Humanized GUCY2C T-cell antigen coupler and uses thereof

Abstract

Provided is a GUCY2C T cell antigen coupler (TAC) polypeptide having (i) an antigen binding domain (e.g., a nanobody) that binds GUCY2C, (ii) an antigen binding domain that binds a protein associated with the TCR complex, and (iii) a T cell receptor signaling domain polypeptide.

Description

[Technical Field]

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 478,862, filed January 6, 2023, and U.S. Provisional Patent Application No. 63 / 594,831, filed October 31, 2023, the entire contents of each of which are incorporated herein by reference in their entirety for all purposes. Summary of the Invention

[0002] In certain embodiments, disclosed herein are GUCY2C (guanylate cyclase 2C) T cell antigen coupler (GUCY2C-TAC) polypeptides, nucleic acids encoding GUCY2C-TAC, T cells comprising GUCY2C-TAC, and methods of using same.

[0003] In certain embodiments, the present specification discloses a guanylate cyclase 2C (GUCY2C) T cell antigen coupler (GUCY2C-TAC) protein, comprising: (a) a first polypeptide comprising a GUCY2C binding domain, the first polypeptide comprising (i) a CDR1 having the amino acid sequence of SEQ ID NO: 72, a CDR2 having the amino acid sequence of SEQ ID NO: 73, and a CDR3 having the amino acid sequence of SEQ ID NO: 74, or (ii) a CDR1 having the amino acid sequence of SEQ ID NO: 75, a CDR2 having the amino acid sequence of SEQ ID NO: 76, and a CDR3 having the amino acid sequence of SEQ ID NO: 77; (b) a second polypeptide comprising an antigen binding domain that binds a protein associated with a TCR complex; and (c) a third polypeptide comprising a TCR co-receptor cytoplasmic domain and a transmembrane domain, wherein the first polypeptide, the second polypeptide, and the third polypeptide are directly fused to each other or connected by at least one linker. In some embodiments, the GUCY2C binding domain comprises a CDR1 having the amino acid sequence of SEQ ID NO: 72, a CDR2 having the amino acid sequence of SEQ ID NO: 73, and a CDR3 having the amino acid sequence of SEQ ID NO: 74. In some embodiments, the GUCY2C binding domain is a nanobody. In some embodiments, the GUCY2C binding domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-62. In some embodiments, the GUCY2C binding domain comprises a CDR1 having the amino acid sequence of SEQ ID NO: 75, a CDR2 having the amino acid sequence of SEQ ID NO: 76, and a CDR3 having the amino acid sequence of SEQ ID NO: 77. In some embodiments, the GUCY2C binding domain is a nanobody.In some embodiments, the GUCY2C binding domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-71. In some embodiments, the GUCY2C binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-71.

[0004] In some embodiments, the first polypeptide, the second polypeptide, and the third polypeptide are in N-terminal to C-terminal order. In some embodiments, the protein associated with the TCR complex is a CD3 protein. In some embodiments, the CD3 protein is a CD3γ protein, a CD3δ protein, and / or a CD3ε protein. In some embodiments, the CD3 protein is a CD3ε protein. In some embodiments, the CD3 protein is a CD3ε protein.

[0005] In some embodiments, the antigen-binding domain that binds a protein associated with the TCR complex is derived from an antibody selected from UCHT1 OKT3, F6A, and L2K. In some embodiments, the antigen-binding domain that binds a protein associated with the TCR complex is a UCHT1 antigen-binding domain. In some embodiments, the UCHT1 antigen-binding domain is a UCHT1 scFV. In some embodiments, the UCHT1 antigen-binding domain comprises a Y to T mutation at a position corresponding to amino acid 182 of SEQ ID NO: 32 (Y182T). In some embodiments, the UCHT1 antigen-binding domain comprises a humanized variant of UCHT1 (huUCHT1). In some embodiments, the UCHT1 antigen-binding domain comprises a humanized variant of UCHT1 comprising a Y to T mutation at a position corresponding to amino acid 177 of SEQ ID NO: 40 (huUCHT1(Y177T)). In some embodiments, the antigen binding domain that binds a protein associated with a TCR complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32 (UCHT1), SEQ ID NO: 44 (UCHT1(Y182T)), SEQ ID NO: 40 (huUCHT1), or SEQ ID NO: 42 (huUCHT1(Y177T)).

