Antibodies and chimeric antigen receptors targeting GCC and methods of use thereof

Abstract

Anti-GCC single domain antibodies (e.g., VHH domain antibodies) and chimeric antigen receptors (CARs) that bind to GCC are provided, wherein the chimeric antigen receptors (CARs) comprise the anti-GCC single domain antibody in an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. Immune cells transduced with the disclosed CAR constructs and / or chimeric receptors can be used in cancer immunotherapy.

Description

[Technical Field]

[0001] cross reference This application claims priority to International Patent Application No. PCT / CN2022 / 122166, filed September 28, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application incorporates by reference the sequence listing submitted with this application in XML file format entitled "IEC230175PCT_SEQUENCE LISTING.xml", created on September 26, 2023, having a size of 96,272 bytes.

[0003] 1. Field The present disclosure relates to the field of antibodies (e.g., single domain antibodies), chimeric antigen receptors and engineered immune cells that target guanylyl cyclase C (GCC), and methods of use thereof. [Background technology]

[0004] 2. Background Guanylyl cyclase C, also known as guanylyl cyclase 2C (GCC, GUC2C, or GUCY2C), is a member of the receptor guanylyl cyclase family and is a transmembrane receptor for the ligands guanylin, uroguanylin, lyphoguanylin, and Escherichia coli heat-stable enterotoxin (STa) (see Proc. Natl. Acad. Sci. USA 93: 14827-14832 (1996) (Non-Patent Document 1); Eur J Cancer 41: 1618-1627 (2005) (Non-Patent Document 2)). GCC is selectively expressed in human intestinal and colorectal tumors and is a relatively specific marker for metastatic cancer cells in extraintestinal tissues (see Proc. Natl. Acad. Sci. USA 93: 14827-14832 (1996) (Non-Patent Document 1)). Therefore, GCC may serve as a sensitive and specific molecular marker for colorectal cancer (CRC) in detecting tumor cells in normal tissues and blood for staging and monitoring CRC (see Gastroenterology 107:1653-1661 (1994) (Non-Patent Document 3), Proc. Natl. Acad. Sci. USA 93: 14827-14832 (1996) (Non-Patent Document 1), Eur J Cancer 41: 1618-1627 (2005) (Non-Patent Document 2)). Recent studies have identified a novel role for GCC as a tumor inhibitor, and GCC, together with its ligand, is involved in regulating the balance between proliferation and differentiation along the intestinal crypt-to-villus axis. Therefore, GCC has become a promising therapeutic target (see Proc. Natl. Acad. Sci. USA 100: 3018-3020 (2003) (Non-Patent Document 4), Expert Rev Clin Pharmacol, 10(5): 549-557 (2017) (Non-Patent Document 5)). The field needs new anti-GCC binding proteins and immunotherapies, such as CAR-T therapies that specifically target GCC. [Prior art documents] [Non-patent literature]

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

[0006] 3. Summary In one aspect, the present specification provides an anti-GCC single domain antibody (sdAb), which comprises: (1) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in SEQ ID NO: 26; (2) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in SEQ ID NO: 27; (3) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in SEQ ID NO: 28; (4) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in SEQ ID NO: 29; (5) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in SEQ ID NO: (6) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 31, (7) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 32, (8) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 33, (9) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 34, (10) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: (11) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 36, (12) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 37, (13) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 38, (14) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO:(14) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in SEQ ID NO: 39, (15) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in SEQ ID NO: 40, or (16) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in SEQ ID NO: 41. In some embodiments, CDR1, CDR2, or CDR3 is defined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, Contact numbering, or any combination thereof.

[0007] In one aspect, the present disclosure provides anti-GCC sdAbs, which include: (1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17; (2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 18; (3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 3, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19; (4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 20; (5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a CDR2 comprising the amino acid sequence of SEQ ID NO: (6) a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (7) a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23; (8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 24; or (9) a CDR1 comprising the amino acid sequence of SEQ ID NO: 8, a CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25.

[0008] In some embodiments, the anti-GCC sdAb provided herein further comprises one or more FR regions set forth in any one of SEQ ID NOs: 26-41.

[0009] In some embodiments, provided herein is an anti-GCC sdAb comprising the amino acid sequence of any one of SEQ ID NOs: 26 to 41. In some embodiments, provided herein is an anti-GCC sdAb comprising or consisting of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 26 to 41.

[0010] In some embodiments, the anti-GCC sdAb is a camelid sdAb. In some embodiments, the anti-GCC sdAb is a humanized sdAb.

[0011] In some embodiments, the anti-GCC sdAb is genetically fused or chemically conjugated to the agent.

[0012] In another aspect, the description provides a fusion protein comprising an anti-GCC sdAb described herein and an Fc region (e.g., human IgG1 Fc or mouse IgG1 Fc). In some embodiments, the Fc region is mouse IgG1 Fc. In some embodiments, the mouse IgG1 Fc comprises the amino acid sequence of SEQ ID NO: 67. In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 42-44.

[0013] In another aspect, the description provides a chimeric antigen receptor (CAR) comprising: (a) an extracellular antigen-binding domain comprising one or more anti-GCC sdAbs according to the description; (b) a transmembrane domain; and (c) an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain comprises one anti-GCC sdAb.

[0014] In some embodiments, the extracellular antigen-binding domain of the CAR further comprises one or more additional antigen-binding domains.

[0015] In some embodiments, the antigen binding domains are fused to each other via a peptide linker.

[0016] In some embodiments, the peptide linker is about 50 amino acids or less in length.

[0017] In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the transmembrane domain is derived from CD8α.

[0018] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell, hi some embodiments, the primary intracellular signaling domain is derived from CD3ζ.

[0019] In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the costimulatory signaling domain is derived from CD137.

[0020] In some embodiments, a CAR according to the present disclosure further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α.

[0021] In some embodiments, a CAR according to the present disclosure further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is from CD8α.

[0022] In some embodiments, the present specification provides a chimeric antigen receptor (CAR), which comprises (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-60, or (ii) an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of SEQ ID NOs: 45-60.

[0023] In yet another aspect, the description provides an isolated nucleic acid, comprising a nucleic acid sequence encoding an anti-GCC sdAb according to the description, a fusion protein according to the description, or a CAR according to the description, or a fragment thereof. In some embodiments, the isolated nucleic acid further comprises a nucleic acid sequence encoding a chimeric receptor, wherein the chimeric receptor comprises a TGFβR and an IL23R. In some embodiments, the chimeric receptor comprises the amino acid sequence of any one of SEQ ID NOs: 64-66.

[0024] In yet another aspect, the description provides a vector comprising an isolated nucleic acid according to the description.

[0025] In yet another aspect, the description provides an engineered immune cell, which comprises a CAR described herein, an isolated nucleic acid described herein, a chimeric receptor and / or a vector described herein. In some embodiments, the engineered immune cell is an engineered immune effector cell. In some embodiments, the engineered immune cell is a T cell, an NK cell, a peripheral blood mononuclear cell (PBMC), a hematopoietic stem cell, a pluripotent stem cell, an embryonic stem cell, or any combination thereof. In some embodiments, the engineered immune cell comprises the amino acid sequence of any one of SEQ ID NOs: 45-60 and 61-63.

[0026] In yet another aspect, the present disclosure provides a method for producing an engineered immune cell, the method comprising introducing a vector according to the present disclosure into a cell.

[0027] In yet another aspect, the description provides a pharmaceutical composition comprising an anti-GCC sdAb according to the present invention, an isolated nucleic acid according to the present invention, a vector according to the present invention, or an engineered immune cell according to the present invention, and a pharmaceutically acceptable excipient.

[0028] In yet another aspect, the description provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of an anti-GCC sdAb according to the description, an engineered immune cell according to the description, or a pharmaceutical composition according to the description. In some embodiments, the disease or disorder is a GCC-associated disease or disorder. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is selected from the group consisting of gastrointestinal cancer, colorectal cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, small intestine cancer, pancreatic cancer, and liver cancer. In some embodiments, the disease or disorder is colorectal cancer. [Brief explanation of the drawings]

[0029] 4. Brief description of the drawings [Figure 1] The CAR expression levels of CAR-T cells are shown. All CAR expression levels are above 40%. UnT refers to T cells that were not transduced with a CAR, which served as a control. [Figure 2A] Figures 2A-2F show the in vitro cytotoxicity of CAR-T cells against GCC-positive cell lines (T84.Luc and SW948.Luc). All CAR-T cells effectively demonstrated cytotoxicity against GCC-positive cells. UnT refers to T cells that were not transduced with a CAR, serving as a control. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 2F] See legend to Figure 2A. [Figure 3A] Figures 3A-3F show the IFNγ and TNFα release of CAR-T cells cocultured with target cells. When cocultured with target cells, the IFNγ and TNFα release of all CAR-T cells was upregulated, whereas that of UnT cells was maintained at baseline levels. UnT refers to T cells not transduced with a CAR, which served as a control. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 3F] See legend to Figure 3A. [Figure 4A] Figures 4A-4C show the in vivo antitumor efficacy of GCC CAR-T cells in a subcutaneous xenograft model of SW948.Luc cells. Mice were evaluated to monitor tumor growth by changes in tumor volume (Figure 4A). At a dose of 0.3 × 10 CAR+T cells / mouse, A2322, C0694, and C0708 CAR-T cells were able to significantly reduce tumor growth. 14 days after injection, CAR-T cells were expanded in mouse peripheral blood (Figure 4B). Mouse body weight was not affected by CAR-T cell injection (Figure 4C). UnT refers to T cells not transduced with a CAR, serving as a control. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 5A]Figures 5A-5D show the binding characteristics of humanized anti-GCC VHH antibodies. Human anti-GCC monoclonal antibody 5F9 (WO 2011050242 A1) served as a reference control. The humanized VHH antibodies showed dose-dependent binding to HEK293T.huGCC.Luc (human GCC) cells and HEK293T.rGCC.Luc (rhesus GCC) cells, but not to HEK293T.mGCC.Luc (mouse GCC) cells or HEK293T.Luc cells. Therefore, these humanized anti-GCC VHH antibodies cross-reacted with human and non-human primate but not mouse targets. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6] The CAR expression levels of CAR-T cells are shown. UnT refers to T cells that were not transduced with a CAR, which served as a control. 5F9 CAR-T cells served as the reference. [Figure 7A] Figures 7A-7H show the results of in vitro cytotoxicity assays of humanized GCC CAR-T cells and their parental CAR-T cells against GCC-positive cell lines (T84.Luc and SW948.Luc) and GCC-negative cell lines (A549.Luc and HEK293T.Luc). 5F9 CAR-T served as a reference control. All CAR-T cells showed specific cytotoxicity against GCC-positive cells but not against GCC-negative cells. UnT refers to T cells that were not transduced with a CAR, which served as a control. 5F9 CAR-T cells served as a reference. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 7F] See legend to Figure 7A. [Figure 7G] See legend to Figure 7A. [Figure 7H] See legend to Figure 7A. [Figure 8A] Figures 8A-8J show the IFNγ release of humanized GCC CAR-T cells and their parental CAR-T cells cocultured with GCC-positive cell lines (T84.Luc and SW948.Luc) and GCC-negative cell lines (A549.Luc and HEK293T.Luc). When cocultured with GCC-positive target cells, the IFNγ release of all CAR-T cells was upregulated, whereas that of UnT cells was maintained at baseline levels. UnT refers to T cells not transduced with a CAR, which served as a control. 5F9 CAR-T cells served as a reference. [Figure 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [Figure 8D] See legend to Figure 8A. [Figure 8E] See legend to Figure 8A. [Figure 8F] See legend to Figure 8A. [Figure 8G] See legend to Figure 8A. [Figure 8H] See legend to Figure 8A. [Figure 8I] See legend to Figure 8A. [Figure 8J] See legend to Figure 8A. [Figure 9A]Figures 9A-9I show the in vivo antitumor efficacy of humanized GCC CAR-T cells in a subcutaneous xenograft model implanted with SW948.Luc cells. Mice were evaluated to monitor tumor growth by changes in tumor volume (Figures 9A-9C). At a dose of 0.3 x 10 CAR+T cells / mouse, A2322, C0694, C0708, and their humanized CAR-T cells were able to significantly reduce tumor growth, with superior efficacy compared to 5F9 CAR-T. 14 days after injection, CAR-T cells were expanded in mouse peripheral blood (Figures 9D-9F). Following CAR-T cell injection, mice maintained normal body weight (Figures 9G-9I). UnT refers to untransduced T cells, which served as a control. 5F9 CAR-T cells served as a baseline. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 9E] See legend to Figure 9A. [Figure 9F] See legend to Figure 9A. [Figure 9G] See legend to Figure 9A. [Figure 9H] See legend to Figure 9A. [Figure 9I] See legend to Figure 9A. [Figure 10] The structures of naked CAR and TF23 armored CAR are shown. SP refers to the signal peptide. TM refers to the transmembrane domain. Binding protein refers to the extracellular antigen-binding domain of tumor-associated antigen or binding protein (i.e., anti-GCC VHH antibody). TF23 refers to the armored domain, which converts TGFβ inhibitory signals into IL-23 activating signals. ECD refers to the extracellular domain. ICD refers to the intracellular domain. [Figure 11] The CAR expression levels of CAR-T cells and their TF23 armored CAR structures are shown. UnT refers to T cells that were not transduced with a CAR, which served as a control. 5F9 CAR-T cells served as a reference. [Figure 12A] Figures 12A-12F show the results of in vitro cytotoxicity assays of armored TF23 GCC CAR-T cells and their naked counterparts against GCC-positive cell lines (SW948.Luc and LS1034.Luc). All CAR-T cells exhibited specific cytotoxicity against GCC-positive cells, and C0708H2TF23 exhibited slightly enhanced cytotoxicity. UnT refers to untransduced T cells, which served as a control. 5F9 CAR-T cells served as a reference. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 12E] See legend to Figure 12A. [Figure 12F] See legend to Figure 12A. [Figure 13A] Figures 13A-13H show the results of in vitro cytotoxicity assays of CAR-T cells against GCC-positive cell lines after five rounds of stimulation in a re-challenge assay. After continued antigen stimulation, the cytotoxic potential of 5F9 and naked GCC CAR-T cells decreased, while armored GCC CAR-T cells still had strong cytotoxic potential against SW948.Luc and LS1034.Luc cell lines. UnT refers to T cells that were not transduced with a CAR, serving as a control. 5F9 CAR-T cells served as a reference. [Figure 13B] See legend to Figure 13A. [Figure 13C] See legend to Figure 13A. [Figure 13D] See legend to Figure 13A. [Figure 13E] See legend to Figure 13A. [Figure 13F] See legend to Figure 13A. [Figure 13G] See legend to Figure 13A. [Figure 13H]See legend to Figure 13A. [Figure 14A] Figures 14A-14I show the in vivo antitumor efficacy of Armored TF23 GCC CAR-T cells in a xenograft model engrafted with LS1034 cells. Mice were evaluated to monitor tumor growth by changes in tumor volume (Figures 14A-14C). At low doses (0.1 × 10 CAR+T cells / mouse), Armored GCC CAR-T cells (A2322H2TF23, C0694H2TF23, and C0708H2TF23) significantly inhibited tumor growth, whereas naked GCC CAR-T cells were unable to inhibit tumor growth. 14 days after injection, Armored CAR-T cells expanded in the mouse peripheral blood, but naked CAR-T cells were not detected in the peripheral blood (Figures 14D-14F). Following CAR-T cell injection, mice maintained normal body weight (Figures 14G-14I). UnT refers to T cells that were not transduced with a CAR, which served as a control. 5F9 CAR-T cells served as a reference. [Figure 14B] See legend to Figure 14A. [Figure 14C] See legend to Figure 14A. [Figure 14D] See legend to Figure 14A. [Figure 14E] See legend to Figure 14A. [Figure 14F] See legend to Figure 14A. [Figure 14G] See legend to Figure 14A. [Figure 14H] See legend to Figure 14A. [Figure 14I] See legend to Figure 14A. DETAILED DESCRIPTION OF THE INVENTION

[0030] 5. Detailed Description The present disclosure is based in part on novel antibodies that bind to GCC, chimeric antigen receptors that bind to GCC, or engineered cells that contain and / or co-express the chimeric receptors, and their improved properties.

[0031] 5.1. Definition The techniques and procedures described or referenced herein generally include those that are well understood and / or commonly employed by those skilled in the art using conventional methods, such as, for example, the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed. 2001), Current Protocols in Molecular Biology (eds. Ausubel et al., 2003), Therapeutic Monoclonal Antibodies: From Bench to Clinic (eds. An, 2009), Monoclonal Antibodies: Methods and Protocols (eds. Albitar, 2010), and Antibody Engineering, Vols. 1 and 2 (eds. Kontermann and Dubel, 2nd ed. 2010). Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. For purposes of interpretation of this specification, the following terminology applies, and where appropriate, terms used in the singular also include the plural, and vice versa. In the event that any explanation of a term provided herein contradicts any document incorporated herein by reference, the explanation of the term provided below shall prevail.

[0032] The terms "antibody," "immunoglobulin," or "Ig" may be used interchangeably herein and are used in the broadest sense, specifically encompassing, for example, monoclonal antibodies (including agonist, antagonist, neutralizing, full-length, or intact monoclonal antibodies), antibody compositions with multiepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain antibodies, and fragments thereof (e.g., domain antibodies), as described below. Antibodies can be human, humanized, chimeric, and / or affinity-matured antibodies, as well as antibodies from other species (e.g., mouse, rabbit, llama, etc.). The term "antibody" is intended to include polypeptide products of B cells in the immunoglobulin family, which have the ability to bind to a specific molecular antigen and are composed of two identical pairs of polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), with the amino-terminal portion of each chain containing a variable region of about 100 to about 130 or more amino acids and the carboxy-terminal portion of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck, ed., 2nd ed. 1995) and Kuby, Immunology (3rd ed. 1997). Antibodies further include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, antibodies including those derived from Camelidae species (e.g., llamas or alpacas) or humanized variants thereof, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigen-binding fragments) of any of the above. These functional fragments refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which they are derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fvs (scFvs) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab') fragments, F(ab') fragments, disulfide-linked Fvs (dsFvs), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies.In particular, antibodies according to the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as molecules containing an antigen-binding domain or site that binds to an antigen (e.g., one or more CDRs of an antibody). For such antibody fragments, see, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (ed. Myers, 1995); Huston et al., 1993, Cell Biophysics 22:189-224; Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2nd ed. 1990). An antibody according to the present invention can be of any class of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibody can be an agonist antibody or an antagonist antibody. The antibody can be neither an agonist nor an antagonist.

[0033] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In some embodiments, the antigen is associated with a cell, e.g., present on or within a cell.

[0034] An "intact" antibody is one that comprises an antigen-binding site and a CL and at least heavy chain constant regions CH1, CH2, and CH3. The constant region may comprise a human constant region or an amino acid sequence variant thereof. In some embodiments, an intact antibody has one or more effector functions.

[0035] A "single-chain Fv" (also abbreviated as "sFv" or "scFv") is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. In some embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Plückthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0036] The term "heavy chain-only antibody" or "HCAb" includes functional antibodies that contain heavy chains but lack the light chains typically found in four-chain antibodies. For example, camelids (e.g., camels, llamas, and alpacas) are known to produce HCAbs.

[0037] As used herein, "single domain antibody" or "sdAb" refers to a single monomeric variable antibody domain and is capable of binding to an antigen (e.g., a single domain antibody that binds to GCC). Single domain antibodies comprise a VHH domain as described herein. Examples of single domain antibodies include, but are not limited to, antibodies that naturally lack light chains, such as antibodies from Camelidae species (e.g., llamas), single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies (e.g., VHH domains) may be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and bovine. For example, as described herein, single domain antibodies may be derived from antibodies produced from Camelidae species, such as camel, llama, dromedary, alpaca, and guanaco. Other species outside of Camelidae can produce heavy chain antibodies that naturally lack light chains, and VHHs derived from such other species are within the scope of this disclosure. In some embodiments, a single domain antibody (e.g., a VHH domain) according to the present invention has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies can be genetically fused or chemically conjugated to another molecule (e.g., an agent) as described herein. A single domain antibody can be part of a larger binding molecule (e.g., a multispecific antibody or a chimeric antigen receptor).

[0038] The terms "binding" or "binding" refer to interactions between molecules, including, for example, forming a complex. The interaction may be a non-covalent interaction, including, for example, hydrogen bonding, ionic bonding, hydrophobic interactions, and / or van der Waals interactions. A complex may further include the association of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the overall non-covalent interactions between a single antigen-binding site on an antibody and a single epitope on a target molecule (e.g., antigen) is the affinity of the antibody or functional fragment for that epitope. The dissociation rate (koff ) and association rate (k on ) and the ratio (k off / k on ) is the dissociation constant K D and is inversely proportional to affinity. D The lower the value, the higher the affinity of the antibody. D The value of varies depending on the antibody-antigen complex, and k on and k off The dissociation constant K of the antibody according to the present invention depends on D can be determined using any method herein or any other method known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When a complex antigen containing multiple repeating antigenic determinants (e.g., a multivalent antigen) comes into contact with an antibody containing multiple binding sites, interaction of the antibody with the antigen at one site increases the likelihood of reaction at a second site. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity.

