting of an immunoglobulin heavy chain variable (VH) region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 28.

[0035] In some embodiments, the GCC binder contains an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 1, 20, 21, 26, 27, or 28. H Includes the region.

[0036] In some embodiments, the GCC binder contains an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 1, 20, 21, 26, 27, or 28. H It consists of regions.

[0037] In some embodiments, the GCC binder contains an amino acid sequence identical to any one of SEQ ID NOs: 1, 20, 21, 26, 27, or 28. H Includes the region.

[0038] In some embodiments, the GCC binder contains an amino acid sequence identical to any one of SEQ ID NOs: 1, 20, 21, 26, 27, or 28. H It consists of regions.

[0039] In some embodiments, the GCC conjugate is selected from the group consisting of IgA antibodies, IgG antibodies, IgE antibodies, IgM antibodies, bi- or multispecific antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, isolated CDRs or sets thereof, single-strand variable fragments (scFv), polypeptide-Fc fusions, single-domain antibodies (sdAb), camel antibodies, mask antibodies, Small Modular ImmunoPharmaceuticals ("SMIPs" trademark), single-strand, tandem diabody, VHH, Anticalin, nanobody, humabody, minibody, BiTE, ankyrin repeat protein, DARPIN, avimer, DART, TCR-like antibodies, Adnectin, Affilin, transbody, Affibody, TrimerX, microproteins, Fynomer, centinlin, and KALBITOR.

[0040] In some embodiments, the GCC conjugate is a single-domain antibody (sdAb). In some embodiments, the GCC conjugate is V H It is a single-domain antibody.

[0041] In some embodiments, the GCC conjugate is an antibody consisting only of the heavy chain.

[0042] In some embodiments, the GCC binder binds to GCC with a KD of approximately 0.3 nanomoles (nM) to approximately 10 nM.

[0043] In some embodiments, the GCC binder binds to GCCs on target cells with an EC50 of approximately 0.5 nM to approximately 8 nM.

[0044] In one embodiment, the present invention provides a method for treating cancer, the method comprising administering a GCC conjugate described herein to a subject in need of treatment.

[0045] In some embodiments, the cancer is selected from gastrointestinal cancer, colorectal cancer, colorectal adenocarcinoma, colorectal leiomyosarcoma, colorectal lymphoma, colorectal melanoma, colorectal neuroendocrine tumor, metastatic colon cancer, gastric cancer, gastric adenocarcinoma, gastric lymphoma, gastric sarcoma, esophageal cancer, squamous cell carcinoma, esophageal adenocarcinoma, or pancreatic cancer.

[0046] In some embodiments, the cancer is gastrointestinal cancer.

[0047] In some embodiments, gastrointestinal cancer is colon cancer, colorectal cancer, stomach cancer, or esophageal cancer.

[0048] In one embodiment, the present invention provides a pharmaceutical composition comprising a GCC binder and a pharmaceutically acceptable carrier, wherein the GCC binder has a heavy chain variable region (V) having complementarity-determining region (CDR) sequences of HYYWS(HCDR1) (SEQ ID NO: 8), RIYPSGSTSYNPSLKS(HCDR2) (SEQ ID NO: 11), and DRSTGWSEWNSDL(HCDR3) (SEQ ID NO: 16). H ), RYWMS(HCDR1)(SEQ ID NO: 9), KIRHDGGEKYYVDSVKG(HCDR2)(SEQ ID NO: 12), and DYTRDV(HCDR3)(SEQ ID NO: 17) are heavy chain variable regions (V) having complementarity determination region (CDR) sequences. H ), RYWMT(HCDR1)(SEQ ID NO: 10), KIKYDGSEKYYADSVKG(HCDR2)(SEQ ID NO: 13), and DYNKDY(HCDR3)(SEQ ID NO: 18) are heavy chain variable regions having complementarity determination region (CDR) sequences (V H ), RYWMT(HCDR1)(SEQ ID NO: 10), KIRHDGGEKYYPDSVKG(HCDR2)(SEQ ID NO: 14), and DYNKDL(HCDR3)(SEQ ID NO: 19) have complementarity-determining region (CDR) sequences in the heavy chain variable region (V H) or heavy chain variable region (V) having complementarity determination region (CDR) sequences of RYWMT(HCDR1)(SEQ ID NO: 10), KIRHDGGEKYYADSVKG(HCDR2)(SEQ ID NO: 15), and DYNKDY(HCDR3)(SEQ ID NO: 18). H ) includes.

[0049] In one embodiment, the present invention provides a method for treating cancer, the method comprising administering a GCC conjugate to a subject in need of treatment, the GCC conjugate having a heavy chain variable region (V) having complementarity-determining region (CDR) sequences HYYWS(HCDR1)(SEQ ID NO: 8), RIYPSGSTSYNPSLKS(HCDR2)(SEQ ID NO: 11), and DRSTGWSEWNSDL(HCDR3)(SEQ ID NO: 16). H ), RYWMS(HCDR1)(SEQ ID NO: 9), KIRHDGGEKYYVDSVKG(HCDR2)(SEQ ID NO: 12), and DYTRDV(HCDR3)(SEQ ID NO: 17) are heavy chain variable regions (V) having complementarity determination region (CDR) sequences. H ), RYWMT(HCDR1)(SEQ ID NO: 10), KIKYDGSEKYYADSVKG(HCDR2)(SEQ ID NO: 13), and DYNKDY(HCDR3)(SEQ ID NO: 18) are heavy chain variable regions having complementarity determination region (CDR) sequences (V H ), RYWMT(HCDR1)(SEQ ID NO: 10), KIRHDGGEKYYPDSVKG(HCDR2)(SEQ ID NO: 14), and DYNKDL(HCDR3)(SEQ ID NO: 19) have complementarity-determining region (CDR) sequences in the heavy chain variable region (V H ) or heavy chain variable region (V) having complementarity determination region (CDR) sequences of RYWMT(HCDR1)(SEQ ID NO: 10), KIRHDGGEKYYADSVKG(HCDR2)(SEQ ID NO: 15), and DYNKDY(HCDR3)(SEQ ID NO: 18). H ) includes.

[0050] In one embodiment, the present invention is identical to any one of sequence numbers 1, 20, 21, 26, 27, or 28. H Provides nucleic acids that encode amino acid sequences.

[0051] In one embodiment, the present invention is identical to any one of sequence numbers 1, 20, 21, 26, 27, or 28. H A vector containing nucleic acid encoding an amino acid sequence is provided.

[0052] In one embodiment, the present invention is identical to any one of sequence numbers 1, 20, 21, 26, 27, or 28. H The present invention provides isolated cells containing a vector containing nucleic acids that encode an amino acid sequence.

[0053] In one embodiment, the present invention provides an anti-guanylate cyclase C (GCC) chimeric antigen receptor (CAR), the anti-GCC CAR comprising an anti-GCC conjugate as described in any one of claims 1 to 10.

[0054] In one embodiment, the present invention provides a method for inducing an immune response, comprising contacting cells with an anti-guanylate cyclase C (GCC) chimeric antigen receptor (CAR), wherein the anti-GCC CAR comprises an anti-GCC conjugate as described in any one of claims 1 to 10.

[0055] In one embodiment, the present invention provides a method for inducing cytotoxicity, comprising contacting cells with an anti-guanylate cyclase C (GCC) chimeric antigen receptor (CAR), wherein the anti-GCC CAR comprises an anti-GCC conjugate as described in any one of claims 1 to 10.

[0056] In one embodiment, the present invention provides a method for detecting the presence of cancer in a mammal, the method comprising (a) contacting a sample containing one or more cells derived from a mammal with an anti-GCC binder according to any one of claims 1 to 10 to form a complex, and (b) detecting the complex, wherein the detection of the complex indicates the presence of cancer in the mammal.

[0057] In some embodiments, the contact is in vitro or in vivo with respect to mammals. In some embodiments, the contact is in vitro.

[0058] Both the above summary and the following detailed description are illustrative and descriptive, and should be understood not as limiting the invention to the claims.

[0059] The drawings included in this specification, which consist of the following figures, are for illustrative purposes only and not to limit. [Brief explanation of the drawing]

[0060] [Figure 1] A and B show exemplary chimeric antigen receptor (CAR) constructs. [Figure 2] This image shows exemplary anti-GCC CAR-T in vitro cell injury performed using four tumor cell lines. A shows HT29-GCC cells (human colorectal cancer cell line HT29 engineered to stably express GCC). B shows HT29-VEC (vector-controlled GCC-negative cell line). For the two tumor cell lines that endogenously express GCC, C shows GSU and D shows LS1034. The bars represent the mean + SD values ​​from three technical replicas. The data are representative of more than three independent experiments performed using anti-GCC CAR T cells derived from more than three donors. CAR T cell injury was determined in the absence of truncated EGFR (tEGFR). [Figure 3] This image shows exemplary anti-GCC CAR-T cell in vitro cytotoxicity performed using four tumor cell lines. A shows HT29-GCC cells (human colorectal cancer cell line HT29 engineered to stably express GCC). B shows HT29-VEC (vector-controlled GCC-negative cell line). For the two tumor cell lines that endogenously express GCC, C shows GSU and D shows LS1034. The bars represent the mean + SD values ​​from three technical replicas. The data are representative of more than three independent experiments performed using anti-GCC CAR T cells derived from more than three donors. CAR T cell injury was determined in the presence of truncated EGFR (tEGFR). [Figure 4]A shows exemplary IFN-γ cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with GCC-expressing (HT29-GCC) tumor cells. B shows exemplary IFN-γ cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with GCC-negative (HT29-VEC) tumor cells. C shows exemplary IFN-γ cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with GSU tumor cells. D shows exemplary IFN-γ cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with LS1034 tumor cells. IFNγ secreted into the supernatant was detected using the Intellicyt QBeads Human PlexScreen kit (Sartorius, 90702). Bars represent mean + SD values ​​from three technical replicas. Data are representative of more than three independent experiments conducted using anti-GCC CAR-T cells derived from more than three donors. Cytokine secretion was determined in the absence of cleaved EGFR (tEGFR). [Figure 5] A shows exemplary IFN-g cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with GCC-expressing (HT29-GCC) tumor cells. B shows exemplary IFN-g cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with GCC-negative (HT29-VEC) tumor cells. C shows exemplary IFN-g cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with GSU tumor cells. D shows exemplary IFN-g cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with LS1034 tumor cells. IFNg secreted into the supernatant was detected using the Intellicyt QBeads Human PlexScreen kit (Sartorius, 90702). Bars represent mean + SD values ​​from three technical replicas. Data are representative of more than three independent experiments conducted using anti-GCC CAR T cells derived from more than three donors. Cytokine secretion was determined in the presence of cleaved EGFR (tEGFR). [Figure 6]A shows exemplary IL-2 cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with GCC-expressing (HT29-GCC) tumor cells. B shows exemplary IL-2 cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with GCC-negative (HT29-VEC) tumor cells. C shows exemplary IL-2 cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with GSU tumor cells. D shows exemplary IL-2 cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with LS1034 tumor cells. IFL-2 secreted into the supernatant was detected using the Intellicyt QBeads Human PlexScreen kit (Sartorius, 90702). Bars represent mean + SD values ​​from three technical replicas. Data are representative of more than three independent experiments conducted using anti-GCC CAR T cells derived from more than three donors. Cytokine secretion was determined in the absence of cleaved EGFR (tEGFR). [Figure 7] A shows exemplary IL-2 cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with GCC (HT29-GCC) tumor cells. B shows exemplary IL-2 cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with GCC-negative (HT29-VEC) tumor cells. C shows exemplary IL-2 cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with GSU tumor cells. D shows exemplary IL-2 cytokine secretion by anti-GCC CAR-T cells co-cultured in vitro with LS1034 tumor cells. IFL-2 secreted into the supernatant was detected using the Intellicyt QBeads Human PlexScreen kit (Sartorius, 90702). Bars represent mean + SD values ​​from three technical replicas. Data are representative of more than three independent experiments conducted using anti-GCC CAR T cells derived from more than three donors. Cytokine secretion was determined in the presence of cleaved EGFR (tEGFR).

[0061] definition To facilitate understanding of the present invention, certain terms are first defined below. Additional definitions of the following terms and other terms are provided herein.

[0062] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless otherwise explicitly indicated in the content. Thus, for example, a reference to “a method” includes one or more methods and / or steps of the kind described herein and / or will be apparent to those skilled in the art when reading this disclosure, etc.

[0063] Administration: As used herein, “administering” a composition to a subject means applying, transferring, or bringing the composition into contact with the subject. Administration can be achieved by any of a number of routes, such as topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, and intradermal.

[0064] Affinity: As used herein, the term "affinity" refers to the characteristics of the binding interaction between a binding site (e.g., an antigen-binding agent (e.g., a variable domain as described herein)) and a target (e.g., an antigen (e.g., GCC)), which indicates the strength of the binding interaction. In some embodiments, the measure of affinity is the dissociation constant (K D ) is represented as. In some embodiments, the binding portion has a high affinity for the target (e.g., about 10 -7 Less than M, approximately 10 -8 Less than M, or about 10 -9 Less than M K D In some embodiments, the binding portion has low affinity for the target (e.g., about 10). -7 Over M, about 10 -6 Over M, about 10 -5 M or more, or about 10 -4 M Super K D ).

[0065] Animals: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to a human being at any stage of development. In some embodiments, “animal” refers to a non-human animal at any stage of development. In certain embodiments, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, an animal may be a transgenic animal, a genetically modified animal, and / or a clone.

[0066] Self: As used herein, the term “self” refers to any material that originates from the same organism in which any material is later reintroduced into the organism.

[0067] Same species: "Same species" refers to material derived from a certain individual that is administered to different individuals (or multiple individuals).

[0068] Antibodies or Antigen Binding Agents: As used herein, the terms “antibody” or “antigen binding agent” refer to polypeptides containing enough classical immunoglobulin sequence elements to confer specific binding to a particular target antigen. Those skilled in the art will understand that these terms may be used interchangeably herein. In some embodiments, as used herein, the terms “antibody” or “antigen binding agent” also refer to “antibody fragments” or “antigen-binding moieties,” which include, for example, portions of an intact antibody such as the antigen-binding region or variable region of an antibody. Examples of “antibody fragments” include Fab, Fab', F(ab')2, and Fv fragments, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, single-domain antibodies, and CDR-containing portions contained in multispecific antibodies formed from antibody fragments. Those skilled in the art will understand that the term “antibody fragment” does not imply, and is not limited to, any particular mode of production. Antibody fragments may be produced by any suitable method, including, but not limited to, cleavage of intact antibodies, chemical synthesis, and recombinant production. As is known in the art, intact antibodies produced in nature are tetrameric drugs of approximately 150 kD, composed of two identical heavy chain polypeptides (each about 50 kD) and two identical light chain polypeptides (each about 25 kD), which associate with each other to form a structure commonly referred to as a "Y-shape." Each heavy chain consists of at least four domains (each about 110 amino acids long), which are amino-terminally variable (V H ) Domain (located at the tip of the Y structure), followed by three stationary domains: C H 1, C H 2, and carboxy-terminal C H 3 (located at the base of the Y-shaped trunk). A short region known as the "switch" connects the heavy chain variable region and the steady region. The "hinge" is C H 2 and C H The three domains are linked to the remainder of the antibody. Two disulfide bonds in this hinge region link the two heavy chain polypeptides together in the intact antibody. Each light chain consists of two domains, which are amino-terminal variable (VL ) domains, separated from each other by another “switch” that follows, carboxyl terminal stationary (C L ) is a domain. An intact antibody tetramer consists of two heavy-light chain dimers, in which the heavy and light chains are linked to each other by one disulfide bond, and two other disulfide bonds link the heavy chain hinge regions to each other, so that the dimers are linked to each other and a tetramer is formed. Naturally produced antibodies are also typically C H Glycosylation occurs on two domains. Each domain in the natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-stranded, 4-stranded, or 5-stranded sheets) compressed together in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as “complementarity-determining regions” (CDR1, CDR2, and CDR3) as well as four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When the natural antibody folds, the FR regions form beta sheets that provide a structural framework to the domain, and the CDR loop regions from both the heavy and light chains come together in three-dimensional space, so they form a single hypervariable antigen-binding site located at the tip of a Y structure. By comparing the amino acid sequences of antibody polypeptide chains, two light chain (κ and λ) classes, several heavy chain (e.g., μ, γ, α, ε, δ) classes, and certain heavy chain subclasses (α1, α2, γ1, γ2, γ3, and γ4) are defined. Antibody classes (IgA [including IgA1 and IgA2], IgD, IgE, IgG [including IgG1, IgG2, IgG3, and IgG4], and IgM) are defined based on the class of the heavy chain sequence used.