[0006] In some embodiments, the CDR sequences of the antigen-binding domain that binds a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), SEQ ID NO: 44 (UCHT1(Y182T)), SEQ ID NO: 40 (huUCHT1), or SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds a protein associated with the TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), SEQ ID NO: 44 (UCHT1(Y182T)), SEQ ID NO: 40 (huUCHT1), or SEQ ID NO: 42 (huUCHT1(Y177T)), and the non-CDR sequences of the antigen-binding domain that binds a protein associated with the TCR complex have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the non-CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), SEQ ID NO: 44 (UCHT1(Y182T)), SEQ ID NO: 40 (huUCHT1), or SEQ ID NO: 42 (huUCHT1(Y177T)).

[0007] In some embodiments, the antigen-binding domain that binds a protein associated with a TCR complex is an OKT3 antigen-binding domain. In some embodiments, the antigen-binding domain that binds a protein associated with a TCR complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34 (OKT3). In some embodiments, the CDR sequences of the antigen-binding domain that binds a protein associated with a TCR complex are 100% identical to the CDR sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the CDR sequences of the antigen-binding domain that binds a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3), and the non-CDR sequences of the antigen-binding domain that binds a protein associated with a TCR complex have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the non-CDR sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the antigen-binding domain that binds a protein associated with a TCR complex is an F6A antigen-binding domain. In some embodiments, the antigen-binding domain that binds a protein associated with a TCR complex comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 36 (F6A). In some embodiments, the CDR sequences of the antigen binding domain that binds a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 36 (F6A).In some embodiments, the CDR sequences of the antigen-binding domain that binds a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 36 (F6A), and the non-CDR sequences of the antigen-binding domain that binds a protein associated with a TCR complex have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the non-CDR sequences of the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the antigen-binding domain that binds a protein associated with a TCR complex is an L2K antigen-binding domain. In some embodiments, the antigen-binding domain that binds a protein associated with a TCR complex comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38 (L2K). In some embodiments, the CDR sequences of the antigen-binding domain that binds a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the CDR sequences of the antigen-binding domain that binds a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 38 (L2K), and the non-CDR sequences of the antigen-binding domain that binds a protein associated with a TCR complex have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the non-CDR sequences of the amino acid sequence of SEQ ID NO: 38 (L2K).

[0008] In some embodiments, the transmembrane domain is a CD4 transmembrane domain and the cytoplasmic domain is a CD4 cytoplasmic domain. In some embodiments, the transmembrane domain and the cytoplasmic domain comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 46 (CD4 transmembrane domain and cytoplasmic domain). In some embodiments, the transmembrane domain is a CD8 transmembrane domain and the cytoplasmic domain is a CD8 cytoplasmic domain. In some embodiments, the component encoded by (a) and the component encoded by (c) are fused to the component encoded by (b). In some embodiments, the component encoded by (b) and the component encoded by (c) are fused to the component encoded by (a). In some embodiments, at least one linker connects the component encoded by (a) to the component encoded by (b). In some embodiments, at least one linker is a glycine- and / or serine-rich linker, a large protein domain, a long helix structure, or a short helix structure. In some embodiments, at least one linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:26 ((G4S)4-based linker), SEQ ID NO:28 (G4S-based linker), SEQ ID NO:14 (CD4-based linker), SEQ ID NO:12 (short helix connector), SEQ ID NO:14 (long helix connector), SEQ ID NO:16 (large domain connector), or SEQ ID NO:24 ((G4S)3-flexible linker). In some embodiments, the GUCY2C-TAC protein does not comprise a costimulatory domain. In some embodiments, the GUCY2C-TAC protein does not comprise an activation domain. In some embodiments, the GUCY2C-TAC protein further comprises a leader sequence.In some embodiments, the leader sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2 (muIgG leader), SEQ ID NO:18 (huIgG leader), SEQ ID NO:20 (huCD8a-1 leader), or SEQ ID NO:30 (huCD8a-2 leader).