[0039] In the context of the binding molecules described herein, terms such as "binds to," "specifically binds to," and similar terms are also used interchangeably herein to refer to binding molecules of an antigen-binding domain that specifically binds to an antigen (e.g., a polypeptide). Binding molecules or antigen-binding domains that bind to or specifically bind to an antigen can be identified, for example, by immunoassays, Octet®, Biacore®, or other techniques well known to those skilled in the art. In some embodiments, a binding molecule or antigen-binding domain binds to or specifically binds to an antigen if it binds to the antigen with higher affinity than any cross-reactive antigen, as measured using laboratory techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Typically, a specific or selective response will be at least twice the background signal or noise and may be more than 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Paul, ed., 2nd ed. 1989). In some embodiments, the extent to which a binding molecule or antigen-binding domain binds to a "non-target" protein is about 10% of the binding of the binding molecule or antigen-binding domain to its specific target antigen, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis or RIA. A binding molecule or antigen-binding domain that binds to an antigen includes, for example, a binding molecule or antigen-binding domain that can bind to an antigen with sufficient affinity so that the binding molecule can be used, for example, as a therapeutic and / or diagnostic agent targeting the antigen. In some embodiments, a binding molecule or antigen-binding domain that binds to an antigen has a dissociation constant (K) of 1 μM, 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM or less. DIn some embodiments, the binding molecule or antigen-binding domain binds to an antigenic epitope that is conserved in antigens from different species.

[0040] In some embodiments, a binding molecule or antigen-binding domain may comprise a "chimeric" sequence, in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567, and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55). Chimeric sequences may also include humanized sequences.

[0041] In some embodiments, binding molecules or antigen-binding domains may comprise portions of "humanized" forms of non-human (e.g., camelid, murine, non-human primate) antibodies, which comprise sequences from a human immunoglobulin (e.g., a recipient antibody) in which native CDR residues are replaced by residues from a corresponding CDR of a non-human species (e.g., a donor antibody) such as camelid, mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some cases, one or more FR region residues of the human immunoglobulin sequence are replaced by corresponding non-human residues. Note that humanized antibodies may also comprise residues that are not found in the recipient or donor antibody. These modifications are made to further improve antibody performance. The humanized antibody heavy or light chain may comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In some embodiments, a humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature 332:323-29 (1988); Presta, Curr. Op. Struct. Biol. 2:593-96 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992); U.S. Patent Nos. 6,800,738, 6,719,971, 6,639,055, 6,407,213, and 6,054,297.

[0042] In some embodiments, a binding molecule or antigen-binding domain may comprise a portion of a "fully human antibody" or "human antibody," where these terms are used interchangeably herein and refer to an antibody comprising a human variable region and, for example, a human constant region. The binding molecule may comprise an antibody sequence. In specific embodiments, the term refers to an antibody comprising variable and constant regions of human origin. In some embodiments, a "fully human" antibody may further encompass antibodies that bind to a polypeptide and are encoded by nucleic acid sequences, which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequences. The term "fully human antibody" includes antibodies having variable and constant regions corresponding to human germline immunoglobulin sequences, e.g., as described by Kabat et al. (See, e.g., Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242). A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by humans and / or produced using any of the techniques for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991)) and yeast display libraries (Chao et al., Nature Protocols 1:755-68 (2006)).Methods that can be used to prepare human monoclonal antibodies are described in Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985), Boerner et al., J. Immunol. 147(1):86-95 (1991), and van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001). Human antibodies can also be prepared by administering antigen to transgenic animals (e.g., mice) that have been modified to produce such antibodies in response to antigen challenge, but in which the endogenous gene locus has been disabled (e.g., Jakobovits, Curr. Opin. Biotechnol. 6(5):561-66 (1995), Bruggemann and Taussing, Curr. Opin. Biotechnol. 8(4):455-58 (1997), and XENOMOUSE). TM (See U.S. Patent Nos. 6,075,181 and 6,150,584 for related technology.) For human antibodies produced by human B cell hybridoma technology, see further, e.g., Li et al., Proc. Natl. Acad. Sci. USA 103:3557-62 (2006).

[0043] In some embodiments, the binding molecule or antigen-binding domain may comprise a portion of a "recombinant human antibody," which term includes human antibodies prepared, expressed, produced, or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from animals (e.g., mice or cows) transgenic and / or transchromosomic for human immunoglobulin genes (see, e.g., Taylor, LD et al., Nucl. Acids Res. 20:6287-6295 (1992)), or antibodies prepared, expressed, produced, or isolated by any other method involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies can have variable and constant regions derived from human germline immunoglobulin sequences (see, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242). However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when transgenic animals of human Ig sequences are used, in vivo somatic mutagenesis) so that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and closely related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo.

[0044] In some embodiments, a binding molecule or antigen-binding domain may comprise a portion of a "monoclonal antibody," a term used herein to refer to an antibody obtained from a population of substantially homogeneous antibodies, each antibody of which is identical except for, for example, possible naturally occurring mutations or well-known post-translational modifications (e.g., amino acid isomerization or deamidation, methionine oxidation, or asparagine or glutamine deamidation) that may be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on an antigen. In specific embodiments, a "monoclonal antibody," as used herein, is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to any particular method for producing the antibody. For example, monoclonal antibodies that can be used in the present disclosure may be prepared by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be produced in bacterial or eukaryotic animal or plant cells using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222:581-97 (1991). Other methods for preparing clonal cell lines and the monoclonal antibodies they express are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al., eds., 5th ed. 2002).

[0045] A typical four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the four-chain unit is usually approximately 150,000 daltons. Each L chain is linked to an H chain via one covalent disulfide bond, while the two H chains are linked to each other via one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus and a constant domain (CL) at its other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain and heavy-chain variable domains. The pairing of a VH and a VL together forms a single antigen-binding site. For the structural characteristics of different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (ed. Stites et al., 8th ed. 1994) and Immunobiology (ed. Janeway et al., 5th ed. 2001).

[0046] The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. A conventional IgG typically contains two Fab regions, each in one of the two arms of the Y-shaped IgG structure. Each Fab region typically consists of one variable region and one constant region from each of the heavy and light chains. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL regions in the Fab region can be arranged in various ways to confer antigen-binding ability according to the present disclosure. For example, similar to the Fab region of a conventional IgG, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on a single polypeptide. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in different orders, as described in more detail in the following sections.

[0047] The terms "variable region," "variable domain," "V region," or "V domain" refer to a portion of an antibody light or heavy chain, typically located at the amino terminus of the light or heavy chain, approximately 120-130 amino acids in length for heavy chains and approximately 100-110 amino acids in length for light chains, that is responsible for the binding and specificity of each particular antibody to its specific antigen. The variable region of a heavy chain may be referred to as "VH." The variable region of a light chain may be referred to as "VL." The term "variable" refers to the fact that some segments of the variable region in an antibody exhibit significant sequence variability. The V region mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed within the 110 amino acids of the variable region. Instead, V regions are composed of less variable (e.g., relatively invariant) segments of about 15-30 amino acids called framework regions (FRs), separated by shorter regions of greater (e.g., extreme) variability called "hypervariable regions," each about 9-12 amino acids in length. The heavy and light chain variable regions each contain four FRs that primarily employ a β-sheet structure, connected by three hypervariable regions that form loops and, in some cases, part of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRs and, together with the hypervariable regions of the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest (5th ed., 1991)). The constant region is not directly involved in binding of an antibody to an antigen, but exhibits various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable region varies widely in sequence among different antibodies. In a specific embodiment, the variable region is a human variable region.

[0048] The terms "variable region residue numbering according to Kabat" or "amino acid position numbering according to Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable regions of the antibody sequences in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening or insertion of variable domain FRs or CDRs. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 (residue 52a according to Kabat) and three inserted residues after residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues in a given antibody can be determined by alignment of the antibody sequence with the "standard" Kabat numbered sequence at the regions of homology. When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system (e.g., Kabat et al., supra) is typically used. When referring to residues in an immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" (e.g., the EU index reported in Kabat et al., supra) is usually used. The "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Other numbering systems are described, for example, by AbM, Chothia, Contact, IMGT, and AHon.

[0049] The term "heavy chain," when used in reference to antibodies, refers to a polypeptide chain of approximately 50 to 70 kDa, in which the amino-terminal portion contains a variable region of approximately 120 to 130 or more amino acids, and the carboxy-terminal portion contains a constant region. The amino acid sequence of the heavy chain constant region can be one of five different types (e.g., isotypes), designated alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). Individual heavy chains vary in size: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these different types of heavy chains each produce one of the five well-known classes (e.g., isotypes) of antibodies: IgA, IgD, IgE, IgG, and IgM, including the four subclasses of IgG: IgG1, IgG2, IgG3, and IgG4.

[0050] The term "light chain" when used in reference to an antibody refers to a polypeptide chain of approximately 25 kDa, in which the amino-terminal portion contains a variable region of about 100 to about 110 or more amino acids, and the carboxy-terminal portion contains a constant region. The light chain is approximately 211 to 217 amino acids in length. There are two different types of amino acid sequences based on the constant domain, called kappa (κ) or lambda (λ).

[0051] As used herein, the terms "hypervariable region," "HVR," "complementarity-determining region," and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of an immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of an antibody VL β-sheet framework. CDR1, CDR2, and CDR3 in a VH domain are also referred to as HCDR1, HCDR2, and HCDR3, respectively. CDR1, CDR2, and CDR3 in a VL domain are also referred to as LCDR1, LCDR2, and LCDR3, respectively. Thus, CDRs are variable region sequences interspersed within framework region sequences.

[0052] CDR regions are well known to those skilled in the art and are defined by well-known numbering systems. For example, Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra; Nick Deschacht et al., J Immunol 2010; 184:5696-5704). Chothia instead refers to the position of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used in Oxford Molecular's AbM antibody modeling software (see, e.g., Antibody Engineering, Vol. 2 (eds. Kontermann and Dubel, 2nd ed. 2010)). The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Another universal numbering system that has been developed and widely adopted is the ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is a comprehensive information system specialized in immunoglobulins (IGs), T cell receptors (TCRs), and major histocompatibility complexes (MHCs) of humans and other vertebrates. As used herein, CDRs refer to amino acid sequences and positions within a light or heavy chain. Because the "positions" of CDRs within the structure of immunoglobulin variable domains are conserved across species and reside in structures called loops, a numbering system that aligns variable domain sequences according to structural features allows CDR and framework residues to be easily identified.This information can be used to graft and replace CDR residues from one species' immunoglobulin onto an acceptor framework, usually from a human antibody. Honegger and Pluckthun, J. Mol. Biol. 309: 657-70 (2001), developed an additional numbering system (AHon). The correspondence between numbering systems, including, for example, Kabat numbering and the IMGT proprietary numbering system, is well known to those skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). Residues from these hypervariable regions or CDRs are illustrated in Table 1 below.

[0053] Table 1: Exemplary CDRs with various numbering systems TIFF2025533779000001.tif87161

[0054] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms "CDR" and "complementarity-determining region" of a given antibody or region thereof (e.g., a variable region), and each CDR of an antibody or region thereof (e.g., CDR-H1, CDR-H2), should be understood to encompass the complementarity-determining region defined by any of the above known schemes. In some cases, a scheme for identifying one or more specific CDRs is specified, such as CDRs defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the specific amino acid sequence of the CDR is provided. It should be noted that a CDR region may be defined by any combination of various numbering systems, such as a combination of the Kabat and Chothia numbering systems, a combination of the Kabat and AbM numbering systems, or a combination of the Kabat and IMGT numbering systems. Thus, a term such as "CDR1 as described in a particular VH" includes, but is not limited to, any CDR1 defined by the above exemplary CDR numbering systems. Given a variable region (eg, VH or VL), one of skill in the art will understand that the CDRs within that region may be defined by various numbering systems or combinations thereof.

[0055] The hypervariable regions may include "extended hypervariable regions" such as 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH.

[0056] The term "constant region" or "constant domain" refers to the carboxy-terminal portions of the light and heavy chains, which are not directly involved in binding an antibody to an antigen but exhibit various effector functions, such as interacting with Fc receptors. The term refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence than other portions of the immunoglobulin, i.e., the variable region, and which contains the antigen-binding site. The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

[0057] The term "framework" or "FR" refers to variable region residues located on either side of a CDR. FR residues are present, for example, in chimeric, humanized, human, domain, diabodies, linear, and bispecific antibodies. FR residues are hypervariable region or variable domain residues other than CDR residues.

[0058] The term "Fc region" herein is intended to define the C-terminal region of an immunoglobulin heavy chain and includes, for example, native-sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226 or Pro230 to its carboxy-terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition can include antibodies with all K447 residues removed, antibodies with K447 residues removed, and antibodies with a mixture of antibodies containing and not containing the K447 residue. A "functional Fc region" possesses the "effector functions" of a native-sequence Fc region. Exemplary "effector functions" include C1q binding (CDC), Fc receptor binding, ADCC, phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and the like. Such effector functions typically require combining the Fc region with a binding region or domain (e.g., an antibody variable region or domain) and can be assessed using a variety of assays well known to those of skill in the art. A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification (e.g., substitution, addition, or deletion). In some embodiments, the variant Fc region comprises at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, or from about 1 to about 5 amino acid substitutions in the native-sequence Fc region or the Fc region of a parent polypeptide. The variant Fc region herein may have at least about 80% homology to a native sequence Fc region and / or the Fc region of a parent polypeptide, or at least about 90% homology thereto, for example, at least about 95% homology thereto.

[0059] As used herein, "epitope" is a term of art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody) can specifically bind. An epitope may be a linear epitope or a conformational, non-linear, or discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope may be consecutive amino acids of a polypeptide (a "linear" epitope), or an epitope may include amino acids from two or more non-contiguous regions of a polypeptide (a "conformational," "non-linear," or "discontinuous" epitope). Those skilled in the art will generally understand that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a set of amino acids, regardless of whether they are folded into a native three-dimensional protein structure. In other embodiments, the binding molecule requires that the amino acid residues that make up the epitope adopt a particular conformation (eg, a bend, twist, turn, or fold) in order to recognize and bind to the epitope.

[0060] "Percent (%) amino acid sequence identity" and "homology" of peptide, polypeptide, or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a particular peptide or polypeptide sequence, after sequence alignment and introduction of gaps, if necessary, to achieve the maximum percent sequence identity, and after any conservative substitutions are not considered as part of the sequence identity. Alignment and percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0061] The term "specificity" refers to the selective recognition of a particular epitope of an antigen by an antigen-binding protein (e.g., a CAR or an antibody). For example, natural antibodies are monospecific. As used herein, the term "multispecific" indicates that an antigen-binding protein (e.g., a CAR or an antibody) has two or more antigen-binding sites, where at least two bind to different antigens. As used herein, "bispecific" indicates that an antigen-binding protein (e.g., a CAR or an antibody) has two different antigen-binding specificities. As used herein, a "monospecific" CAR refers to an antigen-binding protein (e.g., a CAR or an antibody) with one or more binding sites, where each binding site binds to the same antigen.

[0062] As used herein, the term "valent" refers to the presence of a given number of binding sites on an antigen-binding protein (e.g., a CAR or antibody). For example, a natural or full-length antibody has two binding sites and is bivalent. Thus, the terms "trivalent," "tetravalent," "pentavalent," and "hexavalent" refer to the presence of two, three, four, five, and six binding sites on an antigen-binding protein (e.g., a CAR or antibody), respectively.

[0063] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transfer one or more antigens into immune effector cells, such as T cells. Some CARs are also called "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." In some embodiments, a CAR comprises an extracellular antigen-binding domain specific for one or more antigens (e.g., tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptor. "CAR-T cell" refers to a T cell that expresses a CAR.

[0064] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. A polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. These terms further encompass amino acid polymers that have been modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. The definition further includes, for example, polypeptides containing one or more amino acid analogs, including, but not limited to, unnatural amino acids, and other modifications known in the art. It should be understood that, because the polypeptides of the present disclosure may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments, a "polypeptide" can exist as a single chain or two or more associated chains.

[0065] As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase or a synthetic reaction. A polynucleotide may also include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to a short, usually single-stranded, synthetic polynucleotide, usually (but not necessarily) less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Cells producing the binding molecules of the present disclosure may include parent hybridoma cells, as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibody has been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end, and the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of addition of nascent RNA transcripts from 5' to 3' is referred to as the transcription direction. The sequence region 5' of the 5' end of the RNA transcript that has the same sequence on the DNA strand as the RNA transcript is referred to as the "upstream sequence," and the sequence region 3' of the 3' end of the RNA transcript that has the same sequence on the DNA strand as the RNA transcript is referred to as the "downstream sequence."

[0066] An "isolated nucleic acid" is a nucleic acid (e.g., RNA, DNA, or mixture of nucleic acids) that is substantially separated from other genomic DNA sequences and proteins or complexes, such as ribosomes and polymerases, that naturally accompany the native sequence. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the nucleic acid molecule's natural source. Note that an "isolated" nucleic acid molecule, e.g., a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In specific embodiments, one or more nucleic acid molecules encoding the antibodies described herein are isolated or purified. The terms encompass nucleic acid sequences that have been removed from their naturally occurring environment and include recombinant or cloned DNA isolates and chemically synthesized analogs or heterologous biologically synthesized analogs. A substantially pure molecule may include isolated forms of the molecule. Specifically, an "isolated" nucleic acid molecule encoding a CAR or antibody, as described herein, is a nucleic acid molecule that is identified and isolated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced.

[0067] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns, to the extent that the nucleotide sequence encoding the protein may in some forms contain one or more introns.

[0068] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences applicable to prokaryotes include, for example, promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0069] As used herein, the term "operably linked" and similar phrases (e.g., genetically fused), when used to refer to nucleic acids or amino acids, refers to the operable linkage of nucleic acid sequences or amino acid sequences, respectively, which are placed in a functional relationship with each other. For example, operably linking a promoter, enhancer element, open reading frame, 5' and 3' UTRs, and terminator sequence results in the correct production of a nucleic acid molecule (e.g., RNA). In some embodiments, operably linked nucleic acid elements cause transcription of the open reading frame, ultimately resulting in the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operably linked peptide is one whose functional domains are positioned appropriately from each other to confer their intended function.

[0070] The term "vector" refers to a substance for carrying or containing a nucleic acid sequence, such as a nucleic acid sequence encoding a binding molecule (e.g., an antibody) described herein, for introduction into a host cell. Applicable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may contain a selection sequence or a marker capable of stable integration into a host cell chromosome. The vector may also contain one or more selection marker genes and appropriate expression control sequences. The included selection marker genes may, for example, provide resistance to antibiotics or toxins, compensate for nutrient deficiencies, or supply important nutrients missing from the culture medium. Expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as known in the art. When two or more nucleic acid molecules are to be coexpressed (e.g., antibody heavy and light chains or antibody VH and VL), the two nucleic acid molecules may be inserted, for example, into a single expression vector or separate expression vectors. For single vector expression, the encoding nucleic acids can be operably linked to a common expression control sequence or can be linked to different expression control sequences, e.g., one inducible promoter and one constitutive promoter. Introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. These methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblots used to detect gene product expression, or other suitable analytical methods to test for expression of the introduced nucleic acid sequence or its corresponding gene product. One skilled in the art will understand that the nucleic acid molecule is expressed in sufficient amounts to produce the desired product, and will further understand that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0071] As used herein, the term "host" refers to an animal, for example, a mammal (e.g., a human).

[0072] As used herein, the term "host cell" refers to a particular target cell that can be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations or due to integration of the nucleic acid molecule into the host cell genome.

[0073] As used herein, the term "autologous" refers to any material derived from the same individual, where the material is later reintroduced into that individual.

[0074] "Allogeneic" refers to a graft derived from a different individual of the same species.

[0075] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing foreign nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. Such cells include the primary target cell and its progeny.

[0076] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in animals, and more particularly, in humans.

[0077] "Excipient" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption enhancers, antioxidants, adhesives, buffers, carriers, coating agents, colorants, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavoring agents, humectants, lubricants, fragrances, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizers, wetting agents, and mixtures thereof. The term "excipient" may also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), or vehicle.

[0078] In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; proteins such as low molecular weight (e.g., less than about 10 amino acid residues) polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th ed., 1990).

[0079] In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other components of the pharmaceutical formulation and suitable for contact with the tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications, consistent with a reasonable benefit-to-risk ratio. See, e.g., Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th Edition; Rowe et al., eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash, eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd Edition; Gibson, ed., CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, a pharmaceutically acceptable excipient is nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. In some embodiments, a pharmaceutically acceptable excipient is an aqueous pH-buffered solution.

[0080] In some embodiments, the excipient is a sterile liquid, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is an exemplary excipient when the composition (e.g., pharmaceutical composition) is administered intravenously. Saline solutions, aqueous glucose solutions, and glycerin solutions can also be used as liquid excipients, particularly for injectable solutions. The excipient may further include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, ethylene glycol, water, ethanol, or the like. The composition may further contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The composition may take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, or the like. Oral compositions may include formulations and standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

[0081] A composition containing a pharmaceutical compound may include, for example, a binding molecule (eg, an antibody) in isolated or purified form and a suitable amount of an excipient.