[0069] For the purposes of the present invention, in certain embodiments, any polypeptide or polypeptide complex containing a sufficient immunoglobulin domain sequence, such as those found in natural antibodies, may be referred to as an “antibody” or “antigen conjugate” and / or used as such, regardless of whether such polypeptides are naturally produced (e.g., by organisms reacting to an antigen) or produced by recombinant operations, chemical synthesis, or other artificial systems or techniques. In some embodiments, the antibody is monoclonal, and in some embodiments, the antibody is polyclonal. In some embodiments, the antibody has a constant region sequence characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primated, chimeric, etc., as known in the art. Furthermore, the terms “antibody” or “antigen conjugate,” as used herein, are understood to encompass (unless otherwise stated or evident from the context) any construct or format known or developed in the art to acquire the structural and functional characteristics of an antibody in an alternative presentation, in appropriate embodiments. For example, in some embodiments, the term may refer to bipolar or other multispecific (e.g., zybody) antibodies, Small Modular ImmunoPharmaceuticals ("SMIPs" trademark), single-chain antibodies, camel antibodies, and / or antibody fragments. In some embodiments, antibodies may lack covalent modifications (e.g., glycan linkages) that they would have if naturally produced. In some embodiments, antibodies may contain covalent modifications (e.g., glycan linkages, payloads [e.g., detectable portion, therapeutic portion, catalytic portion, etc.], or other pendant groups [e.g., polyethylene glycol, etc.]).

[0070] Approximately or about: As used herein, the terms “approximately” or “about” refer to a value that is similar to the given reference value when applied to one or more values ​​of interest. In certain embodiments, unless otherwise stated or evident from the context (unless such a number would exceed 100% of the possible values), the terms “approximately” or “about” refer to a range of values ​​that fall in either direction (above or below) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the given reference value.

[0071] Complementarity-Determining Region (CDR): The “CDR” of a variable domain is an amino acid residue within a variable region identified according to Kabat, Chothia, the stacking of both Kabat and Chothia, i.e., AbM, contact, and / or structural definitions, or any CDR determination method well known in the art. The CDR of an antibody may be identified as a hypervariable region, first defined by Kabat et al. See, for example, Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington DC. The location of the CDR may also be identified as a structural loop structure, first described by Chothia and others. See, for example, Chothia et al., Nature 342:877-883, 1989. Other approaches to CDR identification include a compromise between Kabat and Chothia, the “AbM definition” derived using Oxford Molecular’s ​​AbM antibody modeling software (now Accelrys®), or the “contact definition” of CDRs based on observed antigen contact sites, as described in MacCallum et al., J.Mol.Biol., 262:732-745, 1996. Another approach, referred herein as the “structural definition” of CDRs, may identify the location of a CDR as a residue that contributes enthalpily to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283:1 156-1166, 2008. Furthermore, other CDR boundary definitions may not strictly follow one of the approaches described above, and the CDR may be shortened or extended in light of predictions or experimental findings that certain residues, groups of residues, or even the entire CDR do not significantly affect antigen binding, but nevertheless overlap with at least a portion of the Kabat CDR.

[0072] Unless otherwise stated, the definition of CDR as used herein shall be as defined in Kabat CDR.

[0073] Effector Function: As used herein, the term “effector function” refers to biological activity such as that resulting from an antigen-binding agent as described herein. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation. “Reduced or minimized” antibody effector function means a reduction of at least 50% (or 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) from a wild-type or unmodified antibody. Determining antibody effector function is readily achievable and measurable by those skilled in the art. In some embodiments, antibody effector functions of complement binding, complement-dependent cytotoxicity, and antibody-dependent cytotoxicity are affected. In some embodiments, effector function is eliminated by mutations in the constant region that exclude glycosylation, e.g., “effectorless mutations.” In one embodiment, the effectorless mutation is the N297A or DANA mutation (D265A+N297A) in the CH2 region. Shields et al., J. Biol. Chem. 276(9):6591-6604 (2001). Alternatively, additional mutations that result in reduced or eliminated effector function include K322A and L234A / L235A (LALA). Alternatively, effector function may be reduced or eliminated by production techniques, such as expression in non-glycosylating host cells (e.g., E. coli) or in host cells that result in alterations of the glycosylation pattern that are ineffective or have a weak effect in promoting effector function (e.g., Shinkawa et al., J. Biol. Chem. 278(5):3466-3473 (2003)).

[0074] Antibody-dependent cell-mediated cytotoxicity, or ADCC, refers to a form of cytotoxicity in which secreted immunoglobulin (Ig) bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) allows these cytotoxic effector cells to specifically bind to antigen-carrying target cells, subsequently killing them using cytotoxicity. "Arming" cytotoxic cells with antibodies is necessary for target cell death through this mechanism. NK cells, the main cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To evaluate the ADCC activity of the molecule of interest, in vitro ADCC assays, such as those described in U.S. Patent No. 5,500,362 or No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as the model disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0075] Antigen: As used herein, the term “antigen” refers to an activating factor that induces an immune response, and / or an activating factor that is bound by a T cell receptor (e.g., when presented by an MHC molecule) or, when exposed to or administered to an organism, binds to an antibody (e.g., produced by a B cell). In some embodiments, an antigen induces a humoral response in an organism (e.g., including the production of an antigen-specific antibody), or, in some embodiments further, an antigen induces a cellular response in an organism (e.g., involving a T cell whose receptor specifically interacts with the antigen). It will be understood by those skilled in the art that a particular antigen may induce an immune response in one or more members of a target organism (e.g., mouse, rabbit, primate, human), but not in all members of the target species. In some embodiments, the antigen elicits an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of members of the target species. In some embodiments, the antigen may or may not bind to antibodies and / or T cell receptors and induce specific physiological responses in the organism. In some embodiments, for example, the antigen may bind to antibodies and / or T cell receptors in vitro, regardless of whether such interactions occur in vivo. In some embodiments, the antigen reacts with specific humoral or cellular immunity products, including those induced by heterologous immunogens. In some embodiments of the disclosed compositions and methods, the GCC protein is the antigen.

[0076] Related to: As used herein, two events or entities are “related” to each other if the presence, level and / or form of one correlates with those of the other. For example, a particular entity (e.g., a polypeptide) is considered related to a particular disease, disorder, or condition if its presence, level and / or form correlates with the incidence and / or susceptibility of that disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “related” to each other if they are physically close to each other and remain close by interacting directly or indirectly. In some embodiments, two or more entities that are physically related to each other are covalently bonded to each other, and in some embodiments, two or more entities that are physically related to each other are not covalently bonded to each other but are non-covalently related by, for example, hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0077] Bonding: The term “bonding,” as used herein, is generally understood to refer to non-covalent associations between or among two or more entities. “Direct” bonding includes physical contact between entities or parts, and indirect bonding includes physical interactions through physical contact with one or more intermediate entities. Bonding between two or more entities can be evaluated in any of a variety of contexts, including when the interacting entities or parts are studied alone or in relation to a more complex system (e.g., when they associate covalently or otherwise with a carrier entity, and / or within a biological system or cell). a "K" refers to the binding rate between a specific binding site and a target for forming a binding site / target complex. As used herein, "K" d "K" refers to the dissociation rate of a specific binding site / target complex. As used herein, "K" D " refers to the dissociation constant, which is K d and K a The ratio to (i.e., K d / K a It is obtained from ) and expressed as molar concentration (M). KD The value can be determined using methods well established in the art, for example, by using surface plasmon resonance or by using a biosensor system such as the Biacore® system.

[0078] Carrier: As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle administered with the composition. In some exemplary embodiments, the carrier may be a sterile solution, such as water and oil, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil. In some embodiments, the carrier is one or more solid components.

[0079] Characteristic parts: As used herein, the term “characteristic parts” is used in its broadest sense to refer to parts of a substance whose presence (or absence) correlates with the presence (or absence) of a particular feature, attribute, or activity of that substance. In some embodiments, a characteristic part of a substance is not a substance that does not share a particular feature, attribute, or activity, but rather a part found in a substance and related substances that share a particular feature, attribute, or activity.

[0080] Codon Optimization: As used herein, a “codon-optimized” nucleic acid sequence refers to a nucleic acid sequence that has been modified so that the translation of the nucleic acid sequence and the expression of the resulting protein are improved for optimization to a particular expression system. A “codon-optimized” nucleic acid sequence encodes the same protein as the non-optimized parent sequence on which the “codon-optimized” nucleic acid sequence is based. For example, a nucleic acid sequence may be “codon-optimized” for expression in mammalian cells (e.g., CHO cells, human cells, mouse cells, etc.), bacterial cells (e.g., E. coli), insect cells, yeast cells, or plant cells.

[0081] Equivalent: As used herein, the term "equivalent" means two or more drugs, entities, situations, states, etc., that are not identical to one another but are similar enough to allow comparison between them, and that conclusions can be reasonably drawn based on observed differences or similarities. Those skilled in the art will understand the degree of identity required for two or more such drugs, entities, situations, states, etc., to be considered equivalent in any given context.

[0082] Corresponding to: As used herein, the term "corresponding to" is often used to specify the position / identity of an amino acid residue in a polypeptide of interest. For the sake of brevity, since residues in a polypeptide are often specified using a classical numbering system based on a reference related polypeptide, for example, the amino acid "corresponding" to the residue at position 190 does not actually have to be the 190th amino acid in a particular amino acid chain, but rather corresponds to the residue found at position 190 in the reference polypeptide, and those skilled in the art will readily understand how to identify the "corresponding" amino acid.

[0083] Origin: As used herein, the terms “derived from the specified sequence” or “specific to the specified sequence” refer to a sequence comprising, for example, a sequence containing a sequence of at least approximately six nucleotides or at least two amino acids, at least about nine nucleotides or at least three amino acids, at least about 10 to 12 nucleotides or four amino acids, or at least about 15 to 21 nucleotides or 5 to 7 amino acids that corresponds to, i.e., is identical to or complementary to, a region adjacent to the specified sequence. In certain embodiments, the sequence comprises all of the specified nucleotide or amino acid sequence. The sequence may be complementary (in the case of a polynucleotide sequence) or identical to a sequence region specific to a particular sequence, as determined by known art. Regions from which the sequence may originate include, but are not limited to, regions encoding a specific epitope, regions encoding a CDR, regions encoding a framework sequence, regions encoding a constant domain region, and regions encoding a variable domain region, as well as untranslated and / or untranscribed regions. The resulting sequence may not necessarily be physically derived from the target sequence under test, but may be generated by any method, including but not limited to chemical synthesis, replication, reverse transcription, or transcription, based on information provided by the base sequence of the region(s) from which the polynucleotide originates. Therefore, the sequence may represent either the sense or antisense direction of the original polynucleotide. In addition, combinations of regions corresponding to the regions of the specified sequence may be modified or combined in ways known in the art to suit the intended use. For example, the sequence may contain two or more consecutive sequences, each containing a portion of the specified sequence, with inserted regions intended to represent a sequence that is not identical to the specified sequence but is derived from it. With respect to antibody molecules, “derived from it” includes antibody molecules functionally or structurally related to the comparative antibody; for example, “derived from it” includes antibody molecules having similar or substantially the same sequence or structure, e.g., having the same or similar CDR, framework, or variable region.The “derived from” an antibody also includes residues, e.g., one or more, e.g., two, three, four, five, six or more residues, which may or may not be consecutive, defined or identified by a numbering scheme or homology or comparison sequence to a general antibody structure, i.e., three-dimensional proximity within a CDR or framework region. The term “derived from” is not limited to physically derived from it, but includes any method of generation, e.g., generation by using sequence information from a comparison antibody to design another antibody.

[0084] Determining: Many of the methods described herein involve a “determining” step. Those skilled in the art will understand, upon reading this specification, that such “determining” may utilize any of the various techniques available to those skilled in the art, including, for example, certain techniques explicitly mentioned herein. In some embodiments, the determination involves the manipulation of a physical sample. In some embodiments, the determination involves the consideration and / or manipulation of data or information, for example, utilizing a computer or other processing unit adapted to perform the relevant analysis. In some embodiments, the determination involves obtaining relevant information and / or material from a source. In some embodiments, the determination involves comparing one or more features of a sample or entity with an equivalent reference.

[0085] Manipulated: The term “manipulated,” as used herein, describes a polynucleotide, polypeptide, or cell that has been designed or modified by a human being, and / or whose presence and production require human intervention and / or activity. For example, a manipulated cell is intentionally designed to induce a particular effect, which differs from the effect of a naturally occurring cell of the same kind. In some embodiments, the manipulated cell expresses a chimeric antigen receptor as described herein. Exemplary manipulative methods are described in the sections on modes and examples for carrying out the invention.

[0086] Epitope: As used herein, the term “epitope” includes any portion that is specifically recognized, either whole or partially, by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope consists of multiple amino acids within an antigen. In some embodiments, such amino acid residues are exposed on the surface when the antigen takes on a relevant three-dimensional structure. In some embodiments, the amino acid residues are physically close to each other in space or conform to each other when the antigen takes on such a structure. In some embodiments, at least some of the amino acids are physically separated from each other (e.g., a non-linear epitope) when the antigen takes on a different structure (e.g., linear).

[0087] Excipients: As used herein, the term “excipient” refers to non-therapeutic agents that may be included in a pharmaceutical composition to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, and ethanol.

[0088] Expression: When used herein in relation to nucleic acids, the terms “expression” or “to be expressed” mean one or more of the following events: (1) production of an RNA transcript from a DNA template (e.g., by transcription), (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end formation), (3) translation of RNA into a polypeptide, and / or (4) post-translational modification of a polypeptide.

[0089] Ex-vivo: As used herein, the term “ex-vivo” refers to an external environment, such as an event occurring outside a multicellular organism. In some embodiments, cells or cell populations are modified outside a multicellular organism (e.g., a non-human primate or mammal such as a human) to express the anti-GCC molecules described herein before such cells or cell populations are administered to a target that requires them.

[0090] Fusion Protein: As used herein, the term “fusion protein” refers to a protein encoded by a nucleic acid sequence manipulated from nucleic acid sequences encoding at least parts of two different (e.g., heterologous) proteins. As those skilled in the art will undoubtedly recognize, to produce a fusion protein, nucleic acid sequences are concatenated such that the resulting reading frame does not contain internal stop codons. In some embodiments, fusion proteins such as those described herein include influenza HA polypeptide or a fragment thereof.

[0091] Guanylate cyclase C (GCC): As used herein, “GCC” is also known as “STAR,” “GUC2C,” “GUCY2C,” or “ST receptor” protein, and refers to mammalian GCC, preferably human GCC protein. Human GCC refers to the protein described in GenBank accession number: NM_004963 and its naturally occurring allele protein variants. Other variants are known in the art. See, for example, accession number Ensp0000261170, Ensembl Database, European Bioinformatics Institute and Wellcome Trust Sanger Institute, U.S. Patent Application No. 20060035852, or GENBANK accession number AAB 19934. Typically, naturally occurring allele variants have an amino acid sequence that is at least 95%, 97%, or 99% identical to the GCC sequence of SEQ ID NO: 5. The transcript codes for a 1073-amino acid protein product and is described in GenBank accession number: NM_004963. GCC proteins are characterized as transmembrane cell surface receptor proteins and are thought to play important roles in maintaining intestinal fluid, electrolyte homeostasis, and cell proliferation.

[0092] Host: The term “host” is used herein to refer to the system (e.g., a cell, an organism, etc.) in which the polypeptide of interest resides. In some embodiments, the host is a system that expresses the specific polypeptide of interest.

[0093] Host cell: As used herein, the term “host cell” refers to a cell into which foreign DNA (recombinant or otherwise) has been introduced. For example, a host cell may be used to produce the polypeptides described herein by standard recombination techniques. Those skilled in the art will understand, upon reading this disclosure, that such a term refers not only to a specific target cell but also to the offspring of such a cell. Such offspring may not be identical to the parent cell in practice, as certain modifications may occur in subsequent generations due to either mutation or environmental influences, but they still fall within the scope of the term “host cell” as used herein. In some embodiments, host cells include any prokaryotic and eukaryotic cells suitable for expressing foreign DNA (e.g., recombinant nucleic acid sequences). Examples of cells include prokaryotic and eukaryotic cells (unicellular or multicellular), bacterial cells (e.g., strains such as E. coli, Bacillus species, and Streptomyces species), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions, such as hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells.In some embodiments, the cells are eukaryotic cells and are selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, HEK293T, 293EBNA, MSR293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC5, Colo205, HB8065, HL-60 (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the above cells. In some embodiments, the cells contain one or more viral genes and are, for example, retinal cells expressing viral genes (e.g., PER.C6® cells).

[0094] Immune response: As used herein, the term “immune response” refers to the response of cells of the immune system, such as B cells, T cells, dendritic cells, macrophages, or polymorphonuclei, to a stimulus, such as an antigen or vaccine. An immune response may include any cells in the body involved in the host defense response, including, for example, epithelial cells that secrete interferon or cytokines. Immune responses include, but are not limited to, innate immune responses and / or adaptive immune responses. As used herein, a protective immune response refers to an immune response that protects a subject from infection (preventing infection or the development of disease associated with infection). Methods for measuring immune responses are well known in the art and include, for example, the measurement of proliferation and / or activity of lymphocytes (such as B cells or T cells), secretion of cytokines or chemokines, inflammation, antibody production, etc.