[0009] In certain embodiments, disclosed herein is a GUCY2C TAC protein comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153.

[0010] In certain embodiments, disclosed herein is a GUCY2C TAC protein comprising an amino acid sequence according to any one of the amino acid sequences set forth in SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153.

[0011] In certain embodiments, disclosed herein is a GUCY2C TAC protein comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:131.

[0012] In certain embodiments, disclosed herein is a GUCY2C TAC protein comprising an amino acid sequence according to the amino acid sequence of SEQ ID NO:131.

[0013] In certain embodiments, disclosed herein are nucleic acid sequences encoding the GUCY2C TAC proteins disclosed herein. In some embodiments, the nucleic acid comprises a sequence having at least 80% sequence identity to any one of the nucleic acid sequences set forth in SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152. In some embodiments, the nucleic acid sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152.

[0014] In certain embodiments, disclosed herein are T cells that express the GUCY2C-TAC proteins disclosed herein.

[0015] In certain embodiments, disclosed herein are T cells comprising a nucleic acid sequence disclosed herein.

[0016] In certain embodiments, disclosed herein are pharmaceutical compositions comprising the T cells disclosed herein and a pharmaceutically acceptable excipient.

[0017] In certain embodiments, disclosed herein are methods for treating a GUCY2C-expressing cancer in an individual in need thereof, comprising administering to the individual a pharmaceutical composition disclosed herein. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is colorectal cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, or pancreatic cancer. In some embodiments, the cancer is primary colorectal cancer, primary gastric cancer, primary gastroesophageal junction cancer, primary esophageal cancer, or primary pancreatic cancer. In some embodiments, the cancer is metastatic colorectal cancer, metastatic gastric cancer, metastatic gastroesophageal junction cancer, metastatic esophageal cancer, or metastatic pancreatic cancer. [Brief explanation of the drawings]

[0018] [Figure 1A] Figure 1A depicts the phenotype of GUCY2C-TAC T cells as measured by flow cytometry. Figure 1A depicts a representative flow plot of T cells double-stained for transduction marker (mStraw) versus TAC expression (anti-myc). [Figure 1B] Figure 1B depicts the phenotype of GUCY2C-TAC T cells as measured by flow cytometry. Figure 1B depicts a bar graph showing the median fluorescence intensity (MFI) of TAC-positive T cells. [Figure 2] 1 depicts quantitative analysis of tumor cell proliferation as a function of GFP area at different E:T ratios for tumor cells grown in the presence of GUCY2C-TAC T cells. [Figure 3] 1 depicts a bar graph showing the proliferative potential, defined as the normalized mitotic index, of GUCY2C-TAC T cells grown in the presence of GUCY2C-expressing leukemia and gastric cancer cells. [Figure 4A-1] Figure 4A shows the effect of nine alternating rounds of co-culture of GUCY2C-TAC T cells with target tumor cells, where co-culture occurs over a period of 3–4 days in each round. Figure 4B depicts a quantitative analysis of the cytotoxicity of GUCY2C3-TAC T cells against tumor cells over nine alternating rounds of co-culture, in which cytotoxicity was assessed as a function of GFP fluorescence and quantified by calculating the area under each GFP curve. [Figure 4A-2] Figure 4A shows the effect of nine alternating rounds of co-culture of GUCY2C-TAC T cells with target tumor cells, where co-culture occurs over a period of 3–4 days in each round. Figure 4B depicts a quantitative analysis of the cytotoxicity of GUCY2C3-TAC T cells against tumor cells over nine alternating rounds of co-culture, in which cytotoxicity was assessed as a function of GFP fluorescence and quantified by calculating the area under each GFP curve. [Figure 4B-1] Figure 4B shows the effect of nine alternating rounds of co-culture of GUCY2C-TAC T cells with target tumor cells, where co-culture occurs over a period of 3–4 days in each round. Figure 4B shows the total amount of T cells recovered after each round of co-culture graphed against the initial total cells seeded at the start of each round. [Figure 4B-2] Figure 4B shows the effect of nine alternating rounds of co-culture of GUCY2C-TAC T cells with target tumor cells, where co-culture occurs over a period of 3–4 days in each round. Figure 4B shows the total amount of T cells recovered after each round of co-culture graphed against the initial total cells seeded at the start of each round. [Figure 4C] Figure 4C depicts the effect of nine alternating rounds of coculture of GUCY2C-TAC T cells with target tumor cells, where coculture occurs over a 3-4 day period in each round. Figure 4C depicts the phenotype of GUCY2C-TAC T cells recovered on day 0 and after rounds 1, 5, and 9, as measured by the percentage of total CD8+ T cells (top left panel), CD4+ T cells (top right panel), mStrawberry+ T cells (middle left panel), mStrawberry- T cells (middle right panel), CD8-gated T cells expressing CD69 (bottom left panel), and CD8-gated, then CD39-gated T cells expressing PD-1 and LAG3 (bottom right panel). [Figure 5A]Figure 5A shows the efficacy of GUCY2C-TAC T cells in mice inoculated with a GUCY2C-expressing liquid tumor model. Figure 5A shows the total tumor burden in mice, as measured by bioluminescence signal over the course of the experiment, after administration of no treatment (NT), non-transduced T cells (NTD), or treatment with GUCY2C-TAC T cells. [Figure 5B] Figure 5B shows the efficacy of GUCY2C-TAC T cells in mice inoculated with a GUCY2C-expressing liquid tumor model. Figure 5B depicts a survival curve showing the survival rate over the course of the experiment. [Figure 6]