[0082] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic molecule, including an antibody or agent and an antibody or pharmaceutical composition according to the present invention, sufficient to bring about a desired result.

[0083] The terms "subject" and "patient" may be used interchangeably. As used herein, in some embodiments, a subject is a mammal, such as a non-primate or a primate (e.g., a human). In specific embodiments, a subject is a human. In one embodiment, a subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In another embodiment, a subject is a mammal, e.g., a human, at risk of developing a disease or disorder.

[0084] "Administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body into the body of a patient, for example, by mucosal, intradermal, intravenous, intramuscular delivery and / or any other physical delivery method described herein or known in the art.

[0085] As used herein, the terms "treat," "treatment," and "treating" refer to reducing or ameliorating the progression, severity, and / or duration of a disease or condition through the administration of one or more therapies. Treatment may be determined by assessing whether one or more symptoms associated with the underlying condition are reduced, ameliorated, and / or alleviated, such that the patient is observed to improve, even though the patient may still have the underlying condition. The term "treatment" includes disease control and amelioration. The term "control" refers to the beneficial effect a subject derives from a therapy that does not necessarily result in a cure of the disease.

[0086] The terms "prevent," "preventing," and "prevention" refer to reducing the likelihood of a disease, disorder, condition, or associated symptom (e.g., diabetes or cancer) occurring (or recurring).

[0087] As used herein, "delaying" cancer progression refers to slowing, inhibiting, decelerating, extending, stabilizing, and / or slowing the progression of the disease. This delay may have different lengths of time depending on the history of the disease and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, since the individual is free from the disease. A method that "delays" cancer progression is one that reduces the probability of disease progression within a given time range and / or reduces the extent of disease within a given time range, compared to the absence of the method. Such comparisons are usually based on clinical trials using a statistically significant number of individuals. Cancer progression can be detected using standard methods, including, but not limited to, computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation studies, arteriography, or biopsy. Progression may also refer to the progression of cancer, which may be initially undetectable, and includes occurrence, recurrence, and seizures.

[0088] As used herein, a "GCC-associated disease or GCC-associated disorder" refers to a disease or disorder involving cells or tissues in which GCC is expressed, selectively expressed, or aberrantly expressed (e.g., overexpressed). In some embodiments, a GCC-associated disease or GCC-associated disorder involves cells in which GCC is selectively expressed. In some embodiments, a GCC-associated disease or GCC-associated disorder involves cells in which GCC is aberrantly expressed. In other embodiments, a GCC-associated disease or GCC-associated disorder involves cells in or lacking at least one activity of GCC. In some embodiments, a GCC-associated disease or GCC-associated disorder is cancer, e.g., colorectal cancer.

[0089] The terms "about" and "approximately" refer to within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1% or less of a given value or range.

[0090] As used in this disclosure and claims, the singular forms "n," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0091] It is to be understood that wherever this specification describes an embodiment with the term "comprising," similar embodiments are also provided where described with "consisting of" and / or "consisting essentially of." It is further to be understood that wherever this specification describes an embodiment with the phrase "consisting essentially of," similar embodiments are also provided where described with "consisting of."

[0092] The term "between" used in the phrase "between A and B" or "between A and B" refers to a range that includes A and B.

[0093] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include A and B, A or B, A alone, and B alone. Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C, A, B or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0094] 5.2. Single Domain Antibodies 5.2.1. Single Domain Antibodies that Bind GCC In one aspect, the present disclosure provides a single domain antibody (e.g., a VHH domain) capable of binding to GCC.

[0095] In some embodiments, a single domain antibody (e.g., a VHH domain) provided herein binds to human GCC. GCC (UniProtKB:P25092) is a surface receptor that plays a role in maintaining intestinal fluid and electrolyte homeostasis and cell proliferation, and is selectively expressed on intestinal epithelial cells and all primary and metastatic colorectal tumors.

[0096] In some embodiments, the anti-GCC single domain antibodies provided herein modulate one or more GCC activities. In some embodiments, the anti-GCC single domain antibodies provided herein are antagonistic antibodies.

[0097] In some embodiments, the anti-GCC single domain antibodies herein have a cytotoxicity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K D ) binds to GCC (e.g., human GCC). Several methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, including RIA, e.g., using the Fab form of the antibody of interest and its antigen (Chen et al., 1999, J. Mol. Biol. 293:865-81), biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques, such as Octet®, e.g., using the Octet® Red 96 system, or Biacore®, e.g., using a Biacore® TM-2000 or Biacore® TM-3000 system. The "association rate" or "k" can also be measured by the same biolayer interferometry (BLI) or surface plasmon resonance (SPR) techniques, e.g., using Octet® Red 96, Biacore® TM-2000, or Biacore® TM-3000 systems.

[0098] In some embodiments, the anti-GCC single domain antibodies according to the present disclosure are VHH domains. Exemplary VHH domains according to the present disclosure are generated as described in Section 6 below, and are designated VHHA2322, VHHA2493, VHHC0464, VHHC0467, VHHC0494, VHHC0524, VHHC0694, VHHC0708, VHHC0806, VHHA2322H1, VHHA2322H2, VHHC0694H1, VHHC0694H2, VHHC0708H1, VHHC0708H2, and VHHC0708H3.

[0099] Thus, in some embodiments, a single domain antibody according to the present disclosure comprises the CDR sequences of any one or more of VHHA2322, VHHA2493, VHHC0464, VHHC0467, VHHC0494, VHHC0524, VHHC0694, VHHC0708, VHHC0806, VHHA2322H1, VHHA2322H2, VHHC0694H1, VHHC0694H2, VHHC0708H1, VHHC0708H2 and VHHC0708H3. In some embodiments, the description provides single domain antibodies that bind to GCC, comprising the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where the CDR sequences are selected from those of VHHA2322, VHHA2493, VHHC0464, VHHC0467, VHHC0494, VHHC0524, VHHC0694, VHHC0708, VHHC0806, VHHA2322H1, VHHA2322H2, VHHC0694H1, VHHC0694H2, VHHC0708H1, VHHC0708H2, and VHHC0708H3.

[0100] In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 26. In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 27. In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 28. In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 29. In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 30. In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 31. In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 32. In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 33. In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 34. In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 35. In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 36. In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 37. In some embodiments, anti-GCC single domain antibodies are provided that comprise one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 38.In some embodiments, an anti-GCC single domain antibody is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 39. In some embodiments, an anti-GCC single domain antibody is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 40. In some embodiments, an anti-GCC single domain antibody is provided that comprises one, two, or all three CDRs of the amino acid sequence of SEQ ID NO: 41. In some embodiments, the anti-GCC single domain antibody is derived from a camelid. In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human shared framework.

[0101] In some embodiments, the single domain antibody has a CDR1 having the amino acid sequence of CDR1 set forth in any one of SEQ ID NOs: 26 to 41. In some embodiments, the single domain antibody has a CDR2 having the amino acid sequence of CDR2 set forth in any one of SEQ ID NOs: 26 to 41. In other embodiments, the single domain antibody has a CDR3 having the amino acid sequence of CDR3 set forth in any one of SEQ ID NOs: 26 to 41. In some embodiments, the single domain antibody has a CDR1 and a CDR2 having the amino acid sequence of CDR1 and CDR2 set forth in any one of SEQ ID NOs: 26 to 41. In some embodiments, the single domain antibody has a CDR1 and a CDR3 having the amino acid sequence of CDR1 and CDR3 set forth in any one of SEQ ID NOs: 26 to 41. In some embodiments, the single domain antibody has a CDR2 and a CDR3, wherein the CDR2 and CDR3 have the amino acid sequences set forth in any one of SEQ ID NOs: 26-41. In some embodiments, the single domain antibody has a CDR1, a CDR2, and a CDR3, wherein the CDR1, CDR2, and CDR3 have the amino acid sequences set forth in any one of SEQ ID NOs: 26-41. CDR sequences can be defined according to well-known numbering systems / schemes. In some embodiments, CDRs are defined according to the IMGT numbering scheme. In some embodiments, CDRs are defined according to the Kabat numbering scheme. In some embodiments, CDRs are defined according to the AbM numbering scheme. In other embodiments, CDRs are defined according to the Chothia numbering scheme. In other embodiments, CDRs defined according to the Contact numbering scheme can be defined according to any combination of the above numbering schemes. In some embodiments, the anti-GCC single domain antibody is from a camelid.In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human shared framework.

[0102] In some embodiments, the present disclosure provides a single domain antibody that binds to GCC, comprising the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where (i) CDR1 comprises the amino acid sequence of any one of SEQ ID NOs: 1-8, (ii) CDR2 comprises the amino acid sequence of any one of SEQ ID NOs: 9-16, and / or (iii) CDR3 comprises the amino acid sequence of any one of SEQ ID NOs: 17-25. In some embodiments, the anti-GCC single domain antibody is derived from a camelid. In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human shared framework.

[0103] In another embodiment, the present disclosure provides a single domain antibody that binds to GCC, comprising the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein (i) CDR1 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-8; and (ii) CDR2 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 9-16; and / or (iii) CDR3 comprises an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 17-25. In some embodiments, the anti-GCC single domain antibody is derived from a camelid. In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, e.g., a human immunoglobulin framework or a human shared framework.

[0104] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 1, CDR2 comprises the amino acid sequence of SEQ ID NO: 9, and CDR3 comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the anti-GCC single domain antibody is from a Camelid. In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human shared framework.

[0105] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 2, CDR2 comprises the amino acid sequence of SEQ ID NO: 10, and CDR3 comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-GCC single domain antibody is from a Camelid. In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human shared framework.

[0106] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 3, CDR2 comprises the amino acid sequence of SEQ ID NO: 11, and CDR3 comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the anti-GCC single domain antibody is from a Camelid. In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human shared framework.

[0107] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 4, CDR2 comprises the amino acid sequence of SEQ ID NO: 11, and CDR3 comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-GCC single domain antibody is from a Camelid. In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human shared framework.

[0108] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 5, CDR2 comprises the amino acid sequence of SEQ ID NO: 12, and CDR3 comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-GCC single domain antibody is from a Camelid. In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human shared framework.

[0109] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 6, CDR2 comprises the amino acid sequence of SEQ ID NO: 13, and CDR3 comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the anti-GCC single domain antibody is from a Camelid. In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human shared framework.

[0110] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 6, CDR2 comprises the amino acid sequence of SEQ ID NO: 14, and CDR3 comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-GCC single domain antibody is from a Camelid. In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human shared framework.

[0111] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 7, CDR2 comprises the amino acid sequence of SEQ ID NO: 15, and CDR3 comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the anti-GCC single domain antibody is from a Camelid. In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human shared framework.

[0112] In some embodiments, CDR1 comprises the amino acid sequence of SEQ ID NO: 8, CDR2 comprises the amino acid sequence of SEQ ID NO: 16, and CDR3 comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the anti-GCC single domain antibody is from a Camelid. In some embodiments, the anti-GCC single domain antibody is humanized. In some embodiments, the anti-GCC single domain antibody comprises a recipient human framework, such as a human immunoglobulin framework or a human shared framework.

[0113] In some embodiments, the single domain antibody further comprises one or more framework regions of VHHA2322, VHHA2493, VHHC0464, VHHC0467, VHHC0494, VHHC0524, VHHC0694, VHHC0708, VHHC0806, VHHA2322H1, VHHA2322H2, VHHC0694H1, VHHC0694H2, VHHC0708H1, VHHC0708H2 and / or VHHC0708H3. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 26. In some embodiments, the single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 27. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 28. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 29. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 30. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 31. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 32. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 33. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 34. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 35.In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 36. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 37. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 38. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 39. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 40. In some embodiments, a single domain antibody comprises one or more frameworks derived from a VHH domain comprising the sequence of SEQ ID NO: 41.

[0114] In some embodiments, a single domain antibody according to the present invention is a humanized single domain antibody. In some embodiments, a humanized single domain antibody may be produced using the methods exemplified in Section 6 below or described in the following sections.

[0115] The framework regions described herein are defined according to the boundaries of the CDR numbering system / scheme. In other words, when the CDRs are defined, for example, by Kabat, IMGT, AbM, or Chothia, the framework regions are the amino acid residues that surround the CDRs in the variable region from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. For example, FR1 is defined as the amino acid residues N-terminal to the CDR1 amino acid residues as defined, for example, by the Kabat numbering system, the IMGT numbering system, the AbM numbering system, the Chothia numbering system, or a combination thereof; FR2 is defined as the amino acid residues between the CDR1 and CDR2 amino acid residues as defined, for example, by the Kabat numbering system, the IMGT numbering system, the AbM numbering system, the Chothia numbering system, or a combination thereof; FR3 is defined as the amino acid residues between the CDR2 and CDR3 amino acid residues as defined, for example, by the Kabat numbering system, the IMGT numbering system, the AbM numbering system, the Chothia numbering system, or a combination thereof; and FR4 is defined as the amino acid residues C-terminal to the CDR3 amino acid residues as defined, for example, by the Kabat numbering system, the IMGT numbering system, the AbM numbering system, the Chothia numbering system, or a combination thereof.

[0116] In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 27. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 28. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 29. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 30. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 31. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 32. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 33. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 33.In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 34. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 35. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 36. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 37. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 38. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 39. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 40. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, an isolated anti-GCC single domain antibody is provided, comprising a VHH domain having the amino acid sequence of SEQ ID NO: 41. In some embodiments, a polypeptide is provided, comprising the amino acid sequence of SEQ ID NO: 41.

[0117] In some embodiments, an antibody or antigen-binding fragment thereof described herein comprises an amino acid sequence having a certain percent identity to any one of antibodies VHHA2322, VHHA2493, VHHC0464, VHHC0467, VHHC0494, VHHC0524, VHHC0694, VHHC0708, VHHC0806, VHHA2322H1, VHHA2322H2, VHHC0694H1, VHHC0694H2, VHHC0708H1, VHHC0708H2, and VHHC0708H3.

[0118] The percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be determined using a mathematical algorithm. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), as modified by Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol. 215:403 (1990). BLAST nucleotide searches can be performed, for example, with the NBLAST nucleotide program parameters set to score=100 and wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches may be performed, for example, with the XBLAST program parameters set to score 50 and word length = 3. To obtain gapped alignments for comparison purposes, Gapped BLAST, described in Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997), can be used. Alternatively, PSI BLAST may be used for an iterated search (Id.), which detects distant relationships between molecules. When using BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the corresponding programs (e.g., for XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov). Another non-limiting example of a mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, CABIOS 4:11-17 (1998). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When comparing amino acid sequences using the ALIGN program, a PAM120 weight residue table can be used, with a gap length penalty of 12 and a gap penalty of 4. Techniques similar to those described above can be used to determine percent identity between two sequences, with or without gaps allowed. When calculating percent identity, typically only exact matches are counted.

[0119] In some embodiments, an anti-GCC single domain antibody is provided, comprising a VHH domain, wherein the VHH domain has at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 26-41. In some embodiments, any one of the VHH sequences having at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a reference sequence contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-GCC single domain antibody comprising the sequence retains the ability to bind to GCC. In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in an amino acid sequence selected from SEQ ID NOs: 26-41. In some embodiments, the substitutions, insertions, or deletions occur in regions other than the CDRs (i.e., FRs). Optionally, the anti-GCC single domain antibody comprises an amino acid sequence selected from SEQ ID NOs: 26-41, including post-translational modifications of the sequence.

[0120] In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 26, wherein the single domain antibodies bind to GCC. In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 27, wherein the single domain antibodies bind to GCC. In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 28, wherein the single domain antibodies bind to GCC. In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 29, wherein the single domain antibodies bind to GCC.In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 30, wherein the single domain antibodies bind to GCC. In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 31, wherein the single domain antibodies bind to GCC. In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 32, wherein the single domain antibodies bind to GCC. In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 33, wherein the single domain antibodies bind to GCC.In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 34, wherein the single domain antibodies bind to GCC. In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 35, wherein the single domain antibodies bind to GCC. In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 36, wherein the single domain antibodies bind to GCC. In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 37, wherein the single domain antibodies bind to GCC.In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 38, wherein the single domain antibodies bind to GCC. In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 39, wherein the single domain antibodies bind to GCC. In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 40, wherein the single domain antibodies bind to GCC. In some embodiments, the single domain antibodies described herein comprise a VHH domain having at least 75%, at least 80%, at least 85%, at least 90%, 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% sequence identity to the amino acid sequence of SEQ ID NO: 41, wherein the single domain antibodies bind to GCC. In some embodiments, functional epitopes can be plotted, for example, by combining alanine scanning, to identify amino acids in a GCC protein that are required for interaction with an anti-GCC single domain antibody according to the present invention.In some embodiments, the conformational and crystal structures of anti-GCC single domain antibodies that bind to GCC can be used to identify epitopes. In some embodiments, the present disclosure provides antibodies that specifically bind to the same epitope as any anti-GCC single domain antibody provided herein. For example, in some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 37.In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, an antibody is provided that binds to the same epitope as an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 41.

[0121] In some embodiments, the present disclosure provides anti-GCC antibodies or antigen-binding fragments thereof that specifically bind to GCC competitively with any one of the anti-GCC single domain antibodies described herein. In some embodiments, competitive binding can be determined by ELISA assay. For example, in some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 27. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 36.In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, an antibody is provided that specifically binds to GCC competitively with an anti-GCC single domain antibody comprising the amino acid sequence of SEQ ID NO: 41.

[0122] In some embodiments, the present specification provides a GCC binding protein comprising any one of the above-described anti-GCC single domain antibodies. In some embodiments, the GCC binding protein is a monoclonal antibody, including a camelid, chimeric, humanized, or human antibody. In some embodiments, the anti-GCC antibody is an antibody fragment, e.g., a VHH fragment. In some embodiments, the anti-GCC antibody is a full-length heavy chain-only antibody comprising an Fc region of any antibody class or isotype (e.g., IgG1 or IgG4). In some embodiments, the Fc region has reduced or minimized effector function. In some embodiments, the GCC binding protein is a fusion protein comprising an anti-GCC single domain antibody described herein. In other embodiments, the GCC binding protein is a multispecific antibody comprising an anti-GCC single domain antibody described herein. Other exemplary GCC binding molecules are described in more detail in the following sections.

[0123] In some embodiments, an anti-GCC antibody (e.g., an anti-GCC single domain antibody) or antigen binding protein according to any of the above embodiments may include any of the features, alone or in combination, as described in Sections 5.2.2-5.2.7 below.

[0124] 5.2.2. Humanized Single Domain Antibodies The single domain antibodies described herein include humanized single domain antibodies. General strategies for humanizing single domain antibodies from Camelidae species have been described (see, e.g., Vincke et al., J. Biol. Chem., 284(5):3273-3284 (2009)), and can be used to produce the humanized VHH domains disclosed herein. The design of humanized single domain antibodies from Camelidae species may include labeled residues in the VHH, such as residues 11, 37, 44, 45, and 47 (residues numbered according to Kabat) (Muyldermans, Reviews Mol Biotech 74:277-302 (2001)).

[0125] Humanized antibodies, such as the humanized single domain antibodies disclosed herein, can be produced using a variety of techniques known in the art, including CDR-grafting (European Patent No. EP 239,400, International Publication No. WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering, or recapitulation (European Patent Nos. EP 592,106 and EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); and Roguska et al., PNAS 91:969-973). (1994)), chain shuffling (U.S. Pat. No. 5,565,332) and, for example, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, WO 9317105, 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. 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). See also U.S. Patent No. US 2005 / 0042664 A1 (February 24, 2005), each of which is incorporated herein by reference in its entirety.

[0126] In some embodiments, the single-domain antibody of the present disclosure may be a humanized single-domain antibody that binds to GCC (including human GCC). For example, a humanized single-chain antibody of the present disclosure may comprise one or more CDRs set forth in SEQ ID NOs: 26-41. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are commonly referred to as "import" residues, and these residues are typically taken from an "import" variable domain. For example, humanization can be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody according to the methods of Jones et al., Nature 321:522-25 (1986), Riechmann et al., Nature 332:323-27 (1988), and Verhoeyen et al., Science 239:1534-36 (1988). In a specific embodiment, the single domain antibodies according to the present invention are humanized as described in Section 6 below.

[0127] In some embodiments, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the CDRs of a parent non-human antibody are grafted onto a human antibody framework. For example, Padlan et al. found that only about one-third of the residues in the CDRs actually contact the antigen, and called these "specificity-determining residues" or SDRs (Padlan et al., FASEB J. 9:133-39 (1995)). In SDR grafting technology, only the SDR residues are grafted onto a human antibody framework (see, for example, Kashmiri et al., Methods 36:25-34 (2005)).

[0128] To reduce antigenicity, the selection of human variable domains for preparing humanized antibodies can be important. For example, the so-called "best-fit" method involves screening the sequence of a non-antibody variable domain against the entire library of known human variable domain sequences. The human sequence closest to the non-human antibody may be selected as the human framework for the humanized antibody (Sims et al., J. Immunol. 151:2296-308 (1993) and Chothia et al., J. Mol. Biol. 196:901-17 (1987)). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular light or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-89 (1992) and Presta et al., J. Immunol. 151:2623-32 (1993)). In some embodiments, the framework is selected from the most abundant human subclass V L 6 Subgroup I(V L 6I) and V H Subgroup III(V H III) In another embodiment, human germline genes are used as the source of the framework regions.