[0095] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment rather than within a multicellular organism, such as in a test tube or reaction vessel, or under cell culture conditions.

[0096] In vivo: As used herein, the term “in vivo” refers to events occurring within multicellular organisms such as humans and non-human animals. In relation to cell-based systems, the term may be used to refer to events occurring within living cells (as opposed to, for example, in vitro or ex vivo systems).

[0097] Isolated: As used herein, the term “isolated” means (1) separated from at least some of the components to which it was originally associated when it was first produced (either in nature and / or in an experimental setting), and / or (2) a substance and / or entity designed, produced, prepared, and / or manufactured by human intervention. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components to which they were originally associated. In some embodiments, the isolated agent is pure to about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99%. As used herein, a substance is “pure” if it substantially contains no other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered “isolated” or even “pure” after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.), and in such embodiments, the isolation percentage or purity percentage of the substance is calculated without including such carriers or excipients. For example, in some embodiments, a biopolymer such as a naturally occurring polypeptide or polynucleotide is considered “isolated” if: a) by its origin or source of induction it is not related to some or all of the components that accompany the polymer in its natural state; b) the polymer substantially does not contain other polypeptides or nucleic acids of the same species that naturally produce it; or c) it is expressed by, or otherwise associated with, components that are not of the species that naturally produce the polymer. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cell system different from the one that naturally produces the polypeptide is considered an “isolated” polypeptide.Alternatively, or further, in some embodiments, a polypeptide subjected to one or more purification techniques may be considered an “isolated” polypeptide insofar as a) the polypeptide has been isolated from other components to which it associates in nature and / or b) the polypeptide to which it associated when it was first produced. In some embodiments, cells may be “isolated” (e.g., purified or separated) from other cells. For example, in some embodiments, genetically modified cells manipulated to express the CARs described herein may be isolated from unmodified cells.

[0098] Nucleic Acid: As used herein, the term “nucleic acid” in its broadest sense refers to any compound and / or substance that is incorporated into or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is incorporated into or can be incorporated into an oligonucleotide chain by a phosphodiester bond. As will be apparent from the context, in some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, “nucleic acid” refers to an oligonucleotide chain containing individual nucleic acid residues. In some embodiments, “nucleic acid” is RNA or contains RNA, and in some embodiments, “nucleic acid” is DNA or contains DNA. In some embodiments, a nucleic acid is one or more native nucleic acid residues, contains them, or consists of them. In some embodiments, a nucleic acid is one or more nucleic acid analogs, contains them, or consists of them. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, contains, or consists of one or more “peptide nucleic acids,” where “peptide nucleic acids” are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, and these are considered to be within the scope of the present invention. Alternatively, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite bonds instead of phosphodiester bonds. In some embodiments, the nucleic acid is, contains, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, inserted bases, and combinations thereof), contains them, or consists of them. In some embodiments, the nucleic acid contains one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to the sugars in natural nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or protein. In some embodiments, the nucleic acid contains one or more introns. In some embodiments, the nucleic acid is prepared by one or more of the following: isolation from natural sources, enzymatic synthesis by polymerization based on complementary templates (in vivo or in vitro), reproduction in recombinant cells or systems, and chemosynthesis. In some embodiments, the nucleic acid has a residue length of at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more. In some embodiments, the nucleic acid is single-stranded, and in some embodiments, the nucleic acid is double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide or is a complement to a sequence encoding a polypeptide.In some embodiments, the nucleic acid has enzymatic activity.

[0099] pharmaceutically acceptable vehicle: The pharmaceutically acceptable carrier (vehicle) useful in this disclosure is conventional. (Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 15) th Edition (1975) describes compositions and formulations suitable for the delivery of one or more therapeutic compositions (e.g., compositions comprising chimeric antigen receptors as described herein), as well as additional pharmaceuticals. Generally, the properties of the carrier will depend on the specific method of administration used. For example, parenteral formulations typically include injectable fluids containing pharmaceutically and physiologically acceptable fluids as a vehicle, such as water, saline, equilibrium salt solution, dextrose aqueous solution, glycerol, etc. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutical composition may contain small amounts of non-toxic adjuncts, such as wetting or emulsifying agents, preservatives, and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0100] Polypeptide: Generally speaking, a "polypeptide" is a set of at least two amino acids linked to one another by peptide bonds. In some embodiments, a polypeptide may contain at least three to five amino acids, each of which is linked to another amino acid by at least one peptide bond. Those skilled in the art will understand that polypeptides may sometimes contain "non-natural" amino acids or other entities, and nevertheless, they can optionally be incorporated into the polypeptide chain. In some embodiments, the term "polypeptide" is used to refer to a particular functional class of polypeptides, such as antibodies, chimeric antigen receptors, or costimulatory domain polypeptides. For each such class, this specification provides several examples of amino acid sequences of known exemplary polypeptides within the class, and / or they are recognized by the art, and in some embodiments, one or more such known polypeptides are reference polypeptides of the class. In such embodiments, the term "polypeptide" refers to any member of the class that exhibits sufficient sequence homology or identity with the relevant reference polypeptide, and those skilled in the art will understand that it should be included within the class. In many embodiments, representative members of the class also share significant activity with the reference polypeptide. For example, in some embodiments, the member polypeptide includes at least one region (i.e., a conserved region, often containing characteristic sequence elements) that exhibits an overall degree of sequence homology or identity with the reference polypeptide, which is at least about 30–40%, and often more than or equal to about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and / or often exhibits very high sequence identity of more than 90%, or even more than 95%, 96%, 97%, 98%, or 99%. Such a conserved region typically contains at least 3–4, often up to 20 or more amino acids, and in some embodiments, the conserved region contains at least one sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acids.

[0101] It is understood that the antibodies and antigen conjugates of the present invention may have additional conserved or non-essential amino acid substitutions, but these do not substantially affect the polypeptide function. Whether a particular substitution is acceptable, i.e., whether it does not adversely affect desired biological properties such as binding activity, can be determined as described in Bowie, JU et al. Science 247:1306-1310 (1990) or Padlan et al. FASEB J.9:133-139 (1995). A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0102] Prevention: As used herein, the term “prevention” refers to prevention, avoidance of disease onset, delay of onset, and / or reduction of the frequency and / or severity of one or more symptoms of a particular disease, disorder, or condition (e.g., infection caused by the influenza virus). In some embodiments, prevention is evaluated on a population basis so that a drug is considered to “prevent” a particular disease, disorder, or condition if a statistically significant reduction in the occurrence, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition.

[0103] Purity: As used herein, an agent or entity is “pure” if it is substantially free of other components. For example, a preparation containing more than about 90% of a particular agent or entity is generally considered a pure preparation. In some embodiments, the agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0104] Recombinant: As used herein, the term “recombinant” is intended to mean polypeptides designed, manipulated, prepared, expressed, produced or isolated by recombinant means (e.g., polypeptides as described herein), such as polypeptides expressed using recombinant expression vectors transfected into host cells, polypeptides isolated from recombinant combinatorial polypeptide libraries, or polypeptides prepared, expressed, produced or isolated by any other means including splicing selected sequence elements together. In some embodiments, one or more such selected sequence elements are found in nature. In some embodiments, one or more such selected sequence elements and / or combinations thereof are designed in silico. In some embodiments, one or more such selected sequence elements are derived from a combination of multiple (e.g., two or more) known sequence elements (e.g., two epitopes derived from two separate HA polypeptides) that do not exist in nature in the same polypeptide.

[0105] Reference: The term “reference” is frequently used herein to describe a standard or control drug, individual, population, sample, sequence, or value compared to a drug, individual, population, sample, sequence, or value of interest. In some embodiments, the reference drug, individual, population, sample, sequence, or value is tested and / or determined substantially concurrently with the test or determination of the drug, individual, population, sample, sequence, or value of interest. In some embodiments, the reference drug, individual, population, sample, sequence, or value is a past reference, optionally embodied in a tangible medium. Typically, as understood by those skilled in the art, the reference drug, individual, population, sample, sequence, or value is determined or characterized under conditions equivalent to those used to determine or characterize the drug, individual, population, sample, sequence, or value of interest.

[0106] Single-domain antibody: As used herein, the terms “single-domain antibody (sdAb),” “variable single-domain,” or “immunoglobulin single variable domain (ISV),” and “single heavy-chain variable domain (VH) antibody” refer to a single variable fragment of an antibody that binds to a target antigen and maintains binding specificity to the antigen in the absence of a light chain or other antibody fragment. These terms are used interchangeably herein. An sdAb is a single antigen-binding polypeptide having three complementarity-determining regions (CDRs). An sdAb can bind to an antigen on its own without pairing with a corresponding CDR-containing polypeptide. A VH single-domain antibody refers to a single-domain antibody having a human heavy-chain variable domain or a domain derived from a human heavy-chain variable domain. In some cases, single-domain antibodies are engineered from camel HCAbs, and those heavy-chain variable domains of camel HCAbs are referred to as “VHH.” Some VHHs may also be known as nanobodies. Camel sdAbs are one of the smallest known antigen-binding antibody fragments (see, for example, Hamers-Casterman et al., Nature 363:446-8 (1993), Greenberg et al., Nature 374:168-73 (1995), and Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). Basic VH or VHH single-domain antibodies have the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1-FR4 refer to framework regions 1-4, respectively, and CDR1-CDR3 refer to complementarity-determining regions 1-3. As described below, some embodiments of various aspects of the present invention relate to a conjugate comprising a single heavy chain variable domain antibody / immunoglobulin heavy chain single variable domain that binds to a GCC antigen in the absence of a light chain.

[0107] Subject: As used herein, the term “subject” means any mammal, including humans. In certain embodiments of the present invention, the subject is an adult, adolescent, or infant. In some embodiments, the terms “individual” or “patient” are used and are intended to be interchangeable with “subject.” The administration of pharmaceutical compositions and / or the implementation of methods of treatment in utero are also contemplated by the present invention. For example, the subject may be a patient (e.g., a human patient or a veterinary patient) with symptoms of cancer (e.g., of gastrointestinal origin), cancer (in which at least a portion of the cells express GCC), or a predisposition to cancer (in which at least a portion of the cells express GCC). The term “non-human animal” in the present invention includes all non-human vertebrates, e.g., non-human mammals and non-mammals, e.g., non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc., unless otherwise noted.

[0108] Substantially: As used herein, the term “substantially” refers to a qualitative state representing the overall or nearly overall degree or extent of the feature or characteristic of the subject. Those skilled in the art of the biological field will understand that biological and chemical phenomena, if any, rarely complete and / or proceed completely, or achieve or avoid certain results. Thus, the term “substantially” is used herein to express the possibility of a lack of completeness inherent in many biological and chemical phenomena.

[0109] Therapeutic Agent: As used herein, the term “therapeutic agent” refers to a biologically active agent (e.g., an antigen-binding agent). This term is used herein to refer to a chemical compound, a mixture of chemical compounds, a biomolecule, or an extract made from a biomolecule. In some embodiments, the therapeutic agent may be an anticancer agent or a chemotherapeutic agent. As used herein, the terms “anticancer agent” or “chemotherapeutic agent” refer to an agent having functional properties that inhibit the development or progression of neoplasms, particularly malignant (cancerous) lesions, such as carcinoma, sarcoma, lymphoma, or leukemia, in humans. Inhibition of metastasis or angiogenesis is often a property of anticancer agents or chemotherapeutic agents. A chemotherapeutic agent may be a cytotoxic agent or a cell proliferation inhibitor. The term “cell proliferation inhibitor” refers to an agent that inhibits or suppresses cell growth and / or cell proliferation. In some embodiments, the therapeutic agent is a genetically modified cell or an antibody. In some embodiments, the therapeutic agent is an anti-GCC CAR. In some embodiments, the therapeutic agent is a cell (e.g., a population of cells) expressing the GCC CAR described herein.

[0110] Transformation: As used herein, refers to any process by which foreign DNA is introduced into a host cell. Transformation may be carried out naturally or artificially using a variety of methods well known in the art. Transformation may rely on any known method aimed at inserting a foreign nucleic acid sequence into a prokaryotic or eukaryotic host cell. In some embodiments, a particular transformation method is selected based on the host cell being transformed, and may include, but is not limited to, viral infection, electroporation, mating, transfection, and lipofection. In some embodiments, the “transformed” cell is stably transformed in such a way that the inserted DNA can replicate either as an autonomously replicating plasmid or as part of a host chromosome. In some embodiments, the transformed cell transiently expresses the introduced nucleic acid for a limited period.

[0111] To treat or to cure: As used herein, the terms “to treat” or “to cure” are defined as administering an anti-GCC antigen conjugate (e.g., an anti-GCC antibody or a fragment thereof) to a subject, e.g., a patient, or to an isolated tissue or cell derived from the subject, e.g., by application to the subject. The anti-GCC antigen conjugate may be administered alone or in combination with a second agent. Treatment may involve curing, alleviating, mitigating, altering, repairing, ameliorating, palliating, improving, or influencing a disorder, symptoms of a disorder, or predisposition to a disorder, e.g., cancer. While we do not wish to be bound by theory, treatment is thought to cause inhibition, removal, or death of cells in vitro or in vivo, or otherwise a reduction in the ability of cells, e.g., abnormal cells, to mediate a disorder, e.g., a disorder as described herein (e.g., cancer).

[0112] Variable Region or Domain: As used herein, the term “variable region” or “variable domain” of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as “VH” and “VL,” respectively. These domains are generally the most variable parts of the antibody (compared to other antibodies of the same class) and contain the antigen-binding site. Antibodies consisting only of a heavy chain have a single heavy-chain variable region.

[0113] Vectors: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which another nucleic acid molecule is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, to which an additional DNA segment can be ligated into a viral genome. Certain vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors can induce the expression of genes to which they are functionally ligated. Such vectors are referred to herein as “expression vectors.” [Modes for carrying out the invention]

[0114] This invention is based on the discovery of novel antigen-binding agents that specifically bind to guanylate cyclase C (GCC) and their use in therapeutic methods. This application provides an anti-GCC single-domain antibody (sdAb).

[0115] The present invention is not limited to specific methods and experimental conditions described, and such methods and conditions may vary. Since the scope of the present invention is limited solely by the appended claims, it should also be understood that, unless otherwise indicated, the terminology used herein is intended solely to describe and not to limit to specific embodiments.

[0116] Guanylate cyclase C Guanylate cyclase C (GCC) (also known as STAR, ST receptor, GUC2C, and GUCY2C) is a transmembrane cell surface receptor that functions in the maintenance of intestinal fluid, electrolyte homeostasis, and cell proliferation (Carrithers et al., Proc Natl Acad Sci USA 100:3018-3020 (2003), Mann et al., Biochem Biophys Res Commun 239:463-466 (1997), Pitari et al., Proc Natl Acad Sci USA 100:2695-2699 (2003)), GenBank accession number NM-004963, each incorporated herein by reference). This function is mediated by guanyline binding (Wiegand et al. FEBS Lett. 311:150-154 (1992)). GCC is also a receptor for thermostable enterotoxins (ST, e.g., having the amino acid sequence NTFYCCELCCNPACAGCY (SEQ ID NO: 29)), which are peptides produced by other infectious organisms in addition to E. coli (Rao, MCCiba Found. Symp. 112:74-93 (1985), Knoop FC and Owens, MJ Pharmacol. Toxicol. Methods 28:67-72 (1992)). The binding of ST to GCC activates a signaling cascade that causes intestinal diseases, such as diarrhea. Nucleotide sequence of human GCC (GenBank accession number NM-004963). Amino acid sequence of human GCC (GenPept accession number NP-004954):

[0117] GCC proteins generally possess several recognized domains, each contributing to the function of the GCC molecule. GCC functions include signaling for protein induction to the cell surface, extracellular ligand binding, tyrosine kinase activity, and guanylate cyclase catalytic activity. In normal human tissues, GCCs are expressed in mucosal cells, for example, in the apical brush border membrane lining the small intestine, large intestine, and rectum (Carrithers et al., Dis Colon Rectum 39:171-181 (1996)). GCC expression is maintained during neoplastic transformation of intestinal epithelial cells and is present in all primary and metastatic colorectal tumors (Carrithers et al., Dis Colon Rectum 39:171-181 (1996), Buc et al. Eur J Cancer 41:1618-1627 (2005), Carrithers et al., Gastroenterology 107:1653-1661 (1994)). Neoplastic cells from the stomach, esophagus, and esophagogastric junction also express GCC (see, e.g., U.S. Patent No. 6,767,704, Debruyne et al. Gastroenterology 130:1191-1206 (2006)). For example, the tissue-specific expression of GCC in gastrointestinal cancers (e.g., colon cancer, gastric cancer, or esophageal cancer) and its association with cancer can be used to justify the use of GCC as a diagnostic marker for this disease (Carrithers et al., Dis Colon Rectum 39:171-181 (1996), Buc et al. Eur J Cancer 41:1618-1627 (2005)).

[0118] As a cell surface protein, GCC can also function as a therapeutic target for receptor-binding proteins such as antibodies or ligands. In normal intestinal tissue, GCC is expressed at the apical end of epithelial cell tight junctions, which form an impermeable barrier between the luminal environment and vascular compartments (Almenoff et al., Mol Microbiol 8:865-873, Guarino et al., Dig Dis Sci 32:1017-1026 (1987)). Therefore, systemic intravenous administration of GCC-binding protein therapies would have minimal effect on intestinal GCC receptors while reaching neoplastic cells of the gastrointestinal system, including invasive or metastatic colon cancer cells, extraintestinal or metastatic colon tumors, esophageal or gastric tumors, and adenocarcinomas of the esophagogastric junction. Furthermore, GCCs are internalized via receptor-mediated endocytosis upon ligand binding (Buc et al. Eur J Cancer 41:1618-1627 (2005), Urbanski et al., Biochem Biophys Acta 1245:29-36 (1995)).

[0119] Polyclonal antibodies generated against the extracellular domain of GCC (Nandi et al. Protein Expr. Purif. 8:151-159 (1996)) inhibited ST peptide binding to human and rat GCC, thereby inhibiting ST-mediated cGMP production by human GCC.

[0120] GCC has been characterized as a protein involved in cancer, including colon cancer. See also Carrithers et al., Dis Colon Rectum 39:171-181 (1996), Buc et al., Eur J Cancer 41:1618-1627 (2005), Carrithers et al., Gastroenterology 107:1653-1661 (1994), and Urbanski et al., Biochem Biophys Acta 1245:29-36 (1995).

[0121] The antigen-binding molecular therapeutics targeting GCC described herein may be used to inhibit GCC-expressing cancer cells. The anti-GCC antigen-binding molecules of the present invention can bind to human GCC. In some embodiments, the anti-GCC antigen-binding molecules of the present invention can inhibit the binding of a ligand, such as guanylin or a thermostable enterotoxin, to GCC.

[0122] antigen binding molecule This invention relates to an anti-GCC antigen-binding molecule. In some embodiments, the anti-GCC molecule of the present invention elicits a cellular response upon binding to GCC on GCC-expressing cells to which it binds. In some embodiments, the anti-GCC antigen-binding agent of the present invention can block ligand binding to GCC.

[0123] The structural unit of naturally occurring mammalian antibodies is represented by a tetramer. Each tetramer consists of two polypeptide chain pairs, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminus of each chain contains a variable region of approximately 100-110 or more amino acids, mainly involved in antigen recognition. The carboxyl-terminus of each chain defines a constant region, mainly involved in effector function. Human light chains can be classified into kappa and lambda light chains. Heavy chains can be classified into mu, delta, gamma, alpha, or epsilon, defining the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 10 or more amino acids. For general information, see Fundamental Immunology Ch.7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). The variable region of each light / heavy chain pair forms the antibody binding site. The preferred isotype of anti-GCC antibody molecules is IgG immunoglobulin, which can be classified into four subclasses with different gamma heavy chains: IgG1, IgG2, IgG3, and IgG4. The majority of therapeutic antibodies are human, chimeric, or humanized antibodies of the IgG1 type. In certain embodiments, the anti-GCC antibody molecule has the IgG1 isotype.

[0124] The variable regions of each heavy and light chain pair form antigen-binding sites. Therefore, an intact IgG antibody has two identical binding sites. However, bifunctional or bispecific antibodies are artificial hybrid constructs that have two different heavy / light chain pairs, resulting in two different binding sites.

[0125] All chains exhibit the same general structure of a relatively conserved framework region (FR) linked by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs of each pair of double-stranded proteins are aligned by the framework region, enabling binding to specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chains contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain follow the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature 342:878-883 (1989). Where used herein, CDRs are referred to according to Kabat for each of the heavy chains (HCDR1, HCDR2, HCDR3) and light chains (LCDR1, LCDR2, LCDR3).

[0126] The anti-GCC antibody molecule may include all of the antibodies described herein, or an antigen-binding subset of the CDR or heavy chain. The amino acid sequences of the anti-GCC antigen-binding agents described herein, including the variable region and CDR, can be found in Tables 1-3.

[0127] Therefore, in one embodiment, the antibody molecule includes one or both of the following: (a) One, two, three, or antigen-binding light chain CDRs (LCDR1, LCDR2 and / or LCDR3) of a human antibody or mouse antibody, such as an antibody derived from a human hybridoma (e.g., the light chain of an anti-GCC antibody described in US20180355062A1, which is incorporated in its entirety by reference). In embodiments, the CDR(s) may include one or more or all of the following amino acid sequences from LCDR1 to 3: LCDR1, or modified LCDR1 (with 1 to 7 amino acids conservatively substituted), LCDR2, or modified LCDR2 (with 1 or 2 amino acids conservatively substituted), or LCDR3, or modified LCDR3 (with 1 or 2 amino acids conservatively substituted), and (b) Heavy chain CDRs (HCDR1, HCDR2 and / or HCDR3) with one, two, three, or antigen-binding counts, as described herein. In embodiments, the CDR(s) may comprise one or more or all of the following amino acid sequences from HCDR1 to HCDR3: HCDR1 or modified HCDR1 (with one or two amino acids conservatively substituted), HCDR2 or modified HCDR2 (with one to four amino acids conservatively substituted), or HCDR3 or modified HCDR3 (with one or two amino acids conservatively substituted).

[0128] In some embodiments, the anti-GCC antibody molecules of the present invention can induce antibody-dependent cell-mediated cytotoxicity (ADCC) in GCC-expressing cells, such as tumor cells. Antibodies having IgG1 and IgG3 isotypes are useful for inducing effector function in antibody-dependent cytotoxicity due to their ability to bind to Fc receptors. Antibodies having IgG2 and IgG4 isotypes are useful for minimizing the ADCC response due to their lower ability to bind to Fc receptors. In relevant embodiments, substitutions in the Fc region or alterations in the glycosylation composition of the antibody may be made, for example, by proliferation in modified eukaryotic cell lines, to enhance the ability of the Fc receptor to recognize and bind to cells to which the anti-GCC antibody binds, and / or to mediate their cytotoxicity (see, for example, U.S. Patents 7,317,091, 5,624,821 and WO00 / 42072, Shields, et al. J. Biol. Chem. 276:6591-6604 (2001), Lazar et al. Proc. Natl. Acad. Sci. USA 103:4005-4010 (2006), and Satoh et al. Expert Opin Biol. Ther. 6:1161-1173 (2006)). In certain embodiments, an antibody or antigen-binding fragment (e.g., a human-derived antibody, human antibody) may include amino acid substitutions or substitutions that alter or modulate its function (e.g., effector function). For example, a human-derived constant region (e.g., a γ1 constant region, a γ2 constant region) may be designed to reduce complement activation and / or Fc receptor binding. (See, for example, U.S. Patent No. 5,648,260 (Winter et al.), U.S. Patent No. 5,624,821 (Winter et al.), and U.S. Patent No. 5,834,597 (Tso et al.), the entire teachings of which are incorporated herein by reference.) Preferably, the amino acid sequence of a human-derived constant region containing such amino acid substitutions or substitutions is at least about 95% identical to the amino acid sequence of an unchanged human-derived constant region over its entire length, and more preferably, at least about 99% identical to the amino acid sequence of an unchanged human-derived constant region over its entire length.Additional anti-GCC antigen-binding molecules are further described in U.S. Patent No. 8,785,600 (Nam et al.), the entire teaching of which is incorporated herein by reference.

[0129] In yet another embodiment, the effector function can also be altered by modifying the glycosylation pattern of the antibody. This alteration means the deletion of one or more carbohydrate moieties found in the antibody, and / or the addition of one or more glycosylation sites that are not present in the antibody. For example, an antibody with enhanced ADCC activity having a mature carbohydrate structure lacking fucose bound to the Fc region of the antibody is described in U.S. Patent Application Publication 2003 / 0157108 (Presta). See also U.S. Patent Application Publication 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Glycofi has also developed yeast cell lines capable of producing antibodies of specific glycoforms.

[0130] Furthermore, or alternatively, antibodies with altered glycosylation patterns can be produced, such as low-fucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bidifferentiated GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC capacity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells having altered glycosylation mechanisms. Cells having altered glycosylation mechanisms have been described in the art and can produce antibodies with altered glycosylation by being used as host cells engineered to express the recombinant antibodies of the present invention. For example, EP1,176,195 by Hang et al. describes a cell line having a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, so that antibodies expressed in such cell lines exhibit low fucosylation. Presta's PCT publication WO03 / 035835 describes Lec13 cells, a variant CHO cell line that exhibits reduced ability to bind fucose to Asn(297)-linked carbohydrates and further results in reduced fucosylation of antibodies expressed in its host cells (see also Shields, R. Let al., 2002 J. Biol. Chem. 277:26733-26740). Umana et al.'s PCT publication WO99 / 54342 describes cell lines engineered to express glycoprotein-modified glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines show increased bidifferentiation of the GlcNac structure, leading to increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

[0131] Humanized antibodies can also be produced using a CDR transplantation approach. Techniques for producing such humanized antibodies are known in the art. Generally, humanized antibodies are produced by obtaining nucleic acid sequences encoding variable heavy chain and variable light chain sequences of an antibody that binds to GCC, identifying complementarity-determining regions or "CDRs" within the variable heavy chain and variable light chain sequences, and transplanting the CDR nucleic acid sequences onto human framework nucleic acid sequences. (See, for example, U.S. Patents 4,816,567 and 5,225,539). The locations of CDRs and framework residues can be determined (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. J. Mol. Biol. 196:901-917 (1987)).

[0132] The anti-GCC antibody molecules described herein have the CDR amino acid sequences and nucleic acid sequences encoding the CDR listed in Tables 5 and 6. In some embodiments, the sequences in Tables 5 and 6 may be incorporated into molecules that recognize GCC for use in the therapeutic or diagnostic methods described herein. The selected human framework means one suitable for in vivo administration, i.e., one that does not exhibit immunogenicity. For example, such a decision may be made by prior experience, including studies of the in vivo use and amino acid similarity of such antibodies. A preferred framework region may be selected from a human-derived antibody having at least about 65% amino acid sequence identity, preferably at least about 70%, 80%, 90%, or 95% amino acid sequence identity, over the length of the framework region within the amino acid sequence of the equivalent portion (e.g., framework region) of the donor antibody, e.g., an anti-GCC antibody molecule (e.g., 3G1). Amino acid sequence identity may be determined using a preferred amino acid sequence alignment algorithm such as CLUSTAL W and default parameters. (Thompson JDet al., Nucleic Acids Res.22:4673-4680(1994).)

[0133] Once the CDRs and FRs of a humanized cloned antibody are identified, the amino acid sequence encoding the CDR is identified, and the corresponding nucleic acid sequence is transplanted onto a selected human FR. This can be done using known primers and linkers, the selection of which is known in the art. All CDRs for a particular human antibody can be replaced with at least some of the non-human CDRs, or only some of the CDRs can be replaced with non-human CDRs. Only the number of CDRs necessary for the humanized antibody to bind to a given antigen needs to be replaced. After the CDRs are transplanted onto the selected human FR, the resulting "humanized" variable heavy chain and variable light chain sequences are expressed to produce a humanized Fv or humanized antibody that binds to GCC. Preferably, the CDR-transplanted (e.g., humanized) antibody binds to the GCC protein with substantially the same or better affinity than the donor antibody. Typically, the humanized variable heavy chain and light chain sequences are expressed as a fusion protein with the human constant domain sequence, thus obtaining an intact antibody that binds to GCC. However, humanized Fv antibodies that do not contain the constant sequence may be produced.

[0134] Antibodies or fragments thereof as described herein, such as humanized antibodies, are also within the scope of the present invention, in which specific amino acids are substituted, deleted, or added within the CDR or framework region. In particular, humanized antibodies may have amino acid substitutions within the framework region to improve binding to an antigen, for example. For example, a select few acceptor framework residues of a humanized immunoglobulin chain may be replaced with corresponding donor amino acids. Possible sites of substitution include amino acid residues adjacent to or interacting with the CDR (see, for example, U.S. Patent No. 5,585,089 or No. 5,859,205). The acceptor framework may be a mature human antibody framework sequence or a consensus sequence. As used herein, the term “consensus sequence” refers to the most frequently occurring sequence, or a sequence derived from the most common residues at each position of the sequence within a region between related family members. Numerous human antibody consensus sequences are available, including consensus sequences from different subgroups of human variable regions (see Kabat, EA, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, US Government Printing Office (1991)). The Kabat database and its applications are freely available online, for example, through IgBLAST at the National Center for Biotechnology Information, Bethesda, Md. (see also Johnson, G. and Wu, TT, Nucleic Acids Research 29:205-206 (2001)).

[0135] In a particular embodiment, the GCC antibody molecule is a human anti-GCC IgG1 antibody. Since such an antibody has the desired binding to the GCC molecule, any one of such antibodies can readily undergo isotype switching to produce, for example, a human IgG4 isotype, while still retaining the same variable region (which defines the antibody's specificity and affinity to some extent). Therefore, when antibody candidates are generated that satisfy the desired “structural” attributes as described above, they can generally possess at least some additional “functional” attributes desired by isotype switching.

[0136] In some embodiments, a portion of the CAR composition of the present invention, including antibody fragments, is humanized or optimized while retaining high affinity for target antigens and other desirable biological properties. According to one embodiment of the present invention, humanized antibodies and antibody fragments are prepared by an analytical process of the parent sequence and various conceptual humanized products using three-dimensional models of the parent sequence and the humanized sequence. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the estimated three-dimensional structures of selected candidate immunoglobulin sequences. Examination of these displays allows for the analysis of the likely roles of residues in the function of the candidate immunoglobulin sequence, for example, the analysis of residues that affect the ability of the candidate immunoglobulin to bind to the target antigen.

[0137] Thus, FR residues can be selected and combined from recipient and transfer sequences to achieve desired antibody or antibody fragment characteristics, such as high affinity for the target antigen. Generally, CDR residues are most directly and substantially involved in the effect on antigen binding.

[0138] Humanized or optimized antibodies or antibody fragments may retain similar antigen specificity to the original antibody, for example, the ability to bind to human GCC in this invention. In some embodiments, the humanized antibody or antibody fragment may have improved affinity and / or specificity for binding to human GCC.

[0139] In some embodiments, the anti-GCC antigen conjugate comprises one or more CDR sequences as provided in Table 1. In some embodiments, the anti-GCC antigen conjugate comprises a heavy chain variable region having CDR1 provided in Table 1. In some embodiments, the anti-GCC antigen conjugate comprises a heavy chain variable region having CDR2 provided in Table 1. In some embodiments, the anti-GCC antigen conjugate comprises a heavy chain variable region having CDR3 provided in Table 1. In some embodiments, the anti-GCC antigen conjugate comprises a heavy chain variable region having CDR1, CDR2, and CDR3 provided in Table 1. In some embodiments, the anti-GCC antigen conjugate comprises a heavy chain variable region having CDR1, CDR2, and CDR3 provided in Table 1. In some embodiments, the anti-GCC antigen conjugate comprises one or more CDR sequences provided in Table 1, where the CDRs contain one, two, or three amino acid substitutions. In one embodiment, the substitutions do not adversely affect the binding of the conjugate to its target.

[0140] [Table 1]

[0141] Anti-GCC antibodies that are not intact antibodies are also useful in this invention. In some embodiments, the anti-GCC antigen conjugate is a single-domain antibody containing heavy chain variable regions having CDR1, CDR2, and CDR3, provided in Table 1. Such antibodies may be derived from any of the antibodies described above. Useful antibody molecules of this type include (i) Fab fragments, i.e., monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, i.e., bivalent fragments containing two Fab fragments linked by disulfide crosslinks in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)); (vii) single-domain functional heavy chain antibodies consisting of a VHH domain (known as nanobodies) (e.g., Cortez-Retamozo, et al., Cancer Res. 64:2853-2857 (2004), and the references cited in that literature); and (vii) isolated CDRs with a framework sufficient to provide antigen-binding fragments, e.g., one or more isolated CDRs. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by synthetic linkers using recombination methods to produce them as a single protein chain where the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv)), see, for example, Bird et al. Science 242:423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988). Such single-chain antibodies are also intended to be included within the term "antigen-binding fragment" of antibodies. These antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antibody fragments such as Fv, F(ab')2, and Fab can be prepared by cleavage of intact proteins, for example, by protease or chemical cleavage.