[0129] An alternative paradigm based on CDR comparison, called superhumanization, does not involve FR homology. This method involves comparing non-human sequences with a functional human germline gene repertoire. Genes encoding the same or closely related canonical structures as the mouse sequences are then selected. Subsequently, genes with the highest homology within the CDRs among genes sharing the canonical structure with non-human antibodies are selected as FR donors. Finally, non-human CDRs are grafted onto these FRs (see, for example, Tan et al., J. Immunol. 169:1119-25 (2002)).

[0130] Typically, it is desirable to humanize antibodies to retain their affinity for the antigen and other favorable biological properties. To achieve this goal, one method involves the preparation of humanized antibodies by analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13:819-24 (2002)), Modeller (Sali and Blundell, J. Mol. Biol. 234:779-815 (1993)), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18:2714-23 (1997)). Inspection of these displays permits analysis of the possible effect of residues on the functioning of the candidate immunoglobulin sequence, for example, analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, by selecting and combining FR residues from the receptor and import sequences, the desired antibody characteristic, such as increased affinity for one or more target antigens, can be obtained. Typically, the hypervariable region residues are directly and most significantly involved in influencing antigen binding.

[0131] Another antibody humanization method is based on a metric of antibody humanization called Human String Content (HSC). This method compares the mouse sequence with the human germline gene repertoire and scores the differences as HSC. The target sequence is then humanized by maximizing the HSC, rather than using a global identity measure, to generate multiple different humanized variants (Lazar et al., Mol. Immunol. 44:1986-98 (2007)).

[0132] In addition to the above methods, empirical methods can also be used to generate and select humanized antibodies. These methods include methods based on generating large libraries of humanized variants and selecting the best clones using enrichment or high-throughput screening techniques. Antibody variants can be isolated from phage, ribosomal, and yeast display libraries and by bacterial colony screening (see, e.g., Hoogenboom, Nat. Biotechnol. 23:1105-16 (2005); Dufner et al., Trends Biotechnol. 24:523-29 (2006); Feldhaus et al., Nat. Biotechnol. 21:163-70 (2003); and Schlapschy et al., Protein Eng. Des. Sel. 17:847-60 (2004)).

[0133] In the FR library approach, a set of residue mutations is introduced at specific positions within the FR, and the library is screened to select the FR that best supports the grafted CDR. The residues to be substituted may include some or all of the "vernier" residues identified as likely to contribute to CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224:487-99 (1992)), or a more limited group of target residues identified by Baca et al., J. Biol. Chem. 272:10678-84 (1997)).

[0134] In FR shuffling, entire FRs are combined with non-human CDRs without creating a combinatorial library of selected residue variants (see, e.g., Dall'Acqua et al., Methods 36:43-60 (2005)). A one-step FR shuffling process may also be used. Such methods prove effective because the resulting antibodies exhibit improved biochemical and physicochemical properties, including enhanced expression, increased affinity, and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44:3049-60 (2007)).

[0135] The "humaneering" method is based on the experimental identification of essential minimal specificity determinants (MSDs), followed by sequential substitution of non-human fragments into a human FR library and evaluation of binding. This method typically results in epitope retention and identification of antibodies from multiple subclasses with different human V-segment CDRs.

[0136] "Human engineering" methods involve engineering non-human antibodies or antibody fragments with specific modifications to their amino acid sequences to produce modified antibodies with reduced immunogenicity in humans, while still retaining the required binding properties of the original non-human antibody. Typically, the techniques involve classifying amino acid residues in non-human antibodies into "low risk," "intermediate risk," or "high risk" residues. Classification is performed using a global risk / reward calculation that assesses the predicted benefit of a particular substitution (e.g., with respect to human immunogenicity) and the risk that the substitution will affect the folding of the resulting antibody. By aligning amino acid sequences from non-human antibody variable regions with corresponding regions of specific or consensus human antibody sequences, specific human amino acid residues to be substituted at given positions (e.g., low risk or intermediate risk) in the non-human antibody sequence can be selected. Amino acid residues at low or intermediate risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence upon alignment. Techniques for producing human engineered proteins are described in Studnicka et al., Protein Engineering 7:805-14 (1994), U.S. Patent Nos. 5,766,886, 5,770,196, 5,821,123 and 5,869,619, and PCT Publication WO 93 / 11794.

[0137] Composite human antibodies can be produced, for example, using Composite Human Antibody™ technology (Antitope Ltd., Cambridge, United Kingdom). To produce composite human antibodies, the variable region sequences are engineered from fragments of multiple human antibody variable region sequences to avoid T-cell epitopes, thereby minimizing the immunogenicity of the resulting antibodies.

[0138] A deimmunized antibody is an antibody from which T cell epitopes have been removed. Methods for producing deimmunized antibodies have been described. See, for example, Jones et al., Methods Mol Biol. 525:405-23 (2009), xiv and De Groot et al., Cell. Immunol. 244:148-153 (2006). A deimmunized antibody comprises a variable region from which T cell epitopes have been deleted and a human constant region. Briefly, the variable region of an antibody is cloned, followed by identification of T cell epitopes by testing overlapping peptides derived from the antibody variable region in a T cell proliferation assay. T cell epitopes are identified using computational methods to identify peptides that bind to human MHC class II. Mutations are introduced into the variable region to eliminate binding to human MHC class II. The mutated variable region is then used to produce a deimmunized antibody.

[0139] 5.2.3. Single Domain Antibody Variants In some embodiments, modifications to one or more amino acid sequences of the single domain antibodies that bind to GCC described herein are contemplated. For example, optimization of the antibody's binding affinity and / or other biological properties may be required, including, but not limited to, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility. Therefore, in addition to the single domain antibodies that bind to GCC described herein, it is also anticipated that variants of the single domain antibodies that bind to GCC described herein may be prepared. For example, single domain antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and / or synthesizing the desired antibody or polypeptide. It will be understood by those skilled in the art that amino acid modifications may alter post-translational processing of single domain antibodies.

[0140] chemical modification In some embodiments, single domain antibodies provided herein are chemically modified, e.g., covalently attached to the single domain antibody via any type of molecule. Antibody derivatives may include antibodies that have already been chemically modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic hydrolysis and cleavage, linkage to cellular ligands or other proteins, or conjugation to one or more immunoglobulin domains (e.g., Fc or portions of Fc). Any one of several chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. The antibody may also contain one or more non-classical amino acids.

[0141] In some embodiments, the antibodies provided herein are modified to increase or decrease the extent of glycosylation of the antibody. Addition or deletion of antibody glycosylation sites can be readily achieved by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0142] When a single-domain antibody provided herein is fused to an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced from mammalian cells typically contain branched bicontact angle oligosaccharides, which are typically linked via N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, and fucose linked to GlcNAc in the "stem" of the bicontact angle oligosaccharide structure. In some embodiments, modifications can be made to the oligosaccharides in a binding molecule provided herein to produce variants with improved properties.

[0143] In other embodiments, when a single domain antibody provided herein is fused to an Fc region, the antibody variant provided herein may have a carbohydrate structure lacking fucose linked (directly or indirectly) to said Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at position Asn 297 relative to the sum of all glycan structures linked to Asn 297 (e.g., complex, hybrid, and high-mannose structures) as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at about position 297 (EU numbering of Fc region residues) in the Fc region; however, due to minor sequence variations in antibodies, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Publication Nos. US 2003 / 0157108 and US 2004 / 0093621. Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, US 2003 / 0115614, US 2002 / 0164328, US 2004 / 0093621, US 2004 / 0132140, US 2004 / 0110704, US 2004 / 0110282, US 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986), U.S. Patent Application No. US 2003 / 0157108, and WO 2004 / 056312, especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004), Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO 2003 / 085107).

[0144] Binding molecules, including single domain antibodies, according to the present disclosure may further comprise isotopic oligosaccharides, e.g., wherein the high-contact angle oligosaccharide attached to the Fc region is isotopic with GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function. Examples of such variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Pat. No. 6,602,684 (Umana et al.), and US 2005 / 0123546 (Umana et al.). Further provided are variants in which the oligosaccharide attached to the Fc region contains at least one galactose residue. Such variants may have improved ADCC function. Such variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0145] In the single domain antibodies and Fc region-containing molecules of the present invention, Fc region variants can be generated by introducing one or more amino acid modifications into the Fc region. The Fc region variants may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that comprises an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0146] In some embodiments, the present disclosure contemplates variants that retain some (but not all) effector functions, making them desirable candidates for the disclosure, where the in vivo half-life of the binding molecule is important, but some effector functions (e.g., complement and ADCC) are unnecessary or deleterious. Reduced / depleted CDC and / or ADCC activity can be confirmed by performing in vitro and / or in vivo cytotoxicity assays. For example, Fc receptor (FcR) binding assays can be performed to ensure that the binding molecule does not have FcγR binding ability (and thus may lack ADCC activity) but retains FcRn binding ability. Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), and U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used, see, for example, the ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity.See, for example, the ELISA for C1q and C3c binding in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays may be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0147] Binding molecules with reduced effector function include antibodies (U.S. Patent No. 6,737,056) with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329. Such Fc mutants include Fc mutants with substitutions of two or more at amino acid positions 265, 269, 270, 297, and 327, including the Fc mutant designated "DANA" in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0148] Several mutants with improved or decreased binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0149] In some embodiments, the variants comprise an Fc region with one or more amino acid substitutions (e.g., substitutions at positions 298, 333, and / or 334 in the Fc region (EU numbering of residues)) that improve ADCC. In some embodiments, modifications occur in the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0150] Binding molecules with extended half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), have been described in US 2005 / 0014934 A1 (Hinton et al.). These molecules comprise an Fc region with one or more amino acid substitutions, where these substitutions improve binding of the Fc region to FcRn. Such Fc variants include those with a substitution at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0151] In some embodiments, this may involve the production of cysteine-engineered antibodies, in which one or more residues of an antibody are replaced by cysteine ​​residues. In some embodiments, the substituted residues occur at accessible sites of the antibody. By replacing those residues with cysteines, reactive thiol groups are positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates, as further described herein.

[0152] Substitutions, deletions, or insertions Mutations may be the substitution, deletion, or insertion of one or more codons encoding the single domain antibody or polypeptide, resulting in an alteration of the amino acid sequence relative to the original antibody or polypeptide. Target sites for substitutional mutagenesis include the CDRs and FRs.

[0153] Amino acid substitutions may result from the replacement of one amino acid with another having similar structural and / or chemical properties, such as replacing leucine with serine, and may be, for example, conservative amino acid substitutions. Standard techniques well known to those of skill in the art may be used to introduce mutations into nucleotide sequences encoding molecules herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In some embodiments, substitutions, deletions, or insertions comprise fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions relative to the original molecule. In specific embodiments, substitutions are conservative amino acid substitutions made at one or more predicted non-essential amino acid residues. Acceptable changes can be determined by systematically making amino acid insertions, deletions, or substitutions in a sequence and testing the resulting variants for activity of the parent antibody.

[0154] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing several residues, as well as intrasequence insertions of single or multiple amino acid residues. An exemplary terminal insertion is an antibody with an N-terminal methionyl residue.

[0155] The present disclosure includes single domain antibodies generated by conservative amino acid substitutions. In conservative amino acid substitutions, an amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. As described above, the art has defined families of amino acid residues with side chains with similar charges. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, e.g., by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed, and the protein's activity can be determined. Conservative substitutions (e.g., within a group of amino acids with similar properties and / or side chains) can be made to preserve or not significantly alter properties. Exemplary substitutions are shown in Table 2 below.

[0156] (Table 2) Amino acid substitutions TIFF2025533779000002.tif84161

[0157] Amino acids can be grouped based on similarities in the properties of their side chains (see, e.g., Lehninger, Biochemistry 73-75 (2nd ed., 1975)): (1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be grouped based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. For example, to improve the oxidative stability of the molecule and prevent aberrant crosslinking, any cysteine ​​residue not involved in maintaining the correct conformation of a single-domain antibody may be substituted with another amino acid, such as alanine or serine. Non-conservative substitutions involve exchanging one member of these classes for another.

[0158] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). The resulting variants, typically selected for further study, are modified (e.g., improved) with respect to some biological property (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and / or substantially retain some biological property of the parent antibody. Exemplary substitutional variants are affinity-matured antibodies, which can be readily generated, for example, by phage-display-based affinity maturation techniques, such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0159] Modifications (e.g., substitutions) can be made in CDRs to, for example, improve antibody affinity. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons frequently mutated during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), where the resulting mutant antibodies or fragments thereof are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any one of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis), and a secondary library is then generated. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR orientation, in which a number of CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. The following section provides a more detailed description of affinity maturation.

[0160] In some embodiments, substitutions, insertions, or deletions may occur in one or more CDRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions according to the present disclosure) may be made in a CDR that do not significantly reduce binding affinity. In some embodiments of the variant VHH sequences according to the present disclosure, each CDR is unaltered or contains no more than one, two, or three amino acid substitutions.

[0161] As described in Cunningham and Wells, Science, 244:1081-1085 (1989), a useful method for identifying antibody residues or regions amenable to targeted mutagenesis is called "alanine scanning mutagenesis." In this method, a target residue or group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether antibody-antigen interactions are affected. Further substitutions at the amino acid position can be introduced to demonstrate functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be determined to identify contact points between the antibody and antigen. Such contact and adjacent residues can be targeted as substitution candidates or removed. Mutants can be screened to determine whether they contain desired properties.

[0162] Amino acid sequence insertions include amino- and / or carboxy-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusing the N- or C-terminus of the antibody to an enzyme (e.g., ADEPT) or a polypeptide which increases the serum half-life of the antigen-binding domain of the antibody.

[0163] Changes may be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. To produce single domain antibody variant DNA, cloned DNA may be subjected to site-directed mutagenesis (see, e.g., Carter, Biochem J. 237:1-7 (1986), and Zoller et al., Nucl. Acids Res. 10:6487-500 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315-23 (1985)), or other known techniques.

[0164] 5.2.4. In vitro affinity maturation In some embodiments, antibody variants with improved properties, such as affinity, stability, or expression level, compared to the parent antibody can be prepared by in vitro affinity maturation. Similar to natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. Antibody libraries are displayed on the surface of organisms (e.g., phage, bacteria, yeast, or mammalian cells) or associated (e.g., covalently or noncovalently) with their encoding mRNA or DNA. Affinity selection of the displayed antibodies allows for the isolation of organisms or complexes carrying the genetic information encoding the antibody. Two or three rounds of mutation and selection using display methods such as phage display typically produce antibody fragments with low affinities in the nanomolar range. Affinity-matured antibodies can have nanomolar or picomolar affinities for target antigens.

[0165] Phage display is a common method for displaying and selecting antibodies. Antibodies are displayed on the surface of Fd or M13 phage as fusions with phage coat proteins. Selection involves binding of antibodies to their target, which is then exposed to antigen to allow phage display, a process called "panning." Phage that bind to the antigen are recovered and used to infect bacteria to produce phage for further rounds of selection. For reviews, see, e.g., Hoogenboom, Methods. Mol. Biol. 178:1-37 (2002), and Bradbury and Marks, J. Immunol. Methods 290:29-49 (2004).

[0166] In yeast display systems (see, e.g., Boder et al., Nat. Biotech. 15:553-57 (1997) and Chao et al., Nat. Protocols 1:755-68 (2006)), antibodies can be fused to the adhesion subunit of the yeast lectin protein Aga2p, which is linked to the yeast cell wall via a disulfide bond with Aga1p. The Aga2p display protein minimizes potential interactions with other molecules on the yeast cell wall by projecting the protein from the cell surface. Magnetic separation and flow cytometry are used to screen libraries and select antibodies with improved affinity or stability. Binding to the soluble antigen of interest is measured by labeling yeast with a biotinylated antigen and a second reagent conjugated to a fluorophore, such as streptavidin. Changes in antibody surface expression can be measured by immunofluorescent labeling of hemagglutinin or c-Myc epitope tags (e.g., scFv) flanking the single-chain antibody. Expression has been shown to correlate with the stability of the displayed protein, and antibodies can therefore be selected to improve stability and affinity (see, e.g., Shusta et al., J. Mol. Biol. 292:949-56 (1999)). An additional advantage of yeast display is that the displayed protein folds in the endoplasmic reticulum of eukaryotic yeast cells, utilizing endoplasmic reticulum chaperones and quality control mechanisms. Once maturation is complete, antibody affinity can be easily "titrated" and displayed on the yeast surface, thereby eliminating the need to express and purify each clone. A theoretical limitation of yeast surface display is the potentially smaller functional display size than other display methods; however, recent methods have demonstrated a size of approximately 10 fold using yeast cell mating systems. 14 (See, e.g., U.S. Patent Publication 2003 / 0186374, and Blaise et al., Gene 342:211-18 (2004)).

[0167] In ribosome display, antibody-ribosome-mRNA (ARM) complexes are produced and used for cell-free selection. A DNA library encoding a specific antibody library is genetically fused to a spacer sequence lacking a termination codon. The spacer sequence remains linked to peptide tRNA during translation, occupying the ribosomal channel and allowing the target protein to protrude from the ribosome and fold. The resulting mRNA, ribosome, and protein complex binds to a surface-bound ligand, allowing the antibody and its encoding mRNA to be captured and isolated by affinity for the ligand. The ribosome-bound mRNA is then reverse transcribed into cDNA, mutagenized, and used for the next round of selection (see, e.g., Fukuda et al., Nucleic Acids Res. 34:e127 (2006)). In mRNA display, puromycin is used as an adapter molecule to establish a covalent link between the antibody and the mRNA (Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-55 (2001)).

[0168] Because these methods are performed entirely in vitro, they offer two major advantages over other selection techniques. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, because there is no need to transform the library after any diversification step, random mutations can be easily introduced after each round of selection, for example, by using a non-proofreading polymerase.

[0169] In some embodiments, a mammalian display system may be used.

[0170] Diversity can also be introduced into the CDRs of an antibody library by targeted or random introduction. Previous methods include sequentially targeting all CDRs of an antibody through high- or low-level mutagenesis, or targeting isolated hotspots for somatic hypermutation (see, e.g., Ho et al., J. Biol. Chem. 280:607-17 (2005)), or suspecting residues that affect affinity for experimental or structural reasons. Diversity can also be introduced by replacing naturally diversified regions through DNA shuffling or similar techniques (see, e.g., Lu et al., J. Biol. Chem. 278:43496-507 (2003); U.S. Patent Nos. 5,565,332 and 6,989,250). Alternative techniques target hypervariable loops extending into framework region residues (see, e.g., Bond et al., J. Mol. Biol. 348:699-709 (2005)) and use loop deletions and insertions in the CDRs, or use hybridization-based diversification (see, e.g., U.S. Patent Publication No. 2004 / 0005709). Other methods for generating diversity in CDRs are disclosed, for example, in U.S. Patent No. 7,985,840. Other methods that can be used for generating antibody libraries and / or antibody affinity maturation are disclosed, for example, in U.S. Pat. Nos. 8,685,897 and 8,603,930 and U.S. Publication Nos. 2014 / 0170705, 2014 / 0094392, 2012 / 0028301, 2011 / 0183855, and 2009 / 0075378, each of which is incorporated herein by reference.

[0171] Screening of the library can be accomplished by a variety of techniques known in the art, for example, single domain antibodies can be immobilized on a solid support, column, needle, or cellulose / poly(vinylidene fluoride) membrane / other filter, expressed in host cells attached to an adsorption plate or used for cell sorting, or conjugated with biotin and captured on streptavidin-coated beads, or used in any other method to pan the display library.

[0172] For reviews of in vitro affinity maturation methods, see, e.g., Hoogenboom, Nature Biotechnology 23:1105-16 (2005), Quiroz and Sinclair, Revista Ingeneria Biomedia 4:39-51 (2010), and references therein.

[0173] 5.2.5. Modification of Single Domain Antibodies Covalent modifications of single domain antibodies are included within the scope of the present disclosure. Covalent modifications include reacting targeted amino acid residues of single domain antibodies with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues of single domain antibodies. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of lysine, arginine, and histidine side chain α-amino groups (see, e.g., Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0174] Other types of covalent modifications of single domain antibodies within the scope of the present disclosure include altering the native glycosylation pattern of the antibody or polypeptide as described above (e.g., Beck et al., Curr. Pharm. Biotechnol. 9:482-501 (2008), and Walsh, Drug Discov. Today 15:773-80 (2010)), and linking the antibody to one of several nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, e.g., by methods described in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337. The single domain antibodies that bind to GCC of the present disclosure can further be genetically fused or conjugated to one or more immunoglobulin constant regions or portions thereof (e.g., Fc) to extend half-life and / or confer known Fc-mediated effector functions.

[0175] The single-chain antibodies that bind GCC of the present disclosure can also be modified to form chimeric molecules, including single-chain antibodies that bind GCC fused to another heterologous polypeptide or amino acid sequence, such as an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60:523-33 (2003)) or the Fc region of an IgG molecule (see, e.g., Aruffo, Antibody Fusion Proteins 221-42 (Chamow and Ashkenazi, eds., 1999)). Single-chain antibodies that bind GCC may also be used to generate chimeric antigen receptors (CARs) that bind GCC, as described in more detail below.

[0176] The present specification further provides fusion proteins, which comprise a single-chain antibody that binds to GCC of the present disclosure and a heterologous polypeptide. In some embodiments, the heterologous polypeptide to which the antibody is genetically fused or chemically conjugated can be used to target the antibody to cells that have GCC expressed on their cell surface.

[0177] The present specification further provides a set of antibodies that bind to GCC antigens. In specific embodiments, the antibody set has different binding rates, different dissociation rates, different affinities for GCC antigens, and / or different specificities for GCC antigens. In some embodiments, the set includes or is composed of about 10 to about 1,000 or more antibodies. The antibody set can be used in assays such as ELISA using, for example, a 96-well or 384-well plate.

[0178] 5.2.6. Preparation of Single Domain Antibodies Methods for preparing single domain antibodies have been described. See, for example, Els Pardon et al., Nature Protocol, 9(3): 674 (2014). Single domain antibodies (e.g., VHHs) can be obtained by methods known in the art, such as immunizing an immunized Camelidae species (e.g., a camel or a llama) and obtaining hybridomas therefrom, or cloning a single domain antibody library using molecular biology techniques known in the art, followed by selection of single clones from an unselected library by ELISA or by using phage display.

[0179] Single domain antibodies according to the present disclosure can be produced by culturing cells transformed or transfected with a vector containing nucleic acid encoding the single domain antibody. Polynucleotide sequences encoding the polypeptide components of the antibodies of the present disclosure can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells, such as hybridoma cells or B cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. Once obtained, the polypeptide-encoding sequence is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in host cells. Many known vectors available in the art can be used for the purposes of the present disclosure. The selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Suitable host cells for expressing the antibodies of the present disclosure include prokaryotes, such as archaebacteria and eubacteria, including gram-negative or gram-positive organisms; eukaryotic microorganisms, such as filamentous fungi or yeast; invertebrate cells, such as insect or plant cells; and vertebrate cells, such as mammalian host cell lines. Host cells are transformed with the above expression vectors and cultured in a conventional nutrient medium, modified appropriately to induce promoters, select transformants, or amplify genes encoding the desired sequences. Antibodies produced by the host cells are purified using standard protein purification methods known in the art.

[0180] For antibody production methods, including vector construction, expression, and purification, see Pluckthun et al., Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation 203-52 (ed. McCafferty et al., 1996), Kwong and Rader, E. coli Expression and Purification of Fab Antibody Fragments, Current Protocols in Protein Science (2009), Tachibana and Takekoshi, Production of Antibody Fab Fragments in Escherichia coli, Antibody Expression and Production (ed. Al-Rubeai, 2011), and Therapeutic Monoclonal Antibodies: From Bench to Clinic (ed. An, 2009).

[0181] Of course, it is anticipated that GCC single domain antibodies may be prepared using alternative methods well known in the art. For example, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis (1969), and Merrifield, J. Am. Chem. Soc. 85:2149-54 (1963)). In vitro protein synthesis may be performed using manual techniques or by automation. Individual portions of an anti-GCC antibody may be chemically synthesized separately and combined by chemical or enzymatic methods to produce the desired anti-GCC antibody. Alternatively, antibodies may be purified from cells or body fluids, e.g., emulsions, of transgenic animals engineered to express antibodies, as disclosed in U.S. Patent Nos. 5,545,807 and 5,827,690.

[0182] Specifically, single domain antibodies or other GCC-binding proteins according to the present invention can be produced by immunizing llamas, sorting single B cells, extracting V genes, cloning the GCC-binding protein (e.g., a VHH-Fc fusion), and small-scale expression and purification. Additional screening can also be performed for single domain antibodies and other molecules that bind to GCC, and results in ELISA-positive, BLI-positive, and a K value of less than 100 nM. D These selection criteria can be combined as described in section 6 below. Alternatively, the ability of a single VHH binding protein (and other molecules that bind GCC) to bind to cells expressing GCC can be measured. Such measurements can be performed by analyzing cells expressing GCC using FACS and measuring the mean fluorescence intensity (MFI) of fluorescently labeled VHH molecules. Each of the above aspects is described in more detail below.

[0183] Polyclonal antibodies Polyclonal antibodies are usually raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Bifunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide ester (conjugated through cysteine ​​residues), N-hydroxysuccinimide (conjugated through lysine residues), glutaraldehyde, succinic anhydride, SOCl or R 1 N=C=NR (where R and R 1 are independently lower alkyl groups) to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin (KLH), serum albumin, thyroid globulin, or soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dimycotic acid ester). The immunization scheme can be selected by one of skill in the art without undue experimentation.

[0184] For example, animals are immunized against the antigen, immunogenic conjugate, or derivative by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1 / 5 to 1 / 10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum antibody titer is measured. The animals are boosted until the titer plateaus. Conjugates can also be produced as protein fusions in recombinant cell culture. Aggregating agents such as alum are suitable for enhancing the immune response.

[0185] Monoclonal antibodies Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., each antibody in the population is identical except for possible, naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of individual antibodies.

[0186] For example, the monoclonal antibodies may be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Patent No. 4,816,567).

[0187] In the hybridoma method, a suitable host animal is immunized to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing protein. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to produce hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0188] The immunizing agent typically contains an antigen protein or a fusion variant thereof. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103. Immortalized cell lines are typically transformed mammalian cells. The hybridoma cells thus prepared are seeded and grown in an appropriate medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. Preferred immortalized myeloma cell lines are those that fuse efficiently, support stable high-level antibody production by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.

[0189] The production of monoclonal antibodies against the antigen is measured in the culture medium in which the hybridoma cells are grown. The presence of monoclonal antibodies against the desired antigen can be measured in the culture medium in which the hybridoma cells are cultured. Such techniques and measurements are known in the art. For example, binding affinity can be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0190] After hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Hybridoma cells can also be grown in vivo as tumors in mammals.

[0191] The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-agarose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0192] Monoclonal antibodies can also be produced by recombinant DNA methods, as described in U.S. Pat. No. 4,816,567 and above. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector and transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or non-immunoglobulin-producing myeloma cells, to synthesize the monoclonal antibodies in such recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Pliickthun, Immunol. Revs. 130:151-188 (1992).

[0193] In a further embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990), Clackson et al., Nature, 352:624-628 (1991), and Marks et al., J. Mol. Biol., 222:581-597 (1991). Subsequent publications have described strategies for constructing very large phage libraries to produce high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)) and combinatorial infection and in vivo recombination (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). As such, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0194] The DNA can be modified, for example, by substituting the coding sequence (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)) or by covalently linking to the coding sequence all or part of a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted to produce a chimeric bivalent antibody containing one antigen-binding site specific for an antigen and another antigen-binding site specific for a different antigen.

[0195] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed by disulfide exchange reactions or thioether bond formation. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0196] Recombinant production in prokaryotic cells Polynucleic acid sequences encoding antibodies of the present disclosure can be obtained by standard recombinant techniques. The desired polynucleic acid sequence can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR technology. Once obtained, the polypeptide-encoding sequence is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many known vectors available in the art can be used for the purposes of the present disclosure. Selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains different components, which depend on its function (amplification or expression of the heterologous polynucleotide, or both) and its compatibility with the host cell in which it is located. Vector components usually include, but are not limited to, an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert, and a transcription termination sequence.

[0197] Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are typically used in conjunction with these hosts. The vector typically contains a replication site, as well as marker sequences capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed with pBR322, a plasmid derived from an E. coli species. Examples of pBR322 derivatives for expressing specific antibodies are described in detail in U.S. Patent No. 5,648,237 to Carter et al.

[0198] Phage vectors containing replicon and control sequences compatible with host microorganisms can be used as transformation vectors in connection with these hosts. For example, phages such as GEM™-11 can be used to produce recombinant vectors, which can then be used to transform susceptible host cells such as E. coli LE392.

[0199] The expression vectors of the present disclosure may contain two or more promoter-cistron pairs, one encoding each polypeptide component. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that controls its expression. Prokaryotic promoters are generally divided into two types: inducible and constitutive. An inducible promoter is a promoter that initiates increased transcription levels of the cistron under its control in response to a change in culture conditions, such as the presence or absence of a nutrient or a change in temperature.

[0200] Numerous promoters compatible with many potential host cells are well known. The selected promoter may be operably linked to the cistronic DNA encoding the antibody of the present invention by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present disclosure. Both the native promoter sequence and multiple heterologous promoters may be used to direct amplification and / or expression of the target gene. In some embodiments, a heterologous promoter is used because it typically allows for greater transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter.

[0201] Suitable promoters for prokaryotic hosts include the PhoA promoter, β-galactosidase and lactose promoter systems, and hybrid promoters such as the tryptophan (trp) promoter system and the tac or trc promoter. However, other promoters that function in bacteria (e.g., other known bacterial or phage promoters) are also suitable. Having disclosed their nucleic acid sequences, one skilled in the art can operably link them to a cistron encoding a target peptide using linkers or adapters (Siebenlist et al., Cell 20: 269 (1980)) and provide any necessary restriction sites.

[0202] In one embodiment, each cistron in a recombinant vector contains a secretory signal sequence component that directs transmembrane translocation of the expressed polypeptide. Typically, the signal sequence may be a component of the vector or may be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present disclosure should be one that can be recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence of a heterologous polypeptide, the signal sequence may be substituted with a prokaryotic signal sequence, e.g., selected from the group consisting of alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leader sequence, LamB, PhoE, PelB, OmpA, and MBP.

[0203] In some embodiments, production of antibodies according to the present disclosure can occur in the cytoplasm of the host cell, so the presence of a secretory signal sequence for each cistron is not required. - The B. difficile strains allow for the correct folding and assembly of expressed protein subunits by providing cytoplasmic conditions that favor the formation of disulfide bonds.

[0204] Prokaryotic host cells suitable for expressing the antibodies of the present disclosure include archaebacteria and eubacteria, such as gram-negative or gram-positive bacteria. Examples of useful bacteria include Escherichia (e.g., Escherichia coli), Escherichia (e.g., Bacillus subtilis), Enterobacter, Pseudomonas (e.g., Pseudomonas aeruginosa), Salmonella typhimurium, Serratia marcescens, Klebsiella, Proteus, Shigella, Rhizobium, Vitreoscilla, or Paracoccus. In some embodiments, gram-negative cells are used. In one embodiment, E. coli cells are used as the host. An example of an E. coli strain is strain W3110 (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219, ATCC Accession No. 27,325) and its derivatives, genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompT A(nmpc-fepE) degP41 kan R Suitable bacterial strains include strain 33D3 (U.S. Pat. No. 5,639,635), which has the nucleotide sequence α-Gly-1, ...

[0205] Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors can ideally be incorporated into the cell culture.

[0206] Host cells are transformed with the expression vector and cultured in a conventional nutrient medium, which is appropriately modified to induce promoters, select transformants, or amplify genes encoding the desired sequences. Transformation refers to the introduction of DNA into a prokaryotic host so that the DNA can replicate as an extrachromosomal element or via chromosomal integrant. Depending on the host cells used, transformation is carried out using standard techniques appropriate for these cells. Calcium treatment using calcium chloride is typically used for bacterial cells that contain large amounts of cell wall barriers. Another transformation method uses polyethylene glycol / DMSO. Another technique used is electroporation.

[0207] Prokaryotic cells for producing the antibodies of the present disclosure may be grown in media known in the art and suitable for culturing the selected host cells. An example of a suitable medium includes Luria Broth (LB) and necessary nutrient supplements. In some embodiments, the medium further contains a selection agent selected based on the construction of the expression vector, thereby selectively allowing growth of prokaryotic cells containing the expression vector. For example, ampicillin may be added to the medium to grow cells expressing the ampicillin resistance gene.

[0208] Any necessary supplements other than carbon, nitrogen, and inorganic phosphate sources may be introduced at appropriate concentrations, either alone or in admixture with another supplement or medium, e.g., a complex nitrogen source. Optionally, the medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol, and dithiothreitol. Prokaryotic host cells are cultured at a suitable temperature and pH.

[0209] When an inducible promoter is used in the expression vector of the present disclosure, protein expression is induced under conditions suitable for promoter activation. In one aspect of the present disclosure, the PhoA promoter is used to control the transcription of polypeptide. Therefore, the transformed host cell is induced by culturing in a phosphate-limited medium. In some embodiments, the phosphate-limited medium is CRAP medium (see, for example, Simmons et al., J. Immunol. Methods 263:133-147 (2002)). Depending on the vector construct used, several other inducers known in the art can be used.

[0210] The expressed antibodies of the present disclosure are secreted into the periplasm of the host cells and recovered therefrom. Protein recovery typically involves disrupting the microorganism, typically using methods such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or intact cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported into the culture medium and isolated therefrom. The produced protein can be further purified by removing the cells from the medium, filtering, and concentrating the culture supernatant. The expressed polypeptide can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0211] Alternatively, proteins can be produced in large quantities by fermentation. Various large-scale fed-batch fermentation procedures can be used to produce recombinant proteins. Various fermentation conditions can be modified to increase the yield and quality of the antibodies of the present disclosure. For example, chaperones have been shown to aid in the correct folding and solubility of heterologous proteins produced in bacterial host cells. See Chen et al., J Bio Chem 274:19601-19605 (1999), U.S. Patent No. 6,083,715, U.S. Patent No. 6,027,888, Bothmann and Pluckthun, J. Biol. Chem. 275:17100-17105 (2000), Ramm and Pluckthun, J. Biol. Chem. 275:17106-17113 (2000), and Arie et al., Mol. Microbiol. 39:199-210 (2001).

[0212] To minimize proteolysis of expressed heterologous proteins (especially those that are sensitive to proteolysis), several host strains lacking proteolytic enzymes can be used in the present disclosure, as described, for example, in U.S. Patent Nos. 5,264,365, 5,508,192, and Hara et al., Microbial Drug Resistance, 2:63-72 (1996). E. coli strains lacking proteolytic enzymes and transformed with plasmids overexpressing one or more chaperones can be used as host cells in expression systems encoding the antibodies of the present disclosure.

[0213] The antibodies produced herein can be further purified to obtain substantially homogeneous preparations for further measurement and use. Standard protein purification methods known in the art can be used. The following procedures are examples of suitable purification procedures: fractionation on an immunoaffinity or ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or cation exchange resins, e.g., DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel filtration, e.g., using Sephadex G-75. Protein A immobilized on a solid phase can be used, for example, in some embodiments for immunoaffinity purification of binding molecules of the present disclosure. The solid phase on which Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled pore glass column or a silicic acid column. In some embodiments, the column is coated with a reagent such as glycerol to prevent nonspecific adhesion of contaminants. The solid phase is then washed to remove contaminants nonspecifically bound to the solid phase. Finally, the antibody of interest is recovered from the solid phase by elution.

[0214] Recombinant production in eukaryotic cells For eukaryotic expression, the vector components usually include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0215] Vectors used in eukaryotic hosts may also contain a signal sequence or other polypeptide insert with a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences and viral secretory leader sequences, such as the herpes simplex gD signal, can be obtained. The DNA of such precursor regions can be ligated in reading frame to DNA encoding the antibodies of the present disclosure.

[0216] Generally, mammalian expression vectors do not need the origin of replication component (the SV40 origin may typically be used because it contains the early promoter).

[0217] Expression and cloning vectors may contain a selection gene, also called a selection marker, that encodes a protein that confers resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), complements an auxotrophic deficiency, or provides a vital nutrient not available from complex media.

[0218] In one form of selectable morphology, a drug is used to inhibit the growth of host cells. Cells successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive in the selectable morphology. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0219] Another example of a suitable selection marker for use in mammalian cells is one that allows the identification of cells capable of incorporating nucleic acids encoding the antibodies of the present disclosure. For example, cells transformed with the DHFR selection gene are identified by first culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is used, an exemplary suitable host cell is a Chinese hamster ovary (CHO) cell line lacking DHFR activity. Alternatively, host cells transformed or co-transformed with another selection marker, such as a DNA sequence encoding a polypeptide, a wild-type DHFR protein, and an aminoglycoside 3'-phosphotransferase (APH), (particularly wild-type hosts containing endogenous DHFR) can be selected by growing the cells in a medium containing the selection agent used for the selection marker, e.g., an aminoglycoside antibiotic.

[0220] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to a nucleic acid encoding a desired polypeptide sequence. Eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Many genes may also contain additional sequences found 70 to 80 bases upstream of the transcription start site. The 3' end of most eukaryotic organisms may be a signal for adding a polyadenylic acid tail to the 3' end of the coding sequence. Any of these sequences can be inserted into a eukaryotic expression vector.

[0221] Transcription of the polypeptide of the vector from a mammalian host cell can be controlled by, for example, a promoter derived from the genome of a virus (e.g., polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, reverse transcription virus, hepatitis B virus, and simian virus 40 (SV40)), a heterologous mammalian promoter (e.g., the actin promoter or an immunoglobulin promoter), or a heat shock promoter, provided that the promoter is compatible with the host cell system.

[0222] Transcription of DNA encoding the antibody of this disclosure by higher eukaryotes is usually increased by inserting an enhancer sequence into the vector. Many enhancer sequences are currently known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). Examples include the SV40 enhancer (bp 100-270), which is located on the late side of the replication origin, the cytomegalovirus early promoter enhancer, and the polyoma and adenovirus enhancers, which are located on the late side of the replication origin. See Yaniv, Nature 297:17-18 (1982) for further information on enhancer elements for activating eukaryotic promoters. The enhancer may be spliced ​​into the vector at a position 5' or 3' from the polypeptide-coding sequence, but is preferably located at a 5' site from the promoter.

[0223] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) also contain sequences necessary for the termination of transcription and stabilization of mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed into polyadenylated fragments in the untranslated portion of the mRNA encoding a polypeptide. A useful transcription termination component is the bovine growth hormone polyadenylation region.

[0224] Suitable host cells for cloning or expressing the DNA in the vectors herein include the higher eukaryotic cells described herein, including vertebrate host cells. Propagation of vertebrate cells in culture (tissue culture) has become a common procedure. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651), the human embryonic kidney line (293 or a 293 cell subclone used for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TR1 cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)), MRC 5 cells, FS4 cells, and a human hepatoma cell line (Hep G2).

[0225] Host cells can be transformed with the above-described expression or cloning vectors to produce the antibody and cultured in conventional nutrient media, modified appropriately to induce promoters, select transformants, or amplify the genes encoding the desired sequences.

[0226] Host cells for producing the antibodies of the present disclosure can be cultured in several media. Commercially available media, such as Ham's F10 (Sigma), minimal essential medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM, Sigma), are suitable for culturing host cells. Any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Pat. Reissue 30,985 can also be used as a culture medium for host cells. Any one of these media can be supplemented, as needed, with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., the drug GENTAMYCIN™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an energy equivalent. It may further contain any other necessary supplements at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., will be those used with the host cell previously selected for expression and will be apparent to those skilled in the art.

[0227] When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If antibodies are produced intracellularly, the first step is to remove particulate debris, host cells, or lysed fragments, for example, by centrifugation or ultrafiltration. If the antibody is secreted into the medium, the supernatant from this expression system is usually first concentrated through a commercially available protein concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). A protease inhibitor, such as PMSF, may be included in any of these steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0228] Protein compositions prepared from cells can be purified by, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The matrix to which the affinity ligand is linked is typically agarose, although other matrices are also available. Compared to agarose, mechanically stable matrices, such as controllable pore size glass or poly(styrene-divinyl)benzene, allow for faster flow rates and shorter processing times. Depending on the antibody to be recovered, other protein purification techniques, such as fractional separation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, silica gel chromatography, heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (e.g., a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, may also be used. After any one or more preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low-pH hydrophobic interaction chromatography.

[0229] 5.2.7. Binding Molecules Including Single Domain Antibodies In another aspect, the present specification provides binding molecules comprising a single domain antibody (e.g., a VHH domain against GCC) according to the present specification. In addition to the chimeric antigen receptors (CARs) according to the present specification, as described in Section 5.3 below, in some embodiments, a single domain antibody against GCC according to the present specification is also part of another binding molecule. Exemplary binding molecules of the present disclosure are described herein.

[0230] Fusion proteins In various embodiments, a single domain antibody according to the present invention may be genetically fused or chemically conjugated to another agent, e.g., a protein-based entity. The single domain antibody may be chemically conjugated to the agent or otherwise non-covalently conjugated to the agent. The agent may be a peptide or an antibody (or fragment thereof).

[0231] Thus, in some embodiments, the present disclosure provides single domain antibodies (e.g., VHH domains) that are recombinantly fused or chemically conjugated (covalently or non-covalently conjugated) to a heterologous protein or polypeptide (or fragment thereof, e.g., a polypeptide of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, or about 500 amino acids or more than 500 amino acids) to produce a fusion protein, and uses thereof. In particular, the present disclosure provides a fusion protein comprising an antigen-binding fragment (e.g., CDR1, CDR2 and / or CDR3) of a single domain antibody in accordance with the present disclosure and a heterologous protein, polypeptide, or peptide.

[0232] Additionally, antibodies herein can be fused to a tag or "tag" sequence (e.g., a peptide) to facilitate purification. In specific embodiments, the tag or tag amino acid sequence can be a hexahistidine peptide, a hemagglutinin ("HA") tag, or a "FLAG" tag.