[0142] Single-domain antibody Single-domain antibodies (sdAbs) differ from conventional four-chain antibodies by possessing a single monomeric antibody variable domain. For example, camelids and sharks produce sdAbs, known as heavy-chain-only antibodies (HcAbs), which naturally lack a light chain. The antigen-binding fragment in each arm of a heavy-chain-only camel antibody possesses a single heavy-chain variable domain (VHH), which can have high affinity for the antigen without the support of a light chain. Camel VHH is known as the smallest functional antigen-binding fragment with a molecular weight of approximately 15 kD. In some embodiments, the antigen-binding agent is a single human heavy-chain variable domain (VH) antibody. Such binding molecules are also referred to as Humabody® and may be used interchangeably herein. Humabody® is a registered trademark of Crescendo Biologics Ltd.

[0143] One aspect of this application provides isolated single-domain antibodies (referred to herein as “anti-GCC sdAb”) that specifically bind to GCC, such as human GCC. In some embodiments, the anti-GCC sdAb modulates GCC activity. In some embodiments, the anti-GCC sdAb is an antagonist antibody. Further provided are antigen-binding fragments derived from any one of the anti-GCC sdAbs described herein, and antigen-binding proteins comprising any one of the anti-GCC sdAbs described herein. In some embodiments, the anti-GCC sdAb comprises one, two, and / or three CDR sequences provided in Table 1. Exemplary anti-GCC sdAbs are listed in Tables 2 and 3. In some embodiments, the anti-GCC sdAb comprises a variable heavy chain domain provided in Table 2 or Table 3. In some embodiments, the anti-GCC sdAb consists of a variable heavy chain domain provided in Table 2 or Table 3.

[0144] In some embodiments, part or all of the CDR sequence, i.e., the VH domain or heavy chain, may be used in another antigen-binding agent, such as a CDR transplant, humanized, or chimeric antibody molecule. Embodiments include an antibody molecule containing enough CDRs to enable binding to cell surface GCCs, for example, all three CDRs derived from one of the heavy chain variable regions described above.

[0145] In some embodiments, the CDR, for example, all HCDRs, are embedded within a human or human-derived framework region(s). Examples of human framework regions include human germline framework sequences, affinity-matured (either in vivo or in vitro) human germline sequences, or synthetic human sequences, such as consensus sequences. In one embodiment, the heavy chain framework is an IgG1 or IgG2 framework.

[0146] In some embodiments, the anti-GCC antigen conjugate of the present invention comprises a heavy chain variable region amino acid sequence provided in Table 2. In some embodiments, the anti-GCC antigen conjugate is an antibody consisting only of a single-domain heavy chain (e.g., an antigen conjugate that does not contain an immunoglobulin light chain).

[0147] [Table 2]

[0148] In some embodiments, the anti-GCC antigen conjugate of the present invention comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the VH sequence provided in Table 2. In some embodiments, the anti-GCC antigen conjugate of the present invention comprises a heavy chain variable region amino acid sequence as shown in Table 2, the sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In one embodiment, the substitutions are outside the CDR region. In some embodiments, the VH anti-GCC antigen conjugate (e.g., a single-domain antibody) comprises a leader sequence. In some embodiments, the VH anti-GCC antigen conjugate comprises a leader sequence including MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 6), MELGLSWVFLVAILEGVQC (SEQ ID NO: 7), or MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 2). In some embodiments, the VH anti-GCC antigen conjugate includes a leader sequence comprising MALPVTALLLPLALLLHAARP (SEQ ID NO: 4).

[0149] In some embodiments, the anti-GCC antigen conjugate of the present invention comprises a heavy chain variable region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the VH sequence provided in Table 3. In some embodiments, the anti-GCC antigen conjugate of the present invention comprises a heavy chain variable region amino acid sequence as shown in Table 3, the sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In one embodiment, the substitutions are outside the CDR region. In some embodiments, the anti-GCC antigen conjugate of the present invention comprises a heavy chain variable region amino acid sequence that is identical to the VH sequence provided in Table 3.

[0150] In some embodiments, the VH anti-GCC antigen conjugate (e.g., a single-domain antibody) includes a leader sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% in Table 3.

[0151] [Table 3-1]

[0152] [Table 3-2]

[0153] Antibody fragments for in vivo therapeutic or diagnostic use may benefit from modifications that improve their serum half-life. Suitable organic moieties intended to increase the in vivo serum half-life of an antibody may include one, two, or more linear or branched moieties selected from hydrophilic polymer groups (e.g., linear or branched polymers (e.g., polyalkane glycols, e.g., polyethylene glycol, monomethoxypolyethylene glycol, etc.), carbohydrates (e.g., dextran, cellulose, polysaccharides, etc.), hydrophilic amino acid polymers (e.g., polylysine, polyaspartic acid, etc.), polyalkane oxides, and polyvinylpyrrolidone), fatty acid groups (e.g., monocarboxylic acids or dicarboxylic acids), fatty acid ester groups, lipid groups (e.g., diacylglycerol groups, sphingolipid groups (e.g., ceramidyl)), or phospholipid groups (e.g., phosphatidylethanolamine groups). Preferably, the organic moiety is bound to a site that does not impair the function of the resulting immune complex (e.g., does not reduce antigen-binding affinity) compared to the uncomplexed antibody moiety. The organic moiety may have a molecular weight of approximately 500 Da to approximately 50,000 Da, preferably approximately 2,000, 5,000, 10,000, or 20,000 Da. Examples and methods for modifying polypeptides, such as antibodies, with the organic moiety can be found, for example, in U.S. Patent Nos. 4,179,337 and 5,612,460, PCT Publications WO95 / 06058 and WO00 / 26256, and U.S. Patent Application Publication 20030026805.

[0154] Chimeric antigen receptor Chimeric antigen receptors (CARs) are hybrid molecules comprising three essential units: (1) an extracellular antigen-binding motif, (2) a ligation / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas R J. Lessons learned from a highly-active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013;2(4):e23621). A general scheme for various embodiments of GCC-specific CARs disclosed herein is shown in Figure 1. In some embodiments, the anti-GCC CAR comprises, from N-terminus to C-terminus, a signal or leader peptide, an antigen-binding domain, a transmembrane and / or hinge domain, a costimulatory domain, and an intracellular domain.

[0155] The present invention provides a CAR (e.g., a CAR polypeptide) comprising an anti-GCC binding domain (e.g., a GCC binding domain as described herein), a transmembrane domain, and an intracellular signaling domain, wherein the anti-GCC binding domain comprises a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 3 (HC CDR3) of any anti-GCC heavy chain binding domain amino acid sequence listed in Table 1 or 8. In some embodiments, the anti-GCC CAR comprises, from N-terminus to C-terminus, a signal or leader peptide, an anti-GCC VH, a CD28 transmembrane and hinge, a CD28 costimulatory domain, and a CD3 zeta intracellular domain.

[0156] The antigen-binding domain may be any protein that binds to an antigen, such as monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including but not limited to single-domain antibodies such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of nanobodies derived from camelids (VHH), as well as other scaffolds known in the art that function as antigen-binding domains, including but not limited to proteins such as recombinant fibronectin domains. In some embodiments, the antigen-binding domain

[0157] The antigen-binding motif of CARs is generally a single-strand fragment variable (ScFv), i.e., formed after the minimal binding domain of an immunoglobulin (Ig) molecule or a single-domain antibody (e.g., WO2018 / 028647A1). Alternative antigen-binding motifs have also been designed, such as receptor ligands (i.e., designed so that IL-13 binds to tumor-expressed IL-13 receptors), intact immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D).

[0158] The linking motif of the CAR may be a relatively stable structural domain, such as the constant domain of IgG, or it may be designed to be an extended, flexible linker. In some embodiments, the anti-GCC binding domain (e.g., a polypeptide containing the sequence provided in Table 1 or Table 8) is linked to a transmembrane domain via a linker, e.g., a linker described herein. In some embodiments, the anti-GCC CAR comprises a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6 (SEQ ID NO: 36).

[0159] Structural motifs, such as those derived from the IgG constant domain, can be used to extend the ScFv binding domain away from the T cell plasma membrane surface. This can be important for some tumor targets where the binding domain is particularly close to the tumor cell surface membrane (e.g., against the disialoganglioside GD2, Orentas et al., unpublished observations). To date, signaling motifs used in CARs have consistently included the CD3-ζ chain because its core motif is a crucial signal for T cell activation. The first reported second-generation CARs featured a CD28 signaling domain and CD28 transmembrane sequence. This motif was similarly used in third-generation CARs containing the CD137(4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009;183(9):5563-74). With the emergence of new technologies, T cell activation by beads linked to anti-CD3 and anti-CD28 antibodies, as well as the presence of the classic "signal 2" from CD28, no longer need to be encoded by the CAR itself. When bead activation was used, in vitro assays showed that third-generation vectors were not superior to second-generation vectors, and they did not offer any clear advantage over second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas R J. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia, Blood. 2013;121(7):1165-74, Kochenderfer JN et al. Blood. 2012;119(12):2709-20).This is supported by the clinical success of CD19-specific CARs in second-generation CD28 / CD3-ζ (Lee DW et al. American Society of Hematology Annual Meeting. New Orleans, La.; Dec. 7-10, 2013) and CD137 / CD3-ζ signaling types (Porter DL et al. N Engl J Med. 2011;365(8):725-33). In addition to CD137, other tumor necrosis factor receptor superfamily members such as OX40 can also provide important sustained signaling in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60). Equally important are the culture conditions under which the CAR T cell population is cultured.

[0160] transmembrane domain With respect to the transmembrane domain, in various embodiments, the CAR may be designed to include a transmembrane domain that binds to the extracellular domain of the CAR (e.g., the anti-GCC antigen-binding domain). The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 15 amino acids of the intracellular region).

[0161] In one embodiment, a transmembrane domain associated with one of the other domains of a CAR is used. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to avoid binding between such domain and the transmembrane domain of the same or different surface membrane protein, for example, to minimize interaction with other members of the receptor complex. In one embodiment, the transmembrane domain can homodimerize with another CAR on the surface of a CAR-expressing cell, e.g., a CART cell. In another embodiment, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interaction with the binding domain of a native binding partner present in the same CAR-expressing cell, e.g., a CART cell.

[0162] As described herein, CARs include transmembrane domains. With respect to transmembrane domains, CARs include one or more transmembrane domains fused to the extracellular GCC antigen-binding domain of the CAR. Transmembrane domains may originate from either natural or synthetic sources. If the source is natural, the domains may originate from any membrane-bound or transmembrane protein.

[0163] Alternatively, the transmembrane domain may be synthetic, in which case it will mainly consist of hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligo or polypeptide linker, preferably 2 to 10 amino acids in length, may form a linkage between the transmembrane domain of the CAR and the cytoplasmic signaling domain. In some embodiments, the linker is a glycine-serine doublet or a triple alanine linker.

[0164] In some embodiments, a transmembrane domain that naturally associates with one of the domains of the CAR is used in addition to the transmembrane domain described above. In some embodiments, the transmembrane domain may be selected by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein in order to minimize interaction with other members of the receptor complex.

[0165] intracellular domain The cytoplasmic domain or, otherwise, the intracellular signaling domain of a CAR is involved in the activation of at least one of the normal effector functions of the immune cell in which the CAR is located. The term “effector function” refers to a specialized function of the cell. For example, the effector function of a T cell may be helper activity, including cytolytic activity or cytokine secretion. Therefore, the term “intracellular signaling domain” refers to the portion of a protein that transmits effector function signals and causes the cell to perform its specialized function. Usually, the entire intracellular signaling domain may be used, but in many cases it is not necessary to use the entire chain. As long as a cleavage portion of the intracellular signaling domain is used, such a cleavage portion may be used in place of the intact chain, as long as it transmits effector function signals. Therefore, the term intracellular signaling domain means that it includes any cleavage portion of the intracellular signaling domain sufficient to transmit effector function signals.

[0166] Examples of intracellular signaling domains for use in CARs include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act cooperatively to initiate signaling after antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences that possess the same functional capabilities. Signals generated by TCRs alone are insufficient for complete T cell activation; secondary or co-stimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation by TCRs (primary cytoplasmic signaling sequences) and sequences that act in an antigen-independent manner and provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0167] Primary cytoplasmic signaling sequences control the primary activation of the TCR complex by either a stimulation or inhibition method. Primary cytoplasmic signaling sequences acting by a stimulation method may contain signaling motifs known as immune receptor tyrosine-based activation motifs or ITAMs. In some embodiments, the ITAM-containing domain within the CAR replicates primary TCR signaling independently of the endogenous TCR complex. In one embodiment, the primary signal is initiated, for example, by the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, and results in mediating T cell responses including, but not limited to, proliferation, activation, and differentiation. Primary cytoplasmic signaling sequences acting by a stimulation method (also referred to as “primary signaling domains”) may contain signaling motifs known as immune receptor tyrosine-based activation motifs or ITAMs.

[0168] In one embodiment, the intracellular signaling domain may include a primary intracellular signaling domain. An exemplary primary intracellular signaling domain may be derived from a primary stimulus or a molecule involved in antigen-dependent simulation. In one embodiment, the intracellular signaling domain may include a co-stimulatory intracellular domain. An exemplary co-stimulatory intracellular signaling domain may be derived from a co-stimulatory signal or a molecule involved in antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain may include the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular signaling domain may include the cytoplasmic sequence from the co-receptor or co-stimulatory molecule.

[0169] In one embodiment, the primary signaling domain includes a modified ITAM domain, for example, a mutant ITAM domain whose activity is altered (e.g., increased or decreased) compared to a natural ITAM domain. In one embodiment, the primary signaling domain includes a modified ITAM-containing primary intracellular signaling domain, for example, an optimized and / or cleaved ITAM-containing primary intracellular signaling domain. In one embodiment, the primary signaling domain includes one, two, three, four or more ITAM motifs.

[0170] Substitutions and variants In some embodiments, amino acid sequence variants of antibodies provided herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of an antibody or antibody fragment, e.g., sdAb. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleic acid sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions of residues in the amino acid sequence of the antibody, and / or substitutions of residues. Any combination of deletions, insertions, and substitutions may be performed to arrive at a final construct, provided that the final construct has the desired properties, e.g., antigen binding.

[0171] a) Substitution, insertion, and deletion variants In some embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVR and FR. Further details regarding classes of amino acid side chains are described below. The amino acid substitution is introduced into the antibody of interest, and the product can be screened for desired activity, such as retention / improvement of antigen binding, reduction of immunogenicity, or improvement of ADCC or CDC.

[0172] Amino acids may be grouped according to their common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basicity: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0173] Non-conservative substitution involves replacing one member of one of these classes with one of another.

[0174] In some embodiments, the antigen-binding domain is humanized. When a non-human antibody is humanized, certain sequences or regions of the antibody are modified to increase its similarity to antibodies or fragments naturally produced in humans. In one embodiment, the antigen-binding domain is humanized. Humanized antibodies (or antigen-binding fragments) may be prepared using various known techniques in the art, for example, CDR implantation (see, for example, European Patent No. EP239,400, International Publication No. WO91 / 09967, and U.S. Patents No. 5,225,539, No. 5,530,101, and No. 5,585,089 (each of which is incorporated herein by reference in whole)), veneering, or surface reconstruction (see, for example, European Patent Nos. EP592,106 and EP519,596, Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498, Studnicka et al., 1994, Protein Engineering, 7(6):805-814, and Roguska et al.) See al., 1994, PNAS, 91:969-973 (each in its entirety is incorporated herein by reference), chain shuffling (see, for example, U.S. Patent No. 5,565,332 (each in its entirety is incorporated herein by reference)), and, for example, U.S. Patent Application Publication No. US2005 / 0042664, U.S. Patent Application Publication No. US2005 / 0048617, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO9317105, Tan et al., J.Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J.Biol.Chem.,272(16):10678-84(1997), Roguska et al.,Protein Eng.,9(10):895-904(1996), Couto et al.,Cancer Res.,55(23 Supp):5973s-5977s(1995), Couto et al.,Cancer Res.This includes, but is not limited to, the techniques disclosed in 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994) (each of which is incorporated herein by reference in its entirety). In many cases, framework residues in the framework region are to be substituted with corresponding residues from a CDR donor antibody to alter, for example, improve antigen binding. These framework substitutions, e.g., conservative substitutions, are identified by methods well known in the art, e.g., modeling the interaction between CDRs and framework residues to identify framework residues important for antigen binding and sequence comparison to identify framework residues rare at specific locations. (See, for example, Queen et al., U.S. Patent No. 5,585,089, and Riechmann et al., 1988, Nature, 332:323 (which is incorporated herein by reference in its entirety).)

[0175] Humanized antibodies or antibody fragments have one or more amino acid residues that remain in those from a non-human source. These non-human amino acid residues are often referred to as “implant” residues, which are typically obtained from “implant” variable domains. As provided herein, humanized antibodies or antibody fragments comprise one or more CDRs from a non-human immunoglobulin molecule and a framework region, where the amino acid residues contained in the framework are derived entirely or largely from the human germline. Several techniques for humanizing antibodies or antibody fragments are well known in the art.