[0233] Methods for fusing or conjugating moieties (including polypeptides) to antibodies are known (see, e.g., Arnon et al., Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy, Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al., eds., 1985); Hellstrom et al., Antibodies for Drug Delivery, Controlled Drug Delivery 623-53 (Robinson et al., eds., 2nd ed., 1987); Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review, Monoclonal Antibodies: Biological and Clinical Applications 475-506 (Pinchera et al., eds., 1985); Analysis, Results, and Future Prospect of the Therapeutic Use of Radiolabeled Antibodies in Cancer Therapy, Monoclonal Antibodies for Cancer Detection and Therapy 475-506 (Pinchera et al., eds., 1985); 303-16 (ed. by Baldwin et al., 1985), Thorpe et al., Immunol. Rev. 62:119-58 (1982), U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946, EP 307434, EP 367,166, EP 394,827, PCT Publications WO 91 / 06570, WO 96 / 04388, WO 96 / 22024, WO 97 / 34631, and WO 99 / 04813, Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-39 (1991), Traunecker et al., Nature, 331: 84-86 (1988), Zheng et al., J. Immunol.154:5590-600 (1995), and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-41 (1992).

[0234] Fusion proteins can be produced, for example, by gene shuffling, motif shuffling, exon shuffling and / or codon shuffling (collectively referred to as "DNA shuffling") techniques. DNA shuffling can be used to alter the activity of single domain antibodies according to the present invention, including, for example, antibodies with high affinity and low dissociation rates (see, e.g., U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998)). Antibodies or the encoded antibodies can be altered prior to recombination by random mutagenesis using error-prone PCR, random nucleotide insertion, or other methods. Polynucleotides encoding antibodies herein can be recombined with one or more components, motifs, segments, portions, domains, fragments, etc., of one or more heterologous molecules.

[0235] In some embodiments, a single domain antibody (eg, a VHH domain) according to the present invention is conjugated to a second antibody to form an antibody heteroconjugate.

[0236] In various embodiments, the single domain antibody is genetically fused to the agent. Genetic fusion can be achieved by placing a linker (e.g., a polypeptide) between the single domain antibody and the agent. The linker can be a flexible linker.

[0237] In various embodiments, the single domain antibody is genetically conjugated to a therapeutic molecule, where the hinge region links the single domain antibody to the therapeutic molecule.

[0238] The present specification further provides methods of producing various fusion proteins according to the present specification. The various methods described in Section 5.2.6 above can be used to produce fusion proteins according to the present specification.

[0239] In a specific embodiment, a fusion protein according to the present disclosure is recombinantly expressed. Recombinant expression of a fusion protein according to the present disclosure may require construction of an expression vector containing a polynucleotide encoding the protein or a fragment thereof. Once a polynucleotide encoding a protein according to the present disclosure or a fragment thereof is obtained, a vector for producing the molecule can be generated by recombinant DNA technology using techniques well known in the art. Thus, described herein are methods for preparing a protein by expressing a polynucleotide containing the encoding nucleotide sequence. Methods well known to those skilled in the art can be used to construct an expression vector containing the coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Further provided are replicable vectors containing a nucleotide sequence encoding a fusion protein according to the present disclosure or a fragment or CDR thereof operably linked to a promoter.

[0240] The expression vector can be transferred to a host cell by conventional techniques and the transfected cells can be cultured by conventional techniques to produce a fusion protein according to the present invention. Thus, the present invention further provides a host cell containing a polynucleotide encoding a fusion protein according to the present invention or a fragment thereof, operably linked to a heterologous promoter.

[0241] A variety of host-expression vector systems can be used to express the fusion proteins according to the present invention. Such host-expression systems represent vehicles by which coding sequences of interest can be produced and subsequently purified, as well as cells that, when transformed or transfected with the appropriate nucleotide coding sequences, can express the fusion proteins according to the present invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing the coding sequence, yeast (e.g., Pastoris) transformed with recombinant yeast expression vectors containing the coding sequence, insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the coding sequence, plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence, or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) containing recombinant expression constructs, which contain promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or from mammalian viruses (e.g., adenovirus late promoters, cowpox virus 7.5K promoters). Bacterial cells such as Escherichia coli, or eukaryotic cells, particularly bacterial cells for expressing complete recombinant antibody molecules, can be used to express recombinant fusion proteins. For example, the combination of mammalian cells such as Chinese hamster ovary cells (CHO) with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus is an effective expression system for antibodies or variants thereof. In specific embodiments, expression of the nucleotide sequence encoding the fusion protein according to the present invention is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0242] In bacterial systems, several expression vectors can be advantageously selected depending on the desired use of the expressed fusion protein. For example, if large quantities of such fusion proteins need to be produced, a vector that directs the expression of high-level fusion protein products that are easily purified to produce pharmaceutical compositions of the fusion protein may be required. These vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO 12:1791 (1983)), in which the coding sequence can be ligated alone in framework with the lacZ coding region to produce the fusion protein, and the pIN vector (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)). pGEX vectors can also be used to express exogenous polypeptides as fusion proteins with glutathione 5-transferase (GST). Typically, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads and elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0243] Many viral-based expression systems can be used in mammalian host cells. When adenovirus is used as an expression vector, the coding sequence of interest can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. The chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) can produce recombinant virus that is viable and capable of expressing the fusion protein in infected hosts (see, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA 8 1:355-359 (1984)). Specific initiation signals may be required for efficient translation of the inserted coding sequence. These signals include the ATG initiation codon and adjacent sequences. Note that the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of sources, both natural and synthetic. The efficiency of expression may be enhanced by including appropriate transcription enhancer elements, transcription terminators, etc. (see, for example, Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

[0244] In addition, a host cell line can be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in a specific fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of expressed exogenous proteins. Therefore, eukaryotic host cells that possess the appropriate cellular machinery for processing the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20, and T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, and HsS78Bst cells.

[0245] Stable expression may be utilized for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express fusion proteins may be engineered. To use expression vectors containing viral origins of replication, host cells may be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) and a selection marker. After introduction of the exogenous DNA, engineered cells may be grown in rich medium for 1-2 days and then transferred to selective medium. The selection marker on the recombinant plasmid confers resistance to selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded to form cell lines. This method may be advantageously used to engineer cell lines that express fusion proteins. Such engineered cell lines are particularly useful for screening and evaluating compositions that interact directly or indirectly with binding molecules.

[0246] Several selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine guanine phosphoribosyltransferase (Lowy et al., Cell 22:8-17 (1980)), genes can be used to select tk-, hgprt-, or aprt-cells, respectively. Furthermore, antimetabolite resistance can be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)), gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)), and neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993)). (1993), and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993), May, TIB TECH 11(5):155-2 15 (1993)), and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the field of recombinant DNA technology can generally be used to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Dracopoli et al. (eds.), Current Protocols in Human Genetics, Chapters 12 and 13, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated herein by reference in their entireties.

[0247] The expression level of a fusion protein can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987)). If the marker in the vector system expressing the fusion protein is amplifiable, increasing the level of inhibitor present in the host cell culture will cause an increase in the copy number of the marker gene. Because the amplified region is associated with the fusion protein gene, production of the fusion protein will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

[0248] A host cell can be co-transfected with multiple expression vectors according to the present invention. The vectors may contain the same selection markers, which allow for equivalent expression of the corresponding encoded polypeptides. Alternatively, a single vector can be used that encodes and expresses multiple polypeptides. The coding sequences may comprise cDNA or genomic DNA.

[0249] Once produced by recombinant expression, the fusion proteins provided herein can be purified by any method known in the art for purifying polypeptides (e.g., immunoglobulin molecules), such as chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen following Protein A, sizing column chromatography, and Kappa select affinity chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Additionally, the fusion protein molecules provided herein can be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

[0250] Immunoconjugates In some embodiments, the present disclosure further provides immunoconjugates, which comprise any of the antibodies described herein (e.g., anti-GCC single domain antibodies) conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope.

[0251] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC), in which the antibody is conjugated to one or more drugs, such as maytansinoids (U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. EP 0 425 235). B1), auristatins, e.g., monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298), dolastatins, calicheamicin or derivatives thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)), anthracyclines such as donomycin or doxorubicin (Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002), and U.S. Patent No. 6,630,579), methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortaxel, trichothecenes, and CC1065.

[0252] In some embodiments, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, midecamycin A chain, α-sarcinin, abrin, dianthin, Phytolacca americana (PAPI, PAPII, and PAP-S), Momordica chalantia inhibitor, toxin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecin.

[0253] In some embodiments, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. Multiple radioisotopes may be used in the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. When a radioconjugate is used for detection, it may contain a radioactive atom used in scintillation studies, such as TC99m or I123, or a spin label used in nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0254] Conjugates of antibodies and cytotoxic agents can be prepared with a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridinedimercapto)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminosulfan (IT), bifunctional derivatives of imidoesters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), disazo compounds (e.g., bis(p-azidobenzoyl)hexanediamine), double nitrogen derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026.

[0255] The linker may be a "cleavable linker" that facilitates release of the conjugate in cells, although non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates of the present disclosure include, but are not limited to, acid-labile linkers (e.g., hydrazone linkers), disulfide bond-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers containing amino acids such as valine and / or citrulline, e.g., citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers, thioether linkers, or hydrophilic linkers designed to circumvent multidrug transport protein-mediated resistance.

[0256] The immunoconjugates or ADCs herein contemplate such conjugates prepared using crosslinkers, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone) vinylsulfone), which are commercially available (e.g., obtained from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0257] In other embodiments, the antibodies herein are conjugated or recombinantly fused with, for example, a diagnostic molecule. Such diagnosis and detection can be accomplished, for example, by coupling the antibody to a detectable substance, including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; conjugated groups, such as, but not limited to, streptavidin / biotin or avidin / biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinamide fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as, but not limited to, luciferase, fluorescein, or aequorin; and chemiluminescent materials, such as 225Ac gamma-, Auger-, beta-, alpha-, or positron-emitting radioisotopes.

[0258] 5.3. Chimeric Antigen Receptors In another aspect, the description provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises a single domain antibody (e.g., VHH) that binds to a GCC in accordance with the present invention. In another aspect, the description provides a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises one or more single domain antibodies (e.g., VHH) that bind to a GCC in accordance with the present invention. Exemplary CARs comprising VHH domains of the present invention (i.e., VHH-based CARs or double VHH-based CARs) are described in Section 6, below.

[0259] In some embodiments, a chimeric antigen receptor (CAR) according to the present disclosure comprises a polypeptide, the polypeptide comprising (a) an extracellular antigen-binding domain comprising a single domain antibody (sdAb) that specifically binds to a GCC according to the present disclosure and, optionally, one or more additional binding domains, (b) a transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, a chimeric antigen receptor (CAR) according to the present disclosure comprises a polypeptide, the polypeptide comprising (a) an extracellular antigen-binding domain comprising one or more single domain antibodies (sdAb) that specifically bind to a GCC according to the present disclosure and, optionally, one or more additional binding domains, (b) a transmembrane domain, and (c) an intracellular signaling domain. Each portion and additional region is described in more detail below.

[0260] 5.3.1. Extracellular Antigen-Binding Domains The extracellular antigen-binding domain of a CAR described herein comprises one or more (e.g., any one of 1, 2, 3, 4, 5, 6, or more) single domain antibodies. In some embodiments, the extracellular antigen-binding domain of a CAR comprises one single domain antibody. In some embodiments, the extracellular antigen-binding domain of a CAR comprises two single domain antibodies. The single domain antibodies can be directly fused to each other via a peptide bond or a peptide linker.

[0261] The CAR of the present disclosure comprises an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises one or more single-domain antibodies. The sdAbs may be of the same or different sources and may have the same or different sizes. In some embodiments, the extracellular antigen-binding domain in accordance with the present disclosure comprises at least one binding domain, and the at least one binding domain comprises a single-domain antibody that binds to GCC in accordance with the present disclosure, e.g., an anti-GCC single-domain antibody described in Section 5.2 above. In some embodiments, the extracellular antigen-binding domain in accordance with the present disclosure comprises one or more binding domains, and each of the one or more binding domains comprises a single-domain antibody that binds to GCC in accordance with the present disclosure, e.g., an anti-GCC single-domain antibody described in Section 5.2 above. In some embodiments, the extracellular antigen-binding domain in accordance with the present disclosure comprises an anti-GCC single-domain antibody.

[0262] In some embodiments, the present disclosure provides a CAR comprising a polypeptide, the polypeptide comprising: (a) an extracellular antigen-binding domain comprising one or more (e.g., one) anti-GCC sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein each anti-GCC sdAb is an anti-GCC sdAb described in Section 5.2 above, e.g., the anti-GCC sdAb comprises a CDR1, a CDR2, and a CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in any one of SEQ ID NOs: 26-41, and the anti-GCC sdAb comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of any one of SEQ ID NOs: 26-41.

[0263] In other embodiments, the extracellular antigen-binding domain comprises two or more antigen-binding domains, at least one of which is a VHH that binds to a GCC in accordance with the present disclosure, and one or more additional binding domains that bind to one or more additional antigens, e.g., one, two, three, four, or more additional single domain antibody binding regions (sdAbs) that target one or more additional antigens. In some embodiments, at least one additional binding domain binds to a GCC in accordance with the present disclosure. In some embodiments, the antigen-binding domains are fused to each other via a peptide linker. In addition to one or more antigen-binding domains in accordance with the present disclosure, the CAR in accordance with the present disclosure may further comprise one or more of a linker (e.g., a peptide linker), a transmembrane domain, a hinge region, a signal peptide, an intracellular signaling domain, or a costimulatory signaling domain, each of which is described in more detail below.

[0264] For example, in some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof. In some embodiments, the costimulatory signaling domain is derived from CD137. In some embodiments, the GCC CAR further comprises a hinge domain (e.g., a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the GCC CAR further comprises a signal peptide (e.g., a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the GCC CAR is monospecific. In some embodiments, the GCC CAR is monovalent. In some embodiments, the GCC CAR is bivalent or bispecific.

[0265] Peptide Linker When multiple antibodies (e.g., multiple antibody fragments) are present in the CAR of the present invention, each antibody can be fused to each other via a peptide linker. In some embodiments, the antibodies are fused directly to each other without any peptide linker. The peptide linkers connecting different antibodies can be the same or different. Different domains of the CAR can also be fused to each other via peptide linkers.

[0266] Depending on the structural and / or functional characteristics of the antibody and / or each domain, each peptide linker in a CAR may have the same or different length and / or sequence. Each peptide linker may be independently selected and optimized. The length, flexibility, and / or other properties of the peptide linker(s) used in a CAR may affect its properties, including, but not limited to, affinity, specificity, or avidity for one or more specific antigens or epitopes. For example, a longer peptide linker may be selected to ensure that two adjacent domains do not spatially interfere with each other. In some embodiments, a short peptide linker may be placed between the transmembrane domain and the intracellular signaling domain of a CAR. In some embodiments, the peptide linker contains flexible residues (e.g., glycine and serine), allowing adjacent domains to move freely relative to each other. For example, a glycine-serine bipartite may be a suitable peptide linker.

[0267] The peptide linker may have any suitable length. In some embodiments, the length of the peptide linker may be at least about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or more amino acids. In some embodiments, the length of the peptide linker may be no more than about any one of 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or fewer amino acids. In some embodiments, the length of the peptide linker can be any one of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0268] The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, the sequence of the hinge region from an antibody having only a heavy chain may be used as a linker. See, for example, WO 1996 / 34103. In some embodiments, the peptide linker is a flexible linker. An exemplary flexible linker is a glycine polymer (G) n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, threonine-serine, and other flexible linkers known in the art. Other linkers known in the art may be included in a CAR according to the present invention, for example, as described in WO 2016014789, WO 2015158671, WO 2016102965, US 20150299317, WO 2018067992, US 7741465, Colcher et al., J. Nat. Cancer Inst. 82:1191-1197 (1990), and Bird et al., Science 242:423-426 (1988), the disclosures of each of which are incorporated herein by reference.

[0269] In some specific embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 75-77.

[0270] 5.3.2. Transmembrane domains The CAR of the present disclosure comprises a transmembrane domain that can be fused directly or indirectly to an extracellular antigen-binding domain. The transmembrane domain may be derived from natural or synthetic sources. As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane (e.g., a eukaryotic cell membrane). A transmembrane domain suitable for the CARs described herein can be obtained from a naturally occurring protein. Alternatively, it may be a synthetic, non-natural protein segment, such as a hydrophobin segment, that is thermodynamically stable in a cell membrane.

[0271] Transmembrane domains are classified based on their three-dimensional structure. For example, transmembrane domains can form an α-helix, a complex of one or more α-helices, a β-barrel, or any other stable structure that can span a cellular phospholipid bilayer. Transmembrane domains can also or alternatively be classified based on their topological structure, including the number of times the transmembrane domain passes through the membrane and the orientation of the protein. For example, single-pass transmembrane proteins pass through the cellular membrane once, while multi-pass transmembrane proteins pass through the cellular membrane at least twice (e.g., 2, 3, 4, 5, 6, 7, or more times). Membrane proteins can be defined as type I, type II, or type III depending on their ends relative to the inside and outside of the cell and the topological structure of one or more transmembrane segments. Type I membrane proteins have a single transmembrane region and are oriented so that the N-terminus of the protein is located on the extracellular side of the cellular lipid bilayer and the C-terminus of the protein is located on the inside of the cell. Type II membrane proteins also have a single transmembrane region, but are oriented so that the C-terminus of the protein is on the extracellular side of the cell's lipid bilayer and the N-terminus of the protein is on the inner side of the cell. Type III membrane proteins have multiple transmembrane segments and can be further classified based on the number of transmembrane segments and the location of the N- and C-termini.

[0272] In some embodiments, the transmembrane domain of a CAR described herein is derived from a type I single-pass membrane protein. In some embodiments, transmembrane domains from multi-pass membrane proteins may be used to adapt a CAR described herein. A multi-pass membrane protein may comprise a complex (at least 2, 3, 4, 5, 6, 7, or more) alpha helix or beta sheet structure. In some embodiments, the N- and C-termini of a multi-pass membrane protein are on opposite sides of a lipid bilayer, e.g., the N-terminus of the protein is on the intracellular side of the lipid bilayer, while the C-terminus of the protein is on the extracellular side.

[0273] In some embodiments, the transmembrane domain of the CAR is selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, tight junction protein-6, IL-2R beta, IL-2R gamma, IL-7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. In some embodiments, the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

[0274] In some specific embodiments, the transmembrane domain is derived from CD8α. In some embodiments, the transmembrane domain is the transmembrane domain of CD8α comprising the amino acid sequence of SEQ ID NO:71.

[0275] The transmembrane domain used in the CARs described herein may further comprise at least a portion of a synthetic, non-naturally occurring protein segment. In some embodiments, the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet. In some embodiments, the protein segment is at least about 20 amino acids, e.g., at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids. Examples of synthetic transmembrane domains are known in the art, e.g., U.S. Pat. No. 7,052,906 and PCT Publication No. WO 2000 / 032776, the relevant disclosures of which are incorporated herein by reference.

[0276] A transmembrane domain according to the present disclosure may comprise a transmembrane region and an intracellular region located C-terminal to the transmembrane domain. The intracellular region of the transmembrane domain may comprise three or more amino acids, and in some embodiments, favors orientation of the transmembrane domain in a lipid bilayer. In some embodiments, one or more cysteine ​​residues are present in the transmembrane region of the transmembrane domain. In some embodiments, one or more cysteine ​​residues are present in the intracellular region of the transmembrane domain. In some embodiments, the intracellular region of the transmembrane domain comprises positively charged amino acids. In some embodiments, the intracellular region of the transmembrane domain comprises the amino acids arginine, serine, and lysine.

[0277] In some embodiments, the transmembrane region of the transmembrane domain comprises hydrophobic amino acid residues. In some embodiments, the transmembrane domain of a CAR according to the present specification comprises a hydrophobic artificial sequence. For example, a triplet of phenylalanine, tryptophan, and valine may be present at the C-terminal position of the transmembrane domain. In some embodiments, the transmembrane region comprises primarily hydrophobic amino acid residues, such as alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or valine. In some embodiments, the transmembrane region is hydrophobic. In some embodiments, the transmembrane region comprises a polyleucine-alanine sequence. The hydrophilic or hydrophobic or hydrophilic characteristics of a protein or protein segment can be assessed by any method known in the art, such as Kyte and Doolittle hydrophilicity analysis.

[0278] 5.3.3. Intracellular signaling domains The CAR of the present disclosure comprises an intracellular signaling domain. The intracellular signaling domain is responsible for activating at least one normal effector function of an immune effector cell expressing the CAR. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or an accessory activity including cytokine secretion. Thus, the term "intracellular signaling domain" refers to a protein portion that transmits an effector function signal and instructs a cell to perform a specific function. Typically, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. Regarding the use of truncated portions of the intracellular signaling domain, such truncated portions may be used instead of the intact chain, so long as they transmit the effector function signal. Thus, the term intracellular signaling domain is intended to refer to any truncated portion that includes a sufficient intracellular signaling domain to transmit the effector function signal.

[0279] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, a CAR comprises an intracellular signaling domain consisting essentially of a primary intracellular signaling domain of an immune effector cell. A "primary intracellular signaling domain" refers to an intracellular signaling sequence that stimulates and induces immune effectors to function. In some embodiments, the primary intracellular signaling domain contains a signaling motif called an immunoreceptor tyrosine-based activation motif, or ITAM. As used herein, "ITAM" is a conserved protein motif typically found in the tails of signaling molecules expressed in immune cells. The motif may comprise two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, where each x is independently any amino acid, producing the conserved motif YxxL / Ix(6-8)YxxL / I. ITAMs in signaling molecules are important for intracellular signaling, which is mediated, at least in part, by phosphorylation of tyrosine residues in ITAMs following activation of the signaling molecule. ITAMs can also serve as docking sites for other proteins involved in signaling pathways. Exemplary ITAM-containing primary intracellular signaling sequences include those derived from CD3ζ, FcRγ (FCER1G), FcRβ (Fcε Rib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.

[0280] In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain consists of the intracellular signaling domain of CD3ζ. In some embodiments, the primary intracellular signaling domain is the intracellular signaling domain of wild-type CD3ζ. In some embodiments, the primary intracellular signaling domain of CD3ζ comprises the amino acid sequence of SEQ ID NO: 73.

[0281] 5.3.4. Costimulatory Signaling Domains In addition to stimulating antigen-specific signals, many immune effector cells require costimulation to promote cell proliferation, differentiation, and survival and to activate cellular effector functions. In some embodiments, a CAR comprises at least one costimulatory signaling domain. As used herein, the term "costimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signaling to induce an immune response (e.g., effector function). The costimulatory signaling domain of the chimeric receptor described herein can be an intracellular signaling domain from a costimulatory protein, which transmits a signal and regulates responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils. A "costimulatory signaling domain" can also be the intracellular portion of a costimulatory molecule. The term "costimulatory molecule" refers to a homologous binding partner in immune cells (e.g., T cells) that mediates a costimulatory response of the immune cell, such as, but not limited to, proliferation and survival, by specifically binding to a costimulatory ligand.

[0282] In some embodiments, the intracellular signaling domain comprises a single costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises two or more (e.g., about any one of 2, 3, 4, or more) costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more of the same costimulatory signaling domains. In some embodiments, the intracellular signaling domain comprises two or more costimulatory signaling domains from different costimulatory proteins (e.g., any two or more costimulatory signaling domains described herein). In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain (e.g., the intracellular signaling domain of CD3ζ) and one or more costimulatory signaling domains. In some embodiments, the one or more costimulatory signaling domains and the primary intracellular signaling domain (e.g., the intracellular signaling domain of CD3ζ) are fused to each other via any peptide linker. The primary intracellular signaling domain and one or more costimulatory signaling domains can be arranged in any suitable order. In some embodiments, the one or more costimulatory signaling domains are located between the transmembrane domain and the primary intracellular signaling domain (e.g., the intracellular signaling domain of CD3ζ). Multiple costimulatory signaling domains can provide additive or synergistic stimulatory effects.

[0283] Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) can induce the cell to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory molecule is applicable to the CARs described herein. The type or types of costimulatory signaling domains are selected based on factors such as, for example, the type of immune effector cell (e.g., T cell, NK cell, macrophage, neutrophil, or eosinophil) on which the effector molecule will be expressed and the desired immune effector function (e.g., ADCC effector). Examples of costimulatory signaling domains used in CARs can be the intracellular signaling domains of costimulatory proteins, including members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6) and members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF, BLyS ... R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, lymphotoxin-α / TNF-β, OX40 / TNFRSF4, OX40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF-α, and TNFRII / TNFRSF1B), SLAM family members (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150), and any other costimulatory molecules, such as CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA These include, but are not limited to, class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C.

[0284] In some embodiments, the one or more costimulatory signaling domains are selected from the group consisting of a ligand that specifically binds to CD27, CD28, CD137, OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0285] In some embodiments, the intracellular signaling domain in a CAR of the present disclosure comprises a costimulatory signaling domain derived from CD137 (i.e., 4-1BB). In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain of CD3ζ and the costimulatory signaling domain of CD137. In some embodiments, the intracellular signaling domain comprises the costimulatory signaling domain of CD137, which comprises the amino acid sequence of SEQ ID NO: 72.

[0286] Mutants of any of the costimulatory signaling domains described herein are also within the scope of the present disclosure, whereby the costimulatory signaling domain can modulate the immune response of an immune cell. In some embodiments, the costimulatory signaling domain comprises up to 10 (e.g., 1, 2, 3, 4, 5, or 8) amino acid residue mutations compared to a wild-type counterpart. Such costimulatory signaling domains comprising one or more amino acid changes may be referred to as mutants. Compared to a costimulatory signaling domain that does not comprise a mutation, mutations of amino acid residues in the costimulatory signaling domain can result in increased signaling and enhanced stimulation of the immune response. Compared to a costimulatory signaling domain that does not comprise a mutation, mutations of amino acid residues in the costimulatory signaling domain can result in decreased signaling and reduced stimulation of the immune response.

[0287] Hinge Area The CAR of the present disclosure may include a hinge domain located between the extracellular antigen-binding domain and the transmembrane domain. A hinge domain is an amino acid segment that is usually expressed between two domains of a protein and can allow one or two flexible domains of a protein to move relative to each other. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen-binding domain relative to the transmembrane domain of the effector molecule can be used.

[0288] The hinge domain may comprise any one of about 10 to 100 amino acids, e.g., about 15 to 75 amino acids, 20 to 50 amino acids, or 30 to 60 amino acids. In some embodiments, the length of the hinge domain may be at least any one of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids.

[0289] In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Any hinge domain of a protein known in the art that contains a hinge domain is applicable to the chimeric receptors described herein. In some embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In some embodiments, the hinge domain is derived from CD8α. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, for example, comprising a fragment of at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. In some embodiments, the hinge domain of CD8α comprises the amino acid sequence of SEQ ID NO: 70.

[0290] The hinge domain of an antibody (e.g., an IgG, IgA, IgM, IgE, or IgD antibody) is also applicable to the pH-dependent chimeric receptor system described herein. In some embodiments, the hinge domain is the hinge domain connecting the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is the hinge domain of an antibody and comprises the hinge domain of an antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the hinge domain of the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the hinge domain of the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

[0291] Non-naturally occurring peptides may be used as the hinge domain of the chimeric receptors described herein. In some embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain and the N-terminus of the transmembrane domain of an Fc receptor is a peptide linker, such as a (GGGGS)n linker (e.g., SEQ ID NO:76), where n can be an integer, such as 1, 2, 3, 4, or more, or a (GxS)n linker (e.g., SEQ ID NO:77), where x and n can independently be integers from 3 to 12, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

[0292] 5.3.6. Signal peptide A CAR of the present disclosure may comprise a signal peptide (also referred to as a signal sequence) at the N-terminal position of the polypeptide. Typically, a signal peptide is a peptide sequence that targets a polypeptide to a desired site within a cell. In some embodiments, the signal peptide targets the effector molecule to the cell's secretory pathway and enables incorporation and anchoring of the effector molecule into the lipid bilayer. Signal peptides applicable to the CARs described herein, including signal sequences of naturally occurring proteins or synthetic, non-natural signal sequences, will be apparent to those of skill in the art. In some embodiments, the signal peptide is derived from a molecule selected from the group consisting of CD8α, GM-CSF receptor α, and IgG1 heavy chain. In some embodiments, the signal peptide is from CD8α. In some embodiments, the signal peptide of CD8α comprises the amino acid sequence of SEQ ID NO: 69.

[0293] 5.3.7. Example CAR binding to GCC The generation of exemplary CARs that bind to GCC is provided in Section 6, below. In some embodiments, the present specification provides a CAR that comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 45-60. In some embodiments, a CAR provided herein comprises an amino acid sequence having a certain percent identity to any one of the exemplary CARs in Section 6, below. In some embodiments, the present specification provides a GCC CAR that comprises a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 45-60.

[0294] In some embodiments, the description provides an isolated nucleic acid encoding any GCC CAR according to the description. More detailed information regarding nucleic acid sequences and vectors is provided below.

[0295] Other exemplary CARs according to the present disclosure further comprise a chimeric receptor. In some embodiments, the chimeric receptor comprises TGFβR and / or IL23R. In some specific embodiments, a CAR according to the present disclosure comprises SEQ ID NO: 61. In other specific embodiments, a CAR according to the present disclosure comprises SEQ ID NO: 62. In other specific embodiments, a CAR according to the present disclosure comprises SEQ ID NO: 63. In some embodiments, the present disclosure provides a CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 61. In some embodiments, provided herein is a CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 62. In some embodiments, provided herein is a CAR comprising a polypeptide having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 63.

[0296] In some embodiments, the description provides isolated nucleic acids encoding any of the CARs and chimeric receptors described herein. More detailed information regarding nucleic acid sequences and vectors is provided below.

[0297] 5.4. Engineered immune cells In yet another aspect, the description provides a host cell (e.g., an immune cell) comprising any one of the CARs described herein. The host cell can be an immune effector cell.

[0298] Thus, in some embodiments, the present disclosure provides an engineered immune cell (e.g., a T cell) comprising a CAR, wherein the CAR comprises a polypeptide, the polypeptide comprising (a) an extracellular antigen-binding domain comprising one or more anti-GCC sdAbs; (b) a transmembrane domain; and (c) an intracellular signaling domain, wherein the anti-GCC sdAb is an anti-GCC sdAb described in Section 5.2 above, including those comprising CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3, respectively, set forth in any one of SEQ ID NOs: 26-41, and wherein the anti-GCC sdAb comprises or consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the sequence of any one of SEQ ID NOs: 26-41. In some embodiments, the extracellular antigen-binding domain further comprises one or more additional antigen-binding domains. In some embodiments, the antigen-binding domains are fused to each other via a peptide linker. In some embodiments, the peptide linker is about 50 amino acids or less in length. In some embodiments, the transmembrane domain is selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and combinations thereof.In some embodiments, the CAR further comprises a hinge domain (e.g., a CD8α hinge domain) located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. In some embodiments, the CAR further comprises a signal peptide (e.g., a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N- to C-terminus, a CD8α signal peptide, an extracellular antigen-binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ.

[0299] In other specific embodiments, the present specification provides an engineered immune cell (e.g., a T cell) comprising a CAR, wherein the CAR comprises a polypeptide containing the amino acid sequence of any one of SEQ ID NOs: 45-63, or an amino acid sequence having at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 45-63.

[0300] In some embodiments, the engineered immune cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. In some embodiments, the engineered immune cells are autologous. In some embodiments, the engineered immune cells are allogeneic.

[0301] The engineered immune cells can further express one or more therapeutic proteins and / or immunomodulatory agents, such as immune checkpoint inhibitors.

[0302] Vectors The present disclosure provides vectors for cloning and expressing any one of the CARs described herein. In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, cowpox vectors, herpes simplex viral vectors, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0303] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, reverse-transcription viruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into reverse-transcription viral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to engineered mammalian cells in vitro or ex vivo. Many reverse-transcription viral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying immunomodulatory agent (e.g., immune checkpoint inhibitor) coding sequences and / or self-inactivating lentiviral vectors carrying chimeric antigen receptors can be packaged using packaging schemes known in the art. Using methods known in the art, the resulting lentiviral vectors can be used to transduce mammalian cells (e.g., primary human T cells). Vectors derived from reverse-transcription viruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term and stable integration of transgenic vectors and propagation in progeny cells. Lentiviral vectors also have low immunogenicity and can transduce non-proliferating cells.

[0304] In some embodiments, the vector comprises any one of the nucleic acids encoding a CAR described herein. The nucleic acid can be cloned into the vector using any molecular cloning method known in the art, including, for example, using a restriction endonuclease site and one or more selectable markers. In some embodiments, the nucleic acid is operably linked to a promoter. A variety of promoters have been explored for gene expression in mammalian cells, and any promoter known in the art can be used in the present disclosure. Promoters can be broadly classified as constitutive promoters or regulatable promoters, such as inducible promoters.

[0305] In some embodiments, the nucleic acid encoding the CAR is operably linked to a constitutive promoter. A constitutive promoter allows for constitutive expression of a heterologous gene (also called a transgenic) in a host cell. Exemplary constitutive promoters contemplated herein include, but are not limited to, the cytomegalovirus (CMV) promoter, human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerokinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken β-actin promoter (CAGG) coupled to a CMV early enhancer. The efficiency of such constitutive promoters for driving transgenic expression has been widely compared in many studies. For example, Michael C. Milone et al. compared the efficiency of CMV, hEF1α, UbiC, and PGK to drive chimeric antigen receptor expression in primary human T cells and concluded that the hEF1α promoter not only induces the highest level of transgenic expression but is also optimally maintained in CD4 and CD8 human T cells (Molecular Therapy, 17(8): 1453-1464 (2009)). In some embodiments, a nucleic acid encoding a CAR is operably linked to the hEF1α promoter.

[0306] In some embodiments, the nucleic acid of the encoding CAR is operably linked to an inducible promoter. Inducible promoters belong to the class of regulated promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, the microenvironment of the engineered immune cells or the physiological state of the engineered immune cells, an inducer (i.e., an inducer), or any combination thereof.

[0307] In some embodiments, the inducing conditions do not induce expression of endogenous genes in the engineered mammalian cell and / or the subject receiving the pharmaceutical composition. In some embodiments, the inducing conditions may be selected from the group consisting of an inducer, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox conditions, the tumor environment, and the activation state of the engineered mammalian cell.

[0308] In some embodiments, the vector further contains a selection marker gene or reporter gene to select cells expressing the CAR from a group of host cells transfected with the lentiviral vector. To enable expression in the host cell, both the selection marker and the reporter gene can be flanked by appropriate regulatory sequences. For example, the vector contains transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the nucleic acid sequence.

[0309] In some embodiments, the vector comprises one or more nucleic acids encoding a CAR. In some embodiments, the vector comprises a nucleic acid containing a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR, wherein the first nucleic acid is operably linked to the second nucleic acid via a third nucleic acid sequence encoding a self-cleaving peptide. In some embodiments, the self-cleaving peptide is selected from the group consisting of T2A, P2A, and F2A.

[0310] 5.4.3. Immune effector cells "Immune effector cell" refers to an immune cell capable of exerting immune effector function. In some embodiments, the immune effector cell expresses at least FcγRIII and exerts ADCC effector function. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

[0311] In some embodiments, the immune effector cells are T cells. The T cells may be αβ T cells or γδ T cells. In some embodiments, these T cells are CD4+ / CD8−, CD4− / CD8+, CD4+ / CD8+, CD4− / CD8−, or combinations thereof. In some embodiments, the T cells produce IL-2, TFN, and / or TNF after expressing a CAR and binding to target cells (e.g., GCC+ tumor cells). In some embodiments, the CD8+ T cells lyse antigen-specific target cells after expressing a CAR and binding to target cells.

[0312] In some embodiments, the immune effector cells are NK cells. In other embodiments, the immune effector cells may be an established cell line, such as NK-92 cells.

[0313] In some embodiments, immune effector cells are differentiated from stem cells (eg, hematopoietic stem cells, pluripotent stem cells, iPS cells, or embryonic stem cells).

[0314] The engineered immune effector cells are prepared by introducing a CAR into immune effector cells, such as T cells. In some embodiments, the CAR is introduced into immune effector cells by transfecting them with any one of the isolated nucleic acids or any one of the vectors described above. In some embodiments, the CAR is introduced into immune effector cells by inserting the protein into the cell membrane and passing the cells through a microfluidic system (e.g., CELL SQUEEZE®) (see, e.g., U.S. Patent Application Publication No. 20140287509).

[0315] Methods for introducing vectors or isolated nucleic acids into mammalian cells are known in the art. The described vectors can be transferred into immune effector cells by physical, chemical, or biological methods.

[0316] Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous 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. In some embodiments, vectors are introduced into cells by electroporation.

[0317] Biological methods for introducing vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human, cells.

[0318] Chemical methods for introducing vectors into immune effector cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0319] In some embodiments, an RNA molecule encoding any of the CARs described herein can be prepared by conventional methods (e.g., in vitro transcription) and introduced into immune effector cells by known methods such as mRNA electroporation. See, e.g., Rabinovich et al., Human Gene Therapy 17:1027-1035 (2006).

[0320] In some embodiments, the transduced or transfected immune effector cells are expanded ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced or transfected immune effector cells are cultured and expanded for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the transduced or transfected immune effector cells are further evaluated or screened to select engineered mammalian cells.

[0321] Reporter genes can be used to identify transfectable cells and evaluate the function of regulatory sequences. Typically, a reporter gene is a gene encoding a polypeptide that is not present or expressed in the recipient organism or tissue and whose expression is indicated by an easily detectable property (e.g., enzymatic activity). Expression of the reporter gene is detected at an appropriate time after DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., FEBS Letters 479: 79-82 (2000)). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.

[0322] Other methods for confirming the presence of a nucleic acid encoding a CAR in an engineered immune effector cell include molecular biological assays well known to those skilled in the art, such as, for example, DNA and RNA blotting, RT-PCR and PCR, and biochemical assays that detect the presence or absence of specific peptides by immunological methods (e.g., ELISA and Western blot).

[0323] 5.4.4. Source of T cells In some embodiments, a source of T cells is obtained from a subject prior to expansion and genetic modification of the T cells. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, multiple T cell lines available in the art can be used. In some embodiments, T cells can be obtained from blood collected from a subject using multiple techniques well known to those of skill in the art (e.g., Ficoll™ isolation). In some embodiments, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, cells collected by apheresis are washed to remove the plasma portion, and the cells may be used for subsequent processing steps in an appropriate buffer or medium. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium, or may lack many (if not all) divalent cations. An initial activation step in the absence of calcium can lead to expanded activation. As one of ordinary skill in the art will readily appreciate, the wash step can be completed by methods known in the art, such as using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be incubated in various biocompatible buffers, such as Ca. 2+ , Mg 2+ Alternatively, the apheresis sample may be removed and the cells resuspended directly in culture medium, such as PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, the undesired components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0324] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by PERCOLL™ gradient centrifugation or counterflow centrifugal elutriation. Specific subgroups of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in some embodiments, T cells are isolated by incubating with anti-CD3 / anti-CD28 (i.e., 3x28) conjugated beads (e.g., DYNABEADS® M-450 CD3 / CD28 T) for a time sufficient to positively select for the desired T cells. In some embodiments, the time period is about 30 minutes. In further embodiments, the time period is 30 minutes to 36 hours or more, and all integer values ​​therebetween. In further embodiments, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the time period is 10 to 24 hours. In some embodiments, the incubation period is 24 hours. For the isolation of T cells from leukemia patients, a longer incubation time (e.g., 24 hours) can increase cell yield. Longer incubation times can be used to isolate T cells in any case where T cells are scarce compared to other cell types, such as tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Longer incubation times can also improve the efficiency of CD8+ T cell capture. Thus, in some embodiments, subpopulations of T cells can be preferentially selected for or deselected at the initiation of culture or at other time points during culture by simply shortening or lengthening the time allowed for binding of T cells to CD3 / CD28 beads and / or increasing or decreasing the bead-to-T cell ratio. Additionally, the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface can be increased or decreased to preferentially select for or deselect for subpopulations of T cells at the initiation of culture or at other desired time points. Those skilled in the art will recognize that multiple rounds of selection can also be used.In some embodiments, it may be desirable to perform a selection procedure and use "unselected" cells for the activation and amplification process, which can then be subjected to further rounds of selection.

[0325] Enrichment of T cell populations by negative selection can be achieved by binding of antibodies to surface markers unique to the negatively selected cells. One method involves cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a mixture of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can enrich for CD4 + To enrich for cells, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, it may be desirable to enrich for or positively select for regulatory T cells, which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in some embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0326] The concentration of cells and surfaces (e.g., particles (e.g., beads)) may be varied to isolate the desired cell population by positive or negative selection. In some embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase cell expansion) to ensure maximum cell-to-bead contact. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells / ml is used. In a further embodiment, a concentration of 1.25 or 1.50 billion cells / ml may be used. The use of a high concentration of cells can result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of a high concentration of cells can enable more effective capture of cells that may weakly express a target antigen of interest (e.g., CD28-negative T cells) or cells from samples containing many tumor cells (i.e., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are expected to be available. In some embodiments, the use of a high concentration of cells enables more effective selection of CD8+ T cells, which normally have weak CD28 expression.

[0327] In some embodiments, it may be desirable to use a lower concentration of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and the cells are minimized. This selects for cells that express high amounts of the desired antigen for binding to the particles. For example, CD4+ T cells express higher levels of CD28 and are captured more effectively than dilute concentrations of CD8+ T cells. In some embodiments, the concentration of cells used is 5×10 6In some embodiments, the concentration used is about 1 x 10 5 pieces / mL~1×10 6 The value can be any integer value between 0.01 and 0.15, and can be any integer value between 0.01 and 0.15.

[0328] In some embodiments, cells may be incubated on a rotator at various speeds for various lengths of time at 2°C to 10°C or at room temperature.

[0329] T cells for stimulation may be frozen after a washing step. Without being bound by theory, the freezing and subsequent thawing step may provide a more uniform product by removing granulocytes and, to some extent, monocytes from the cell population. After the washing step, which removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are useful in such cases, one method uses PBS containing 20% ​​DMSO and 8% human serum albumin, or a medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or a medium containing 31.25% plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1°C / min and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods and uncontrolled immediate freezing at -20°C or in liquid nitrogen can be used.