[0176] Certain types of substitution variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) to certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-mature antibodies, which may be readily generated using, for example, phage display-based affinity maturation techniques, such as those described herein. Briefly, after mutating one or more HVR residues and presenting the variant antibody on a phage, it is screened for specific biological activities (e.g., binding affinity).

[0177] Modifications (e.g., substitutions) may be made in HVRs to improve antibody affinity, for example. Such modifications may be made in HVR "hotspots," i.e., residues encoded by codons that mutate frequently during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al.'s Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. Next, the library is screened to identify any antibody variant with the desired affinity. Another way to introduce diversity involves an HVR-specific approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, for example, using alanine scanning mutagenesis or modeling. Often, CDR-H3 and CDR-L3 are targeted in particular.

[0178] In some embodiments, substitutions, insertions, or deletions may be made within one or more HVRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made within the HVR. Such modifications may be outside the HVR "hotspot" or CDR. In some embodiments of the variant VH sequences provided above, each HVR is either unmodified or contains one, two, or three or fewer amino acid substitutions.

[0179] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. This method identifies target residues or groups (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and determines whether they are substituted with neutral or charged amino acids (e.g., alanine or polyalanine) to affect the antibody-antigen interaction. Further substitutions may be introduced at amino acid positions that are functionally sensitive to the initial substitution. Alternatively, the crystal structure of the antigen-antibody complex may be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues may be targeted or excluded as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0180] Amino acid sequence insertions include intrasequence insertions of single or multiple amino acid residues, in addition to amino-terminus and / or carboxyl-terminus fusions, which are polypeptides ranging in length from 1 to 100 or more residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminus or C-terminus of an antibody with an enzyme (e.g., for ADEPT) or with a polypeptide that increases the serum half-life of the antibody.

[0181] b) Glycosylated variants In some embodiments, the antibodies provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be easily carried out by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0182] If an antibody contains an Fc region, the carbohydrate to which it is bound may be altered. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides, generally bound by an N-bond to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the branched oligosaccharide structure. In some embodiments, the modification of oligosaccharides in the antibodies of this application may be performed to produce antibody variants with certain improved properties.

[0183] In some embodiments, antibody variants are provided having carbohydrate structures lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1%–80%, 1%–65%, 5%–65%, or 20%–40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans at Asn297, compared to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) bound to Asn297, when measured by MALDI-TOF mass spectrometry, as described in WO2008 / 077546. Asn297 refers to the asparagine residue located approximately 297th (EU numbering of Fc region residues) within the Fc region, although Asn297 may also be located approximately ±3 amino acids upstream or downstream of 297, i.e., between 294 and 300, due to slight sequence variability in the antibody. Such fucosylated variants may possess improved ADCC function. See, for example, U.S. Patent Publications US2003 / 0157108 (Presta, L.) and US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, and US2004 / 01321. References include 40, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J.Mol.Biol.336:1239-1249(2004), and Yamane-Ohnuki et al. Biotech.Bioeng.87:614(2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986), U.S. Patent Application No. US2003 / 0157108A1, Presta, L., and WO2004 / 056312A1, Adams et al.), as well as knockout cell lines, such as alpha-1,6-fucosyltransferase gene knockout CHO cells, i.e., FUT8 knockout cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004), Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO2003 / 085107).

[0184] Antibody variants, for example, further have a bifid oligosaccharide, where the bifid oligosaccharide bound to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and U.S.2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.), WO1998 / 58964 (Raju, S.), and WO1999 / 22764 (Raju, S.).

[0185] CARs (including their functional parts and functional variants) can be obtained by methods known in the art. CARs may be prepared by any suitable method for producing polypeptides or proteins. Suitable methods for the novel synthesis of polypeptides and proteins are described in references such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Polypeptides and proteins can also be recombinantly produced using standard recombination methods and nucleic acids as described herein. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some CARs (including their functional parts and functional variants) may be isolated and / or purified from sources such as plants, bacteria, insects, mammals, such as rats and humans. Methods of isolation and purification are well known in the art. Alternatively, the CARs (including their functional parts and functional variants) described herein may be commercially synthesized by companies. In this regard, CARs may be synthesized, recombinant, isolated, and / or purified.

[0186] Detectable markers and tags The antibody-antigen binding fragment is specific to one or more of the antigens disclosed herein and can be expressed together with (e.g., co-expressed with) a tag protein. In some embodiments, the furin recognition site and the downstream 2A self-cleaving peptide sequence are designed for simultaneous bicistronic expression of the tag sequence and the antibody sequence. In some embodiments, the 2A sequence comprises the nucleic acid sequence GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 3). In some embodiments, the furin and P2A sequences comprise nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 3. In some embodiments, the P2A tag comprises the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0187] In some embodiments, the antibody or its antigen-binding fragment is specific to one or more of the antigens disclosed herein and may be expressed together with EGFR. In some embodiments, the antibody or its antigen-binding fragment is specific to one or more of the antigens disclosed herein and is expressed together with (e.g., co-expressed with) cleaved EGFR (tEGFR). In some embodiments, tEGFR comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 35. tEGFR:MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM(Sequence ID:35)

[0188] Antibodies or their antigen-binding fragments are specific to one or more of the antigens disclosed herein and may be combined with detectable markers, such as those detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (e.g., computed tomography (CT), computed transverse tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance imaging (MTR), ultrasound, fiber optics, and laparoscopy). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). Examples of useful detectable markers include fluorescent compounds, such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and lanthanide phosphors. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), are also useful.

[0189] Antibodies or their antigen-binding moieties can also be conjugated with enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When an antibody or its antigen-binding moiety is conjugated with a detectable enzyme, it can be detected by adding additional reagents used by the enzyme to produce a distinguishable reaction product. For example, in the presence of the activating factor horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine yields a visually detectable colored reaction product. Antibodies or their antigen-binding moieties can also be conjugated with biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated with enzymes or fluorescent labels.

[0190] Antibodies, or their antigen-binding portions, can be conjugated with paramagnetic materials such as gadolinium. Paramagnetic materials such as superparamagnetic iron oxide are also useful as labels. Antibodies can also be conjugated with lanthanides (such as europium and dysprosium), as well as manganese. Antibodies or antigen-binding fragments can also be labeled with predetermined polypeptide epitopes (such as leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, and epitope tags) that are recognized by a secondary reporter.

[0191] Antibodies, or their antigen-binding moieties, can also be complexed with radiolabeled amino acids. Radiolabeling can be used for both diagnostic and therapeutic purposes. For example, radiolabeling can be used to detect one or more of the antigens and antigen-expressing cells disclosed herein by X-ray, emission spectroscopy, or other diagnostic techniques. Furthermore, radiolabeling can be used therapeutically as a toxin for the treatment of tumors in a subject, such as neuroblastoma. Examples of polypeptide labeling include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111In, 125 I, 131 I.

[0192] Means for detecting such detectable markers are well known to those skilled in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent markers can be detected using a photodetector that detects emitted illumination. Enzyme labels are usually detected by providing a substrate to an enzyme and detecting the reaction product produced by the enzyme's action on the substrate, and colorimetric labels are detected simply by visualizing the colored label.

[0193] nucleic acids, expression vectors, and host cells Embodiments of the present invention further provide nucleic acids comprising nucleotide sequences encoding antibodies described herein, or the antigen-binding portion thereof (including its functional portion and functional variants). The nucleic acids of the present invention may comprise nucleotide sequences encoding any of the leader sequences, antigen-binding domains, transmembrane domains, and / or intracellular T cell signaling domains described herein. In one embodiment, the present invention comprises a recombinant nucleic acid construct comprising a nucleic acid molecule encoding an antibody or a fragment thereof, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an anti-GCC-binding domain. In one embodiment, the present invention provides nucleic acids encoding a VH amino acid sequence by SEQ ID NOs: 1, 20, 21, 26, 27, or 28, or a sequence having at least 75%, 80%, 90%, or 95% sequence identity with SEQ ID NOs: 1, 20, 21, 26, 27, or 28. In some embodiments, the nucleic acids comprise sequences that are at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 30-34.

[0194] In some embodiments, nucleotide sequences may be codon-modified. While not bound by any particular theory, codon optimization of nucleotide sequences is thought to increase the translation efficiency of mRNA transcripts. Codon optimization of nucleotide sequences may involve substituting native codons with other codons encoding the same amino acid, which can improve translation efficiency by being translated by tRNA that is more readily available in the cell. Optimization of nucleotide sequences may also improve translation efficiency by reducing secondary mRNA structures that interfere with translation.

[0195] In one embodiment, the present invention relates to a vector comprising a nucleic acid molecule as described herein, for example, a nucleic acid molecule encoding an antibody or antigen-binding fragment as described herein. In one embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenovirus vector, or retroviral vector.

[0196] In one embodiment, the vector is a lentiviral vector. In one embodiment, the vector further includes a promoter. In one embodiment, the promoter is an EF-1 promoter.

[0197] Expression vectors include plasmids, retroviruses, cosmids, YACs, and EBV-derived episomes. A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites manipulated to allow for the easy insertion and expression of any VH or VL sequence. In such vectors, splicing typically occurs between the splice donor site of the inserted J region and the splice acceptor site prior to the human C region, as well as in splice regions occurring within the human CH exon. A suitable expression vector may contain multiple components, such as an origin of replication, selectable marker genes, one or more expression regulatory elements, such as transcriptional regulatory elements (e.g., promoters, enhancers, or terminators), and / or one or more translational signals, signal sequences, or reader sequences. Polyadenylation and transcription termination occur in native chromosome regions downstream of the coding region. The resulting chimeric antibody can be bound to any potent promoter. Examples of suitable vectors that may be used include those suitable for mammalian hosts and based on viral replication systems, such as Simian virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus 2, bovine papillomavirus (BPV), papovavirus BK variant (BKV), or mouse and human cytomegalovirus (CMV), as well as Moloney's mouse leukemia virus (MMLV), and natural Ig promoters. Various suitable vectors are known in the art and include vectors maintained in a single copy or multiple copies, or vectors that are integrated into host cell chromosomes, for example, via LTR or via artificial chromosomes manipulated at multiple integration sites (Lindenbaum et al. Nucleic Acids Res. 32:e172 (2004), Kennard et al. Biotechnol. Bioeng. Online May 20, 2009). Additional examples of suitable vectors are listed in later sections.

[0198] The present invention also includes RNA constructs that can be directly transfected into cells. A method for generating mRNA for use in transfection comprises template in vitro transcription (IVT) using specially designed primers, followed by poly-A addition, to produce a construct containing 3' and 5' untranslated sequences ("UTR"), a 5' cap and / or internal ribosome entry site (IRES), the nucleic acid to be expressed, and a poly-A tail, typically 50 to 2000 nucleotides in length. RNA thus produced can efficiently transfect different types of cells. In one embodiment, the template includes a CAR sequence. In one embodiment, the RNA CAR vector is transduced into cells, such as T cells or NK cells, by electroporation.

[0199] Accordingly, the present invention provides an expression vector comprising an antibody, an antigen-binding fragment of an antibody (e.g., a human, humanized, chimeric antibody, or any of the aforementioned antigen-binding fragments), an antibody chain (e.g., a heavy chain, a light chain), or a nucleic acid encoding the antigen-binding portion of an antibody chain that binds to a GCC protein.

[0200] Expression in eukaryotic host cells is useful because such cells are more likely to assemble and secrete properly folded, immunoactive antibodies than prokaryotic cells. However, any antibodies produced that are inactive due to improper folding may be regenerative according to known methods (Kim and Baldwin, “Specific Intermediates in the Folding Reactions of Small Proteins and the Mechanism of Protein Folding”, Ann. Rev. Biochem. 51, pp. 459-89 (1982)). Host cells may produce portions of intact antibodies, such as light chain dimers or heavy chain dimers, which are also antibody homologs according to the present invention.

[0201] In one embodiment, nucleic acids may be incorporated into a recombinant expression vector. In this regard, embodiments provide recombinant expression vectors comprising any of the nucleic acids. For the purposes of this specification, the term “recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct that enables the expression of mRNA, protein, polypeptide, or peptide by a host cell, where the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector comes into contact with the cell under conditions sufficient to have mRNA, protein, polypeptide, or peptide expressed within the cell. Vectors as a whole are not naturally occurring.

[0202] However, some vector components may be naturally occurring. Recombinant expression vectors may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, partially synthesized or obtained from natural sources, and may contain natural, unnatural, or altered nucleotides. Recombinant expression vectors may contain nucleotide-nucleotide bonds that are naturally occurring, or not naturally occurring, or both types of bonds. Preferably, nucleotides or nucleotide-nucleotide bonds that are not naturally occurring or altered do not interfere with the transcription or replication of the vector.

[0203] In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector that can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for proliferation and expansion, or for expression, or both, such as plasmids and viruses. Vectors may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.).

[0204] Bacteriophage vectors such as λ, λZapII (Stratagene), EMBL4, and λNMI149 may also be used. Examples of plant expression vectors include pBIO1, pBI101.2, pBHO1.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-C1, pMAM, and pMAMneo (Clontech). Recombinant expression vectors may be viral vectors, such as retroviral vectors or lentiviral vectors. Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, and include self-inactivated lentiviral vectors in particular, as provided in Milone et al., Mol.Ther.17(8):1453-1464 (2009). Other examples of lentiviral vectors that can be used in clinical settings include, but are not limited to, Oxford BioMedica plc's LENTIVECTOR® gene delivery technology and Lentigen's LENTIMAX® vector system. Non-clinical lentiviral vectors are also available and are known to those skilled in the art.

[0205] Numerous transfection techniques are generally known in the art (see, for example, Graham et al., Virology, 52:456-467 (1973), Sambrook et al., above, Davis et al., Basic Methods in Molecular Biology, Elsevier (1986), and Chu et al., Gene, 13:97 (1981)).

[0206] Transfection methods include calcium phosphate coprecipitation (e.g., see Graham et al., above), direct microinjection into cultured cells (e.g., see Capecchi, Cell, 22:479-488 (1980)), electroporation (e.g., see Shigekawa et al., BioTechniques, 6:742-751 (1988)), liposome-mediated gene transfer (e.g., see Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transduction (e.g., see Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid delivery using high-speed microprojectiles (e.g., see Klein et al., Nature, 327:70-73 (1987)).

[0207] In one embodiment, the recombinant expression vector may be prepared using, for example, standard recombinant DNA techniques described above in Sambrook et al. and Ausubel et al. The circular or linear expression vector construct may be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. The replication system may be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0208] Recombinant expression vectors may contain regulatory sequences such as transcription and translation start and stop codons, which are specific to the host cell type into which the vector is introduced (e.g., bacteria, fungi, plants, or animals), taking into account whether the vector is DNA or RNA-based. Recombinant expression vectors may also contain restriction sites to facilitate cloning.

[0209] Recombinant expression vectors may contain one or more marker genes, which allow for the selection of transformed or transfected host cells. Examples of marker genes include those for biocide resistance, such as resistance to antibiotics and heavy metals, and complementarity to provide protrophotrophy in trophically dependent hosts. Examples of marker genes suitable for the expression vectors of the present invention include neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0210] Recombinant expression vectors may include native or non-native promoters functionally linked to a nucleotide sequence encoding an antibody or its antigen-binding fragment, or to a nucleotide sequence complementary to or hybridizing with the nucleotide sequence encoding the antibody or its antigen-binding fragment. For example, the selection of strong, weak, inducible, tissue-specific, and development-specific promoters is within the scope of the art. Similarly, combinations of nucleotide sequences and promoters are also within the scope of the art. Promoters may be non-viral promoters or viral promoters, such as cytomegalovirus (CMV) promoters, SV40 promoters, RSV promoters, EF1 alpha promoters, or promoters found in long terminal repeats of mouse stem cell viruses.

[0211] Recombinant expression vectors can be designed for transient expression, stable expression, or both. They can also be constructed for constitutive or inducible expression.

[0212] Furthermore, recombinant expression vectors may be constructed to contain suicide genes. As used herein, the term “suicide gene” refers to a gene that kills cells that express a suicide gene. A suicide gene may be a gene that confers sensitivity to a drug, for example, a drug, to cells that express the gene, causing the cells to die when they come into contact with or are exposed to the drug. Suicide genes are known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase, and nitroreductase.