[0330] In some embodiments, cryopreserved cells are thawed, washed, and allowed to stand at room temperature for one hour before activation, as described herein.

[0331] The present disclosure also contemplates collecting a blood sample or apheresis product from a subject at a time prior to the potential need for the expanded cells described herein. Thus, a cell source for expansion can be collected at any desired time, and the desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy, such as for various diseases or conditions that would benefit from T cell therapy, as described herein. In one embodiment, the blood sample or apheresis blood component is obtained from a normal, healthy subject. In some embodiments, the blood sample or apheresis blood component is obtained from a normal, healthy subject who is at risk for, but does not have, a disease, and ocular cells are isolated and frozen for later use. In some embodiments, the T cells can be expanded, frozen, and later used. In some embodiments, the sample is obtained from a patient shortly after diagnosis of a particular disease, as described herein, but prior to any treatment. In further embodiments, cells are isolated from a subject's blood sample or apheresis blood component prior to multiple associated therapeutic modalities, including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506), antibodies or other immunoablative agents (e.g., CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228), and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important in growth factor-induced signal transduction (Liu et al., Cell 66:807-815 (1991); Henderson et al., Immun 73:316-321 (1991); Bierer et al., Curr. Opin. Immun. 5:763-773 (1993)).In a further embodiment, the patient's cells are isolated and frozen for later use in conjunction with (e.g., before, concurrently with, or after) bone marrow or stem cell transplantation, T cell ablative therapy using chemotherapeutic agents (e.g., fludarabine), external beam radiotherapy (XRT), cytoxan, or antibodies (e.g., OKT3 or CAMPATH).

[0332] In some embodiments, T cells can be obtained directly from a patient after treatment. In this regard, it has been observed that after some cancer treatments, particularly treatment with drugs that damage the immune system, the quality of the obtained T cells may be optimal or their ex vivo expansion capacity may be improved immediately after treatment, during the period when the patient would normally be recovering from treatment. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Therefore, in the context of the present disclosure, it is contemplated to collect blood cells during this recovery period, including T cells, dendritic cells, or other hematopoietic cells. Furthermore, in some embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning schemes can be used to create conditions in the subject that favor the repletion, recirculation, regeneration, and / or expansion of cell types, particularly for a defined time window after treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0333] 5.4.5. T cell activation and expansion In some embodiments, before or after genetic modification of the T cells with a CAR described herein, the T cells can be activated and expanded, typically using methods described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005.

[0334] Typically, T cells can be expanded by contacting them with a surface bearing a reagent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the T cell surface. Specifically, as described herein, T cell populations can be stimulated by contact with an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody immobilized on a surface, or by a protein kinase C activator (e.g., bryostatin) bound to a calcium ion vector. A ligand that binds to a helper molecule can also be used to costimulate helper molecules on the T cell surface. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. An anti-CD3 antibody and an anti-CD28 antibody can also be used to stimulate proliferation of CD4+ T cells or CD8+ T cells. Examples of anti-CD3 antibodies include UCHT1, OKT3, and HIT3a (BioLegend, San Diego, US), and may be used as well as other methods known in the art (Graves J et al., J. Immunol. 146:2102 (1991); Li B et al., Immunology 116:487 (2005); Rivollier A et al., Blood 104:4029 (2004)). Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), as well as other methods known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, (1998); Haanen et al., J. Exp. Med. 190(9):13191328, (1999); Garland et al., J. Immunol Meth. 227(1-2):53-63 (1999)).

[0335] In some embodiments, the primary stimulatory signal and the costimulatory signal for T cells can be provided by various schemes. For example, the agents providing each signal can be in solution or coupled to a surface. If coupled to a surface, the agents can be coupled to the same surface (i.e., in a "cis" configuration) or to a separate surface (i.e., in a "trans" configuration). Alternatively, one agent can be coupled to a surface and another agent in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface, and the agent providing the primary activation signal is in solution or coupled to a surface. In some embodiments, both agents can be in solution. In another embodiment, the agents can be in soluble form and cross-linked to a surface, such as a cell expressing an Fc receptor or an antibody or other binding agent that will bind the agent. In this regard, see, e.g., U.S. Patent Application Publication Nos. 20040101519 and 20060034810, which describe artificial antigen-presenting cells (aAPCs), which are contemplated for use in activating and expanding T cells in some embodiments of the present disclosure.

[0336] In some embodiments, T cells are combined with agent-coated beads, followed by separation of the beads and cells and culturing the cells. In alternative embodiments, the agent-coated beads and cells are not separated prior to culturing, but are cultured together. In further embodiments, the beads and cells are first concentrated by applying a force, such as a magnetic force, to increase ligation of cell surface markers, thereby inducing cell stimulation.

[0337] For example, cell surface proteins can be linked by allowing the T cells to contact paramagnetic beads (3x28 beads) to which anti-CD3 and anti-CD28 are attached. In one embodiment, cells (e.g., 10 4 ~4×10 8T cells) and beads (e.g., anti-CD3 / CD28 MACSiBead particles with a recommended titer of 1:100) are combined in a buffer, preferably PBS (without divalent cations such as calcium and magnesium). One of skill in the art will readily appreciate that any cell concentration can be used. For example, the target cells may be present in very small amounts in the sample, comprising as little as 0.01% of the sample, or the entire sample (i.e., 100%) may contain the desired target cells. Thus, any cell number is within the scope of the present disclosure. In some embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase cell enrichment) to ensure maximum cell-particle contact. For example, in one embodiment, a concentration of approximately 2 billion cells / mL is used. In another embodiment, greater than 100 million cells / mL is used. In further embodiments, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL are used. In yet other embodiments, cell concentrations of 75, 80, 85, 90, 95, or 100 million cells / mL are used. In further embodiments, concentrations of 1.25 or 1.50 billion cells / mL may be used. The use of higher concentrations may result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of higher cell concentrations may allow for more effective capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and are anticipated to be available in some embodiments. For example, the use of higher cell concentrations may allow for more effective selection of CD8+ T cells, which typically have weak CD28 expression.

[0338] In some embodiments, the mixture can be cultured for any integer time between a few hours (about 3 hours) and about 14 days, or both. In another embodiment, the mixture can be cultured for 21 days. In one embodiment, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several stimulation cycles may be necessary, as T cell culture times can be 60 days or longer. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)), which may contain factors essential for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-β, and TNF-α, or any other additives for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, surfactants, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. The culture medium may be RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-vivo 15, or X-vivo 20, or an optimization agent supplemented with amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a set of defined hormones, and / or cytokines in amounts sufficient to grow and expand T cells. Antibiotics (e.g., penicillin and streptomycin) are included only in experimental cultures and are not included in cell cultures to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2). T cells exposed to different stimulation times may exhibit various characteristics. For example, a typical blood or peripheral blood mononuclear cell product of an apheresis blood component will have more helper T cell populations (TH, CD4+) than cytotoxic or suppressor T cell populations (TC, CD8).Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors produces a T cell population that is primarily composed of TH cells before about day 8-9, and after about day 8-9, the T cell population includes an increasingly abundant TC cell population. Therefore, for therapeutic purposes, it may be advantageous to infuse a T cell population that is primarily composed of TH cells into a subject. Similarly, if an antigen-specific subpopulation of TC cells is isolated, it may be beneficial to expand that subpopulation to a greater extent.

[0339] It should be noted that in addition to CD4 and CD8 markers, other phenotypic markers also vary significantly but are highly reproducible during cell expansion, allowing for the customization of activated T cell products for specific purposes.

[0340] 5.4.6. Exogenously introduced TGFβR and IL23R-expressing CAR-T cells In some embodiments, the T cells herein further express an exogenously introduced chimeric receptor comprising TGFβR and IL23R (also referred to herein as "TF23").

[0341] More specifically, in some embodiments, CAR-T cells expressing exogenously introduced TGFβR and IL23R can be produced by introducing one or more nucleic acids encoding polypeptides comprising both TGFβR and IL23R.

[0342] The CAR, TGFβR, and IL23R can each be introduced into a T cell individually as a single polypeptide. For example, a nucleic acid encoding a CAR according to the present specification, a nucleic acid encoding a TGFβR, and a nucleic acid encoding an IL23R may each be introduced into a T cell.

[0343] Alternatively, any two or all three may be introduced into T cells together as a single polypeptide via one nucleic acid, which is cleaved during translation within the cell. For example, a nucleic acid encoding a polypeptide comprising a CAR according to the present disclosure and a TGFβR linked via a self-cleaving peptide linker is introduced into T cells, and a nucleic acid encoding an IL23R is separately introduced into T cells. Similarly, a nucleic acid encoding a polypeptide comprising a CAR according to the present disclosure and an IL23R linked via a self-cleaving peptide linker is introduced into T cells, and a nucleic acid encoding a TGFβR is separately introduced into T cells. In some embodiments, a nucleic acid encoding a polypeptide comprising all three of a CAR, a TGFβR, and an IL23R linked to each other via a self-cleaving peptide linker may be introduced into T cells. Self-cleaving peptide linkers are described in more detail above. In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of F2A, E2A, P2A, T2A, or variants thereof. In some embodiments, the self-cleaving peptide is a 2A self-cleaving peptide P2A fragment comprising the amino acid sequence of SEQ ID NO:74 or SEQ ID NO:78.

[0344] Alternatively, CAR-T cells according to the present disclosure can be produced from a polynucleotide comprising multiple regions, e.g., a region encoding a CAR, a region encoding a TGFβR, and / or a region encoding an IL23R. The different regions can be controlled by the same promoter. For example, in some embodiments, the present disclosure uses an internal ribosome entry site (IRES) to express multiple genes from a single promoter. In other embodiments, the different regions are controlled by a single promoter.

[0345] In some specific embodiments, a CAR-T cell described herein has been exogenously introduced with a TF23 chimeric receptor, the TF23 chimeric receptor comprising a first extracellular domain comprising the extracellular domain of TGFβR1, a first transmembrane domain comprising the transmembrane domain of IL-12Rβ1, a first intracellular domain comprising the intracellular domain of IL-12Rβ1, a 2A self-cleaving peptide, a second extracellular domain comprising the extracellular domain of TGFβR2, a second transmembrane domain comprising the transmembrane domain of IL-23R, and a second intracellular domain comprising the intracellular domain of IL-23R, as shown in FIG. 10 . In some specific embodiments, a CAR-T cell described herein expresses an exogenously introduced polypeptide, the polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 64 to 66. In another specific embodiment, a CAR-T cell described herein has been exogenously introduced with a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 64. In other specific embodiments, the CAR-T cells herein comprise the amino acid sequence of any one of SEQ ID NOs: 61-63.

[0346] Polynucleotides In some embodiments, the present disclosure provides polynucleotides encoding antibodies of the invention that bind GCC (e.g., VHH domain antibodies), and fusion proteins comprising antibodies that bind GCC as described herein. The polynucleotides of the present disclosure may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or non-coding (antisense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0347] In some embodiments, the present disclosure provides a polynucleotide encoding a GCC-linked CAR according to the present disclosure. The polynucleotide of the present disclosure may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or non-coding (antisense) strand. In some embodiments, the polynucleotide is in the form of cDNA. In some embodiments, the polynucleotide is a synthetic polynucleotide.

[0348] The present disclosure further relates to variants of the polynucleotides described herein, where the variants encode, for example, fragments, analogs, and / or derivatives of an antibody or CAR that binds GCC of the present disclosure. In some embodiments, the present disclosure provides polynucleotides, including polynucleotides having a nucleotide sequence that is at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding an antibody or CAR that binds GCC of the present disclosure. As used herein, the phrase "a polynucleotide having a nucleotide sequence at least, e.g., 95% "identical" to a reference nucleotide sequence" is intended to mean that the nucleotide sequence of the polynucleotide has identity to the reference sequence, except that the polynucleotide sequence may contain up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with alternative nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur anywhere at or between the 5' or 3' terminal positions of the reference nucleotide sequence, interspersed among single nucleotides in the reference sequence, or in one or more contiguous groups within the reference sequence.

[0349] Polynucleotide variants may contain alterations in coding regions, non-coding regions, or both. In some embodiments, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants contain silent substitutions, which do not result in a change in the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants may be produced for a variety of reasons, such as to optimize codon expression for a particular host (i.e., changing the codons of human mRNA to those preferred by a bacterial host, such as E. coli). In some embodiments, polynucleotide variants contain at least one silent mutation in a non-coding or coding region of the sequence.

[0350] In some embodiments, polynucleotide variants have been generated to modulate or alter expression (or expression levels) of an encoded polypeptide. In some embodiments, polynucleotide variants have been generated to increase expression of an encoded polypeptide. In some embodiments, polynucleotide variants have been generated to decrease expression of a polypeptide. In some embodiments, polynucleotide variants exhibit increased expression of an encoded polypeptide compared to the parent polynucleotide sequence. In some embodiments, polynucleotide variants exhibit decreased expression of an encoded polypeptide compared to the parent polynucleotide sequence.

[0351] Further provided are vectors comprising the nucleic acid molecules described herein. In embodiments, the nucleic acid molecules may be incorporated into a recombinant expression vector. The present disclosure provides recombinant expression vectors comprising any of the nucleic acids of the present disclosure. As used herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that allows expression of mRNA, protein, polypeptide, or peptide by a host cell when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector contacts a cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vectors described herein are not entirely naturally occurring; however, some vectors may be naturally occurring. The described recombinant expression vectors may comprise any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthetic or derived in part from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may compr...

Claims

1. An anti-GCC single domain antibody (sdAb), (1) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 26, respectively; (2) CDR1, CDR2, and CDR3, each having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 27; (3) CDR1, CDR2, and CDR3, each having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 28; (4) CDR1, CDR2, and CDR3, each having the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 29; (5) CDR1, CDR2, and CDR3, each having the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 30; (6) CDR1, CDR2, and CDR3, each having the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 31; (7) CDR1, CDR2, and CDR3, each having the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 32; (8) CDR1, CDR2, and CDR3, each having the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 33; (9) CDR1, CDR2, and CDR3, each having the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 34; (10) CDR1, CDR2, and CDR3, each having the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 35; (11) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 36, respectively; (12) CDR1, CDR2, and CDR3, each having the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 37; (13) CDR1, CDR2, and CDR3, each having the amino acid sequences of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 38; (14) CDR1, CDR2, and CDR3, each having the amino acid sequence of CDR1, CDR2, and CDR3 set forth in SEQ ID NO: 39; (15) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 40, respectively; or (16) CDR1, CDR2, and CDR3 having the amino acid sequences of CDR1, CDR2, and CDR3 shown in SEQ ID NO: 41, respectively. The anti-GCC single domain antibody (sdAb) comprising:

2. 2. The anti-GCC sdAb of claim 1, wherein the CDR1, CDR2, or CDR3 is defined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or any combination thereof.

3. (1) CDR1 comprising the amino acid sequence of SEQ ID NO: 1, CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and CDR3 comprising the amino acid sequence of SEQ ID NO: 17; (2) CDR1 comprising the amino acid sequence of SEQ ID NO: 2, CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and CDR3 comprising the amino acid sequence of SEQ ID NO: 18; (3) CDR1 comprising the amino acid sequence of SEQ ID NO: 3, CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and CDR3 comprising the amino acid sequence of SEQ ID NO: 19; (4) CDR1 comprising the amino acid sequence of SEQ ID NO: 4, CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and CDR3 comprising the amino acid sequence of SEQ ID NO: 20; (5) CDR1 comprising the amino acid sequence of SEQ ID NO: 5, CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and CDR3 comprising the amino acid sequence of SEQ ID NO: 21; (6) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (7) CDR1 comprising the amino acid sequence of SEQ ID NO: 6, CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23; (8) CDR1 comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and CDR3 comprising the amino acid sequence of SEQ ID NO: 24, or (9) CDR1 comprising the amino acid sequence of SEQ ID NO: 8, CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and CDR3 comprising the amino acid sequence of SEQ ID NO:

25.

2. The anti-GCC sdAb of claim 1, comprising:

4. 4. The anti-GCC sdAb of any one of claims 1 to 3, further comprising one or more FR regions as set forth in any one of SEQ ID NOs: 26 to 41.

5. The anti-GCC sdAb according to any one of claims 1 to 4, comprising the amino acid sequence of any one of SEQ ID NOs: 26 to 41.

6. 5. The anti-GCC sdAb of any one of claims 1 to 4, comprising or consisting of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of SEQ ID NOs: 26 to 41.

7. 2. The anti-GCC sdAb of claim 1 which is a camelid sdAb.

8. 2. The anti-GCC sdAb of claim 1, which is a humanized sdAb.

9. 9. The anti-GCC sdAb of any one of claims 1 to 8, which is genetically fused or chemically conjugated to an agent.

10. The anti-GCC sdAb according to any one of claims 1 to 9, which is fused to an Fc region.

11. 11. The anti-GCC sdAb of claim 10, wherein the Fc region is human IgG1 Fc or murine IgG1 Fc, optionally wherein the murine IgG1 Fc comprises the amino acid sequence of SEQ ID NO:

67.

12. A fusion protein comprising the anti-GCC sdAb of any one of claims 1 to 8 and mouse IgG1 Fc, and comprising any one of the amino acid sequences of SEQ ID NO: 42 to 44.

13. (a) an extracellular antigen-binding domain comprising one or more of the anti-GCC sdAbs according to any one of claims 1 to 9; (b) a transmembrane domain; and (c) an intracellular signaling domain; A chimeric antigen receptor (CAR) comprising:

14. The CAR of claim 13, wherein the extracellular antigen-binding domain comprises one anti-GCC sdAb.

15. The CAR of claim 13 or 14, wherein the extracellular antigen-binding domain further comprises one or more additional antigen-binding domains.

16. The CAR of claim 15, wherein the antigen-binding domains are fused to each other via a peptide linker.

17. The CAR of claim 16, wherein the peptide linker is about 50 amino acids or less in length.

18. The CAR according to any one of claims 13 to 17, wherein the transmembrane domain is derived from a molecule selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1.

19. The CAR of claim 18, wherein the transmembrane domain is derived from CD8α.

20. The CAR of any one of claims 13 to 19, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell.

21. The CAR of claim 20, wherein the primary intracellular signaling domain is derived from CD3ζ.

22. The CAR of claim 20 or 21, wherein the intracellular signaling domain further comprises a costimulatory signaling domain.

23. The CAR of claim 22, wherein the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand for CD83, and a combination thereof.

24. The CAR of claim 23, wherein the costimulatory signaling domain is derived from CD137.

25. The CAR of any one of claims 13 to 24, further comprising a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

26. The CAR of claim 25, wherein the hinge domain is derived from CD8α.

27. The CAR according to any one of claims 13 to 26, further comprising a signal peptide located at the N-terminus of the polypeptide.

28. The CAR of claim 27, wherein the signal peptide is derived from CD8α.

29. A chimeric antigen receptor (CAR) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 45-60.

30. 13. An isolated nucleic acid comprising a nucleic acid sequence encoding the anti-GCC sdAb of any one of claims 1 to 11 or a nucleic acid sequence encoding the fusion protein of claim 12.

31. An isolated nucleic acid comprising a nucleic acid sequence encoding the CAR of any one of claims 13 to 29.

32. 32. The isolated nucleic acid of claim 31, further comprising a nucleic acid sequence encoding a chimeric receptor, wherein the chimeric receptor comprises a TGFβR and an IL23R, and optionally, the chimeric receptor comprises the amino acid sequence of any one of SEQ ID NOs: 64-66.

33. A vector comprising the isolated nucleic acid of any one of claims 30 to 32.

34. An engineered immune cell comprising the CAR of any one of claims 13 to 29, the isolated nucleic acid of any one of claims 30 to 32, or the vector of claim 33.

35. the immune cells are immune effector cells; Optionally, the immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, or any combination thereof.

35. The engineered immune cell of claim 34.

36. 36. The engineered immune cell of claim 35, comprising any one of the amino acid sequences of SEQ ID NOs: 45-60 and 61-63.

37. 34. A method for producing an engineered immune cell, comprising introducing the vector of claim 33 into a cell.

38. 37. A pharmaceutical composition comprising an anti-GCC sdAb according to any one of claims 1 to 11, an isolated nucleic acid according to any one of claims 30 to 32, a vector according to claim 33, or an engineered immune cell according to any one of claims 34 to 36, and a pharmaceutically acceptable excipient.

39. 39. A method of treating a disease or disorder in a subject, the method comprising administering to the subject an effective amount of an anti-GCC sdAb according to any one of claims 1 to 11, an engineered immune cell according to any one of claims 34 to 36, or a pharmaceutical composition according to claim 38.

40. 40. The method of claim 39, wherein the disease or disorder is a GCC-associated disease or disorder.

41. 40. The method of claim 39, wherein the disease or disorder is cancer.

42. 42. The method of claim 41, wherein the disease or disorder is selected from the group consisting of gastrointestinal cancer, colorectal cancer, gastric cancer, esophageal cancer, gastroesophageal junction cancer, small intestine cancer, pancreatic cancer, and liver cancer.

43. 43. The method of claim 41 or 42, wherein the disease or disorder is colorectal cancer.

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