[0213] Embodiments further provide host cells containing any of the recombinant expression vectors described herein. As used herein, the term “host cell” refers to any cell type that may contain the recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, e.g., a plant, animal, fungus, or algae, or a prokaryotic cell, e.g., a bacterium or protist. The host cell may be a cultured cell or a primary cell, i.e., a cell directly isolated from an organism, e.g., a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in a suspension state. Suitable host cells are known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, HEK293 T cells, and the like. For the purpose of amplifying or replicating the recombinant expression vector, the host cell may be a prokaryotic cell, e.g., a DH5a cell. For the purpose of producing recombinant antibodies or their antigen-binding fragments, the host cell may be a mammalian cell. The host cell may be a human cell. The host cell may be of any cell type, originate from any tissue type, and be at any developmental stage, but it may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0214] One aspect of this application provides engineered immunoeffector cells comprising any one of the antibodies or antigen-binding fragments described herein, any one of the isolated nucleic acids described above, or any one of the vectors described above.

[0215] A cell population comprising at least one host cell as described herein is also provided by one embodiment. The cell population may be a heterogeneous population comprising at least one other cell, e.g., a host cell (e.g., a T cell), in addition to a host cell containing one of the recombinant expression vectors described herein, and not comprising any recombinant expression vector or cells other than T cells, e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the cell population may be a substantially homogeneous population, in which case the population mainly comprises host cells containing (e.g., essentially consisting of) a recombinant expression vector. The population may also be a clonal cell population, in which case all cells in the population are clones of a single host cell containing a recombinant expression vector, such that all cells in the population contain a recombinant expression vector. In one embodiment of the present invention, the cell population is a clonal population comprising a host cell containing a recombinant expression vector as described herein.

[0216] Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, such as screening a library from cells expressing the gene, inducing a gene in a vector known to contain the same, or directly isolating the same from cells and tissues using standard techniques.

[0217] Alternatively, the target gene may be produced synthetically rather than by cloning. The present invention also provides vectors into which the DNA of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow for stable, long-term integration of the transgene and its proliferation in daughter cells. Lentiviral vectors have further advantages over vectors derived from onchoretroviruses, such as mouse leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0218] The expression of natural or synthetic nucleic acids encoding anti-GCC conjugates described herein can be achieved by functionally ligating the nucleic acid encoding the anti-GCC conjugate polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for eukaryotic replication and incorporation. A typical cloning vector contains transcription and translation terminators, start sequences, and promoters useful for controlling the expression of desired nucleic acid sequences.

[0219] The expression constructs of the present invention may also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patents 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0220] Nucleic acids can be cloned into a wide variety of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Of particular interest are expression vectors, replication vectors, probe-generating vectors, and sequencing vectors. Furthermore, expression vectors may be provided to cells in the form of viral vectors.

[0221] 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), as well as in other virology and molecular biology manuals. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains a functional origin of replication, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers in at least one organism (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0222] Numerous virus-based systems have been developed for gene delivery into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors using techniques known in the art and packaged in retroviral particles. Recombinant viruses can then be isolated and delivered to target cells either in vivo or ex vivo. Numerous retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. Numerous adenovirus vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0223] Additional promoter elements, such as enhancers, control the frequency of transcription initiation. Typically, they are located 30–110 bp upstream of the initiation site, but recently, many promoters have been shown to also contain functional elements downstream of the initiation site. The spacing between promoter elements is often flexible so that promoter function is maintained even if the elements invert or move relative to each other. In thymidine kinase (TK) promoters, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, individual elements are thought to function either cooperatively or independently to activate transcription.

[0224] A suitable example of a promoter is the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence that can induce high levels of expression of any polynucleotide sequence functionally ligated to it.

[0225] Another example of a suitable promoter is elongation growth factor-1a (EF-1a). However, other constitutive promoter sequences may also be used, including the Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, and Roussarcoma virus promoter, as well as human gene promoters, such as, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also intended as part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence to which the promoter is functionally linked when such expression is desired, or turn off when such expression is undesirable. Examples of inductive promoters include, but are not limited to, the metallothione promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0226] To evaluate the expression of an anti-GCC conjugate (e.g., a single-domain antibody) polypeptide or a portion thereof, the expression vector introduced into cells may also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected by a viral vector. In other embodiments, the selectable marker may hold a separate DNA fragment and be used in a co-transfection procedure.

[0227] Both selectable markers and reporter genes may be positioned adjacent to appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo. Reporter genes are used to identify potential transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and whose polypeptide expression manifests as some readily detectable characteristic, such as enzymatic activity. Reporter gene expression is assayed at a suitable time after the DNA has been introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and may be prepared using known techniques or commercially available. Generally, a construct with the smallest 5' flanking region exhibiting the highest level of reporter gene expression is identified as a promoter. Such promoter regions may be ligated to a reporter gene and used to evaluate drugs for their ability to regulate promoter-driven transcription.

[0228] Methods for introducing and expressing genes into cells are known in the art. In relation to expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means. Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle impact, microinjection, and electroporation. Methods for preparing cells containing vectors and / or foreign nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0229] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, are the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, for example. See, for example, U.S. Patents 5,350,674 and 5,585,362. Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0230] An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). When a non-viral delivery system is utilized, the exemplary delivery vehicle is nanoparticles, e.g., liposomes or other suitable submicron-sized delivery systems. The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, nucleic acids may be associated with lipids. Lipid-associated nucleic acids may be encapsulated within the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, bound to liposomes via linking molecules associated with both liposomes and oligonucleotides, encapsulated within liposomes, complexed with liposomes, dispersed in a lipid-containing solution, mixed with lipids, combined with lipids, contained as a suspension in lipids, containing or complexed with micelles, or otherwise associated with lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they may exist as micelles or in bilayer structures with a "broken-down" structure. They may also simply be scattered in solution, forming aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. For example, lipids include not only naturally occurring lipid droplets in the cytoplasm, but also a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0231] Suitable lipids for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposomes" is a general term encompassing various monolayer and multilayer lipid vehicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized by having a phospholipid bilayer membrane and a vesicle structure with an aqueous medium inside. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-reorganization before forming a sealed structure, encapsulating water and dissolved solutes between the lipid bilayers (Ghosh et al, 1991 Glycobiology 5:505-10).

[0232] However, the invention also includes compositions having structures in solution that differ from the usual vesicle structure. For example, lipids may take the form of micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine nucleic acid complexes are also considered. Various assays may be performed to confirm the presence of recombinant DNA sequences in host cells, regardless of the method used to introduce foreign nucleic acids into host cells or otherwise expose cells to the inhibitors of the invention. Such assays include, for example, “molecular biological” assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, “biochemical” assays, such as immunological means (ELISA and Western blotting), or assays described herein for identifying drugs that fall within the scope of the invention, to detect the presence or absence of specific peptides.

[0233] The present invention further provides a vector comprising a nucleic acid molecule encoding an anti-GCC conjugate (e.g., a single-domain antibody). In one embodiment, the anti-GCC conjugate (e.g., single-domain antibody) vector can be directly transduced into cells, such as T cells. In one embodiment, the vector includes, but is not limited to, cloning or expression vectors, such as one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double microchromosomes), retroviruses, and lentiviral vector constructs. In one embodiment, the vector can express the anti-GCC conjugate construct in mammalian T cells. In one embodiment, the mammalian T cells are human T cells.

[0234] In some embodiments, nonviral methods may be used to deliver nucleic acids encoding anti-GCC binders described herein to cells, tissues, or subjects. In some embodiments, nonviral methods involve the use of transposons (also called transposable elements). In some embodiments, transposons are DNA fragments that can insert themselves into a location in the genome, for example, DNA fragments that can self-replicate and insert copies of themselves into the genome, or DNA fragments that can be spliced ​​from a longer nucleic acid and inserted into another location in the genome.

[0235] Additional and exemplary transposon and nonviral delivery methods are described on pages 196-198 of International Application WO2016 / 164731, filed 8 April 2016, and are incorporated in their entirety by reference.

[0236] Treatment method The present invention relates to a therapeutic method comprising administering an anti-GCC antigen-binding molecule, such as those described herein, to a target. In some embodiments, the anti-GCC antigen-binding molecules disclosed herein (e.g., single-domain antibodies) may be used in a method for treating or preventing a mammalian disease. In this regard, one embodiment provides a method for treating or preventing a mammalian cancer, the method comprising administering to a mammal an antigen-binding molecule (e.g., a single-domain antibody), nucleic acid, recombinant expression vector, host cell, cell population, antibody and / or its antigen-binding moiety, and / or a pharmaceutical composition in an amount effective to treat or prevent a mammalian cancer. The present invention also relates to an anti-GCC antigen-binding molecule, such as those described herein (e.g., sdAb), for use in the treatment of a disease. The present invention also relates to an anti-GCC antigen-binding molecule, such as those described herein (e.g., sdAb), for use in the treatment of cancer. The present invention also relates to an anti-GCC antigen-binding molecule, such as those described herein (e.g., sdAb), for use in the manufacture of a pharmaceutical intended for the treatment of cancer.

[0237] The compositions described herein may be administered by any convenient method, including aerosol inhalation, injection, ingestion, blood transfusion, implantation, or transplantation. The compositions described herein may be administered to a patient by arterial, subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular, intravenous (IV) injection, or intraperitoneal injection. In one embodiment, for example, a composition described herein, comprising antigen-binding molecule (e.g., single-domain antibody) expressing cells, is administered to a patient by intradermal or subcutaneous injection. In one embodiment, for example, a composition described herein, comprising antigen-binding molecule (e.g., single-domain antibody) expressing cells, is administered by IV injection. For example, a composition described herein, comprising antigen-binding molecule (e.g., single-domain antibody) expressing cells, may be injected directly into a tumor, lymph node, or site of infection.

[0238] For methods of administering host cells or cell populations, the cells may be homogeneous or self-derived cells to the mammal. Preferably, the cells are self-derived to the mammal. As used herein, homogeneous means any substance originating from different animals of the same species as the individual into which the substance is introduced. Two or more individuals are said to be homogeneous if one or more of their genes are not identical at one or more loci. In some embodiments, homogeneous substances originating from individuals of the same species may be genetically distinct enough to interact antigenically. As used herein, the term “self” means any substance originating from the same individual into which any substance is subsequently reintroduced.

[0239] Mammals referred to herein may be any mammal. As used herein, the term “mammal” refers to any mammal, including, but not limited to, rodents such as mice and hamsters, and lagomorphs such as rabbits. Mammals may be from the order Carnivora, including the families Felidae (cats) and Canidae (dogs). Mammals may be from the order Artiodactyla, including the families Bovidae (cats) and Swine (pig), or from the order Perissodactyla, including the family Equidae (horse). Mammals may be from the orders Primates, Ceboids, or Simoids (monkeys) or Haplorhini (humans and apes). In some embodiments, the mammal is a human.

[0240] Regarding this method, cancers include acute lymphoblastic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's lymphoma, hypopharyngeal cancer, and kidney cancer. It may be any cancer, including any of the following: laryngeal cancer, leukemia, humoral neoplasm, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, chronic lymphocytic leukemia B, hairy cell leukemia, acute lymphoblastic leukemia (ALL), Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, retinal cancer, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0241] In certain embodiments, the cancer is gastrointestinal cancer. In some embodiments, the cancer is stomach cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer has abnormal expression of GCC.

[0242] The terms “to treat” and “to prevent,” as well as words derived therefrom, when used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that a person skilled in the art would recognize as having potential benefits or therapeutic effects. In this regard, the present method may provide treatment or prevention of any amount or any level of cancer in mammals.

[0243] Furthermore, the treatment or prevention provided by this method may include the treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented, such as cancer. Also, for the purposes of this specification, “prevention” may include delaying the onset of the disease, or its symptoms or conditions.

[0244] Another embodiment provides a method for detecting the presence of cancer in a mammal, the method comprising (a) contacting a sample containing one or more cells derived from a mammal with an antigen-binding molecule (e.g., a single-domain antibody), its antigen-binding portion, or a pharmaceutical composition to form a complex, and (b) detecting the complex, wherein the detection of the complex indicates the presence of cancer in the mammal. In some embodiments, the contact may be performed in vitro or in vivo with respect to the mammal. In some embodiments, the contact is in vitro.

[0245] The sample may be obtained by any preferred method, e.g., biopsy or autopsy. A biopsy is the removal of tissue and / or cells from an individual. Such removal may involve collecting tissue and / or cells from the individual for experimentation on the removed tissue and / or cells. This experiment may include determining whether the individual has and / or suffers from a particular condition or disease. The condition or disease may be, for example, cancer.

[0246] With regard to embodiments of methods for detecting the presence of proliferative disorders in mammals, such as cancer, a sample containing mammalian cells may include whole cells, their lysates, or fractions of whole cell lysates, such as the nuclear or cytoplasmic fraction, the total protein fraction, or the nucleic acid fraction. If the sample contains whole cells, the cells may be any cells of a mammal, such as blood cells or endothelial cells, or cells of any organ or tissue.

[0247] Furthermore, detection of the complex can be performed by any number of methods known in the art. For example, the antibodies described herein, or their antigen-binding moieties, can be labeled with detectable labels such as those disclosed above, e.g., radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0248] Methods for testing antigen-binding molecules (e.g., single-domain antibodies) for their ability to recognize target cells and their antigen specificity are known in the art. For example, Clay et al., J.Immunol, 163:507-513 (1999) teach a method for measuring the release of cytokines (e.g., interferon-γ, granulocyte / monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α), or interleukin-2 (IL-2)).

[0249] Another embodiment brings to light the use of the antigen-binding molecules (e.g., single-domain antibodies or their antigen-binding moieties) and / or pharmaceutical compositions of the present invention for the treatment or prevention of proliferative disorders in mammals, such as cancer. The cancer may be any of the cancers described herein.

[0250] Any method of administration, including local and systemic administration, may be used for the disclosed therapeutic agents. For example, local, oral, intravascular, such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration may be used. The specific method and regimen of administration will be selected by the attending clinician, taking into account the details of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic). If two or more drugs or compositions are administered, one or more routes of administration may be used; for example, chemotherapeutic agents may be administered orally, and antibodies or antigen-binding fragments or complexes or compositions may be administered intravenously. Methods of administration include injection in which the antibody, antigen-binding fragment, or composition is delivered in a non-toxic, pharmaceutically acceptable carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixative oil, ethyl oleate, or liposomes. In some embodiments, topical administration of the disclosed compounds may be used, for example, by applying an antibody or antigen-binding fragment to an area of ​​tissue from which a tumor has been removed, or to an area suspected of being prone to tumor development. In some embodiments, sustained intratumoral (or near-tumor) release of a pharmaceutical preparation containing a therapeutically effective amount of antibody or antigen-binding fragment may be beneficial. In other examples, the complex is applied topically to the cornea as eye drops, or intraocularly from within the vitreous humor.

[0251] The disclosed therapeutic agents can be formulated in unit dosage forms suitable for individual administration of precise doses. In addition, the disclosed therapeutic agents can be administered in single-dose or multi-dose schedules. A multi-dose schedule is one in which the primary course of treatment consists of two or more distinct doses, e.g., 1 to 10 doses, followed by other doses at subsequent time intervals as needed to maintain or enhance the effect of the composition. Treatment may involve daily or multi-day doses of the compound(s) over several days, months, or even years. Therefore, the administration regimen will also be determined, at least in part, based on the specific needs of the person being treated and will depend on the judgment of the administering physician.

[0252] In one embodiment, the present disclosure provides a pharmaceutical composition comprising at least one therapeutic agent of the present disclosure (e.g., a therapeutic agent of the present disclosure) or a pharmaceutically acceptable salt thereof, either alone or in combination with other anti-cancer agents, together with a pharmaceutically acceptable carrier suitable for administration to a human or animal subject.

[0253] In one embodiment, the present disclosure provides a method of treating a human or animal subject suffering from a cell proliferative disease such as cancer. The present disclosure provides a method of treating a human or animal subject that requires such treatment, the method comprising administering to the subject a therapeutically effective amount of a therapeutic agent of the present disclosure or a pharmaceutically acceptable salt thereof, either alone or in combination with other anti-cancer agents.

[0254] In particular, the compositions are formulated together as combination therapies or administered separately. In combination therapies, the compounds of the present disclosure and other anti-cancer agent(s) can be administered either simultaneously, concurrently or sequentially without a specific time limit, such administration providing therapeutically effective levels of the two compounds in the patient's body.

[0255] In some embodiments, the compounds of the present disclosure and other anti-cancer agent(s) are generally administered sequentially by injection or orally in any order. The dosing regimen may vary depending on the stage of the disease, the physical strength of the patient, the safety profile of the individual drugs, and the resistance of the individual drugs, as well as other criteria well known to the attending physician and physician(s) administering the combination.

[0256] The compounds of the present disclosure may be used, in particular as radiosensitizers, particularly for the treatment of tumors that show low sensitivity to radiotherapy.

[0257] In another embodiment, the present invention provides a kit comprising an antibody, such as a single-domain antibody, as described herein, for detecting GCC for, for example, the treatment or prevention of a disease or immune response, and / or for the diagnosis, prognosis, or monitoring of the disease. Such a kit may contain other components, packaging, instructions, or materials that are useful for detecting the GCC protein. The kit may contain a labeled single-domain antibody or a binder and one or more compounds for detecting the label, as described herein.

[0258] Unless otherwise stated, all technical and scientific terms and phrases used herein have the same meaning as those commonly understood by those skilled in the art. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described below. All publications referenced herein are incorporated herein by reference.

[0259] Standard techniques may be used for recombinant DNA and oligonucleotide synthesis, as well as for tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques may be carried out according to the manufacturer's specifications, as is commonly done in the art, or as described herein. The aforementioned techniques and procedures may generally be carried out according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for all purposes.

[0260] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The present invention will be more fully understood by reference to the following embodiments. [Examples]

[0261] These examples are provided to aid in understanding the present invention, but are not intended to limit the scope of the invention, nor should they be construed as such. The examples do not include detailed descriptions of conventional methods (such as molecular cloning techniques) that are well known to those skilled in the art.

[0262] Example 1. GCC V H Antibody isolation and selection A single-domain heavy chain antibody (V) that specifically targets human GCC. H ) was generated (for example, the anti-GCC binders provided in Tables 1-3). As shown in Table 5, recombinant single-domain anti-GCC V H The constructs were evaluated in vitro for their binding affinity to GCC. Binding rates were determined using the Octet binding assay. These were measured using the real-time biolayer interferometer-based biosensor Octet (ForteBio). All binding tests were performed with HBS-ET Octet dynamic buffer. The biosensor was always washed with Octet dynamic buffer between different steps. Each V H A 2x dilution series of seven steps was prepared. The contact time for each binding step was 300 seconds, and the dissociation step was varied between 400 and 600 seconds. The dynamic binding (ka) and dissociation (kd) rate constants were determined by processing the data using ForteBio Analysis software and fitting it to a 1:1 binding model. The calculated affinity and rate constants are shown in Table 4.

[0263] The binding of single-domain antibodies to CT26 cells was measured using flow cytometry. CT26 cells were obtained by serial dilution V. H The cells were incubated with [substance name] and fluorescence was measured by flow cytometry. Anti-GCC V HAs shown in Table 4, the binding of the antibody to CT26 cells was confirmed by a FACS dose-response assay to obtain the EC50 (nM) at cell binding.

[0264] [Table 4]

[0265] To determine stability, V H The construct was subjected to size exclusion chromatography (SEC). In short, the purified V H The samples were stored overnight at 4°C in PBS buffer at various concentrations and then analyzed at different time points using an SEC column. The samples were injected into sodium phosphate buffer. Data were collected over time, and the area of ​​the monomer peaks remaining after storage was calculated compared to that present at the start (T=0). Stability results were obtained as shown in Table 5 below.

[0266] [Table 5]

[0267] An ELISA was performed to measure VH binding to human light chains. The binding value of each VH molecule (measured at OD450nm) was less than 0.1, which is considered a background signal for this assay.

[0268] Example 2. Design and characterization of GCC chimeric antigen receptors Chimeric antigen receptor constructs are manipulated to include an extracellular binding domain (e.g., an anti-GCC binding sequence) containing the single-domain antibody described above (e.g., VH provided in Table 2 or Table 3). CAR T constructs are generated by ligating the binding sequence in-frame to the CD28 hinge / transmembrane domain, as well as the costimulatory domain and the CD3 zeta-1xx signaling domain. Schematic diagrams of exemplary CAR constructs are shown in Figures 1A and 1B.

[0269] Nucleic acids encoding CAR construct sequences were cloned into the retroviral plasmid backbone. Retroviral vector-containing supernatant was generated by transient transfection of phenix ampho cells (ATCC CRL-3213), and the retroviral vector-containing supernatant was collected and stored at -80°C.

[0270] Human primary T cells from healthy donors were purified from peripheral blood mononuclear cells (PBMCs) isolated from leukopak (purchased from a commercial supplier with the written consent of the donor) using immunomagnetic bead selection of CD3+ cells according to the manufacturer's protocol (EasySep® Human T Cell Isolation Kit, Stem Cell Technologies #17951). The T cells were cultured at a density of 1 million cells / ml in X-vivo 15 medium (Lonza #04-744Q) supplemented with 10% penicillin-streptomycin (Gibco 15140-122) and 2 ng / ml of IL-2 (Milteyni 130-097-743). Cells were activated with CD3 / CD28 MACS® T Cell TransAct reagent (Miltenyi Biotec MACS #130-111-160), and transduced overnight with a retroviral vector encoding the CAR construct on day 2 or 3. The following day, the CAR-T cell cultures were transferred to G-Rex6® well plates (WilsonWolf P / N 80240M) and grown in X-vivo 15 medium (Lonza #04-744Q) supplemented with 10% penicillin-streptomycin (Gibco 15140-122) and 2 ng / ml IL-2 (Milteyni 130-097-743) until harvest on days 7-10. Medium changes and IL-2 supplementation were performed every 2-3 days.

[0271] CAR T cell expression was evaluated by flow cytometry using an anti-EGFR antibody (R&D systems: FAB9577R) or a recombinant GCC extracellular domain protein for CAR surface expression.

[0272] Example 3. GCC CAR T cell activity in vitro This example describes anti-GCC CAR T cell activity in vitro. CAR-T cell cytotoxicity against GCC-expressing and GCC-negative target cancer cell lines was examined. The target cancer cell lines included GSU, LS1034, and HT55 (which endogenously express GCC), as well as HT29-GCC, a human colorectal cancer cell line engineered to stably express GCC, and its vector control cell line, HT29-vec, which is GCC-negative. Each target cell line was seeded in a 384-well plate, and GCC CAR-T or non-target CAR-T cells (negative control) were added at effector-to-target (E:T) ratios of 10:1, 3:1, 1:1, and 0.3:1. Wells containing only target cells and wells containing only effector cells were included as controls. After 2 days, cell viability was measured using the CellTiter-Glo® One Solution Assay (Promega, G8462). The viability of target cells was calculated from the luminescence signal of the co-culture wells by first subtracting the signal of the wells containing only effector cells and then dividing by the signal of the wells containing only target cells. The killing rate was calculated by subtracting the viability of target cells from 100% of the GCC CAR-T cells.

[0273] V HAnti-GCC conjugates induced cell death against GCC-expressing target cell lines, in contrast to non-targeted CD19 CAR-T cells (1928z-1xx) used as a control. As shown in Figures 2A-2D, CAR-T cells expressing anti-GCC CAR in the absence of cleaved EGFR (tEGFR) showed in vitro cytotoxicity against GCC-expressing cells such as HT29-GCC cells (human colorectal cancer cell line HT29 manipulated to stably express GCC) (Figure 2A), as well as endogenously GCC-expressing cell lines GSU (Figure 2C) and LS1034 (Figure 2D). As shown in Figures 3A-3D, CAR-T cells expressing anti-GCC CAR in the presence of cleaved EGFR (tEGFR) also showed in vitro cytotoxicity against GCC-expressing cells such as HT29-GCC (Figure 3A), GSU ​​(Figure 3C), and LS1034 (Figure 3D). The bars represent the mean + SD values ​​from three technical replicas. The data are representative of more than three independent experiments conducted using anti-GCC CAR T cells derived from more than three donors. GCC CAR-T cells did not exhibit cell death against GCC-negative HT29-vec cells (Figures 2B and 3B), indicating that the killing activity of GCC CAR-T cells was antigen-dependent.

[0274] In addition to antigen-dependent cell death, the in vitro activity of GCC CAR-T cells was also evaluated by assessing antigen-dependent secretion of IFNγ and IL2. Anti-GCC V H GCC CAR-T cells with binding agents were co-cultured with GCC-expressing and GCC-negative target cancer cell lines at E:T ratios of 10:1, 3:1, 1:1, and 0.3:1. Supernatants were collected after 2 days of co-culture. IFNγ and IL2 secreted into the supernatant were detected using the Intellicyt QBeads Human PlexScreen kit (Sartorius, 90702). All VH-binding GCC CAR-T cells secreted IFNγ in both the presence (5A-5D) and absence (4A-4D) of tEGFR when co-cultured with GCC-expressing target cells, but did not secrete IFNγ when co-cultured with GCC-negative target cells (Figures 4B and 5B), indicating antigen-dependent cytokine release. HWhen GCC CAR-T cells containing a binder were co-cultured with GCC-expressing target cells, they secreted IL2 in both the presence (7A-7D) and absence (6A-6D) of tEGFR. However, they did not secrete IL2 when co-cultured with GCC-negative target cells (Figures 6B and 7B), indicating antigen-dependent cytokine release.

[0275] Having described several aspects of at least one embodiment of the present invention, it will be readily apparent to those skilled in the art that various changes, modifications, and improvements will be readily apparent. Such changes, modifications, and improvements are intended to be part of this disclosure and to be within the spirit and scope of the present invention. Accordingly, the foregoing description and drawings are merely examples, and the present invention is described in detail by the following claims.

[0276] Sequence List Table 6 below provides the names and sequences disclosed herein.

[0277] [Table 6-1]

[0278] [Table 6-2]

[0279] [Table 6-3]

[0280] [Table 6-4]

[0281] The doctrine of equality To modify these claims, the use of common terms such as “First,” “Second,” and “Third” within the claims does not, in itself, imply any priority, right of way, or order of one claim to another, nor does it imply any chronological order in which the actions of the method are performed, but is used merely as a distinguishing mark to differentiate one claim having a particular name from another claim having the same name (apart from the use of common terms).

[0282] The articles “a” and “an,” as used herein in this specification and in the claims, should be understood to include multiple referents unless otherwise explicitly indicated. Claims or descriptions containing “or” between one or more members of a group are considered satisfied unless otherwise explicitly indicated or otherwise evident from the context, if one, two or more, or all of the members of the group are present in, used in, or otherwise related to a given product or process. The present invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or process. The present invention also includes embodiments in which two or more, or all, members of the group are present in, used in, or otherwise related to a given product or process. Furthermore, unless otherwise indicated, or unless it would be obvious to those skilled in the art that such a change would result in a contradiction or inconsistency, the present invention should be understood to encompass all modifications, combinations, and permutations introduced in another claim dependent on the same basic claim (or any other such claim) of one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the enumerated claims. Where elements are presented as a list (e.g., a group of Markouches or similar format), each subgroup of the elements should also be disclosed, and it should be understood that any element(s) may be excluded from the group. In general, where the present invention or an aspect of the present invention is deemed to include certain elements, features, etc., it should be understood that certain embodiments or aspects of the present invention consist of, or are essentially composed of, such elements, features, etc. For brevity, in no case are those embodiments specifically described herein in numerous terms. It should also be understood that any embodiment or aspect of the present invention may be expressly excluded from the claims, regardless of whether specific exclusions are enumerated herein. Publications, websites, and other reference materials mentioned herein to describe the background of the present invention and to provide additional details relating to its implementation are incorporated herein by reference.

Claims

1. An anti-guanylate cyclase C (GCC) agent, a heavy chain variable region (VH) having the complementarity determining region (CDR) sequences: RYWMT (HCDR1) (SEQ ID NO: 10), KIRHDGGEKYYADSVKG (HCDR2) (SEQ ID NO: 15) and DYNKDY (HCDR3) (SEQ ID NO: 18); A heavy chain variable region (V) having the complementarity determining region (CDR) sequences of HYYWS (HCDR1) (SEQ ID NO: 8), RIYPSGSTSYNPSLKS (HCDR2) (SEQ ID NO: 11), and DRSTGWS EWNSDL (HCDR3) (SEQ ID NO: 16). Ｈ ), A heavy chain variable region (V) having the complementarity determining region (CDR) sequences of RYWMS (HCDR1) (SEQ ID NO: 9), KIRHDGGEKYYVDSVKG (HCDR2) (SEQ ID NO: 12), and DYTRDV (HCDR3) (SEQ ID NO: 17). Ｈ ), A heavy chain variable region (V) having the complementarity determining region (CDR) sequences of RYWMT (HCDR1) (SEQ ID NO: 10), KIKYDGSEKYYADSVKG (HCDR2) (SEQ ID NO: 13), and DYNKDY (HCDR3) (SEQ ID NO: 18). Ｈ ),or A heavy chain variable region (V) having the complementarity determining region (CDR) sequences of: RYWMT (HCDR1) (SEQ ID NO: 10), KIRHDGGEKYYPDSVKG (HCDR2) (SEQ ID NO: 14), and DYNKDL (HCDR3) (SEQ ID NO: 19). Ｈ ) The anti-GCC agent comprising:

2. An immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 28; An immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:1 or SEQ ID NO:20; An immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 21; An immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:26; or An immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence at least 90% identical to SEQ ID NO:

27. The anti-GCC agent according to claim 1 ,

3. An anti-guanylate cyclase C (GCC) agent, an immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence at least 90% identical to SEQ ID NO:28; An immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:1 or SEQ ID NO:20; An immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 21; An immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:26; or An immunoglobulin heavy chain variable (VH) region comprising an amino acid sequence at least 90% identical to SEQ ID NO:

27. The anti-GCC agent comprising:

4. The V Ｈ The anti-GCC agent of any one of claims 1 to 3, wherein the region comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 20, 21, 26, 27, or 28.

5. The V Ｈ The anti-GCC agent of any one of claims 1 to 4, wherein the region comprises an amino acid sequence identical to any one of SEQ ID NOs: 1, 20, 21, 26, 27, or 28.

6. The anti-GCC agent is selected from the group consisting of an IgA antibody, an IgG antibody, an IgE antibody, an IgM antibody, a bi- or multispecific antibody, a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fd' fragment, an Fd fragment, an isolated CDR or set thereof, a single chain variable fragment (scFv), a polypeptide-Fc fusion, a single domain antibody (sdAb), a camelid antibody, a masked antibody, a small modular antibody, 6. The anti-GCC agent according to any one of claims 1 to 5, selected from the group consisting of ImmunoPharmaceuticals ("SMIPs™"), single chain, tandem diabodies, VHH, Anticalin, nanobodies, humabodies, minibodies, BiTEs, ankyrin repeat proteins, DARPINs, avimers, DARTs, TCR-like antibodies, Adnectins, Affilins, transbodies, Affibodies, TrimerX, microproteins, Fynomers, centirins, and KALBITOR.

7. The anti-GCC agent according to any one of claims 1 to 6, wherein the anti-GCC agent is a single domain antibody (sdAb).

8. The anti-GCC agent according to any one of claims 1 to 6, wherein the anti-GCC agent is a heavy chain-only antibody.

9. The anti-GCC agent has a K Ｄ The anti-GCC agent according to any one of claims 1 to 8, which binds to GCC at

10. The anti-GCC agent has an EC ５０ The anti-GCC agent according to any one of claims 1 to 9, which binds to GCC on a target cell.

11. A therapeutic agent for cancer, comprising an anti-GCC agent according to any one of claims 1 to 10.

12. 12. The method of claim 11, wherein the cancer is selected from gastrointestinal cancer, colorectal cancer, colorectal adenocarcinoma, colorectal leiomyosarcoma, colorectal lymphoma, colorectal melanoma, colorectal neuroendocrine tumor, metastatic colon cancer, gastric cancer, gastric adenocarcinoma, gastric lymphoma, gastric sarcoma, esophageal cancer, squamous cell carcinoma, adenocarcinoma of the esophagus, or pancreatic cancer.

13. The method of claim 12, wherein the cancer is gastrointestinal cancer.

14. The method of claim 13, wherein the gastrointestinal cancer is colon cancer, colorectal cancer, gastric cancer, or esophageal cancer.

15. A pharmaceutical composition comprising an anti-GCC agent according to any one of claims 1 to 10 and a pharma- ceutical acceptable carrier.

16. A cancer therapeutic agent comprising the composition described in claim 15.

17. A nucleic acid encoding the V H amino acid sequence contained in the anti-GCC agent according to any one of claims 1 to 10.

18. The nucleic acid of claim 17, wherein the V H amino acid sequence is identical to any one of SEQ ID NOs: 1, 20, 21, 26, 27, or 28.

19. A vector comprising the nucleic acid of claim 17 or 18.

20. 20. An isolated cell comprising the vector of claim 19.

21. 11. An anti-guanylate cyclase C (GCC) chimeric antigen receptor (CAR), the anti-GCC CAR comprising an anti-GCC agent according to any one of claims 1 to 10.

22. An immune response inducer comprising the anti-GCC CAR described in claim 21.

23. An inducer of cytotoxicity comprising the anti-GCC CAR described in claim 21.

24. A method for detecting the presence of cancer in a mammal, comprising the steps of: detecting the presence of cancer in a mammal, the method comprising the steps of: detecting the presence of cancer in a mammal, the method comprising the steps of: (a) contacting a sample comprising one or more cells derived from said mammal with said anti-GCC agent to form a complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of the cancer in the mammal. The detection agent comprising:

25. 25. The detection agent of claim 24, wherein said contacting is in vitro or in vivo with respect to said mammal.

26. 26. The detection agent of claim 25, wherein the contacting is in vitro.

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