[0023] Figure 1 shows the efficacy of GUCY2C-TAC T cells in mice inoculated with a GUCY2C-expressing solid tumor model. Total tumor volume in mice, measured by weekly caliper measurements over the course of the experiment, was monitored for animals that received no treatment (NT), non-transduced T cells (NTD), or GUCY2C-TAC T cells. [Figure 7A] Figure 7A shows the binding specificity of the G22H8 nanobody. Figure 7A shows the binding of G22H8-GFP to target HEK or QT6 cells engineered with either a GUCY2C-expressing plasmid, a Protein A-expressing plasmid control, or an empty vector control, as measured by mean fluorescence (y-axis) versus concentration of fusion protein (x-axis). [Figure 7B] Figure 7B shows the binding specificity of the G22H8 nanobody. Figure 7B shows a membrane proteome array showing G22H8-GFP target binding (y-axis) versus human membrane proteins (x-axis). [Figure 8-1] 1 shows the expression levels of GUCY2C in cancer cells that naturally and ectopically express GUCY2C as measured by flow cytometry. [Figure 8-2] 1 shows the expression levels of GUCY2C in cancer cells that naturally and ectopically express GUCY2C as measured by flow cytometry. [Figure 9]1 shows the expression levels of GUCY2C in cancer cells that naturally and ectopically express GUCY2C, as measured by mRNA analysis using droplet digital polymerase chain reaction (ddPCR). [Figure 10] Representative flow plots depicting double staining of the transduction marker (mStrawberry) versus the CD69 early activation marker in GUCY2C-TAC T cells co-cultured with antigen-positive (HCT116-GUCY2C) or antigen-negative (HCT116) target cells are shown. [Figure 11] 1 depicts a bar graph showing normalized levels of the CD69 early activation marker expressed in GUCY2C-TAC T cells co-cultured with antigen-positive or antigen-negative target cells as measured by flow cytometry. [Figure 12] Representative flow plots depicting double staining of interferon gamma (IFNγ) versus tumor necrosis factor alpha (TNFa) in permeabilized GUCY2C-TAC T cells after co-culture with antigen-positive (HCT116GUCY2C or N87GUCY2C) or antigen-negative (HCT116 or N87) target cells are shown. [Figure 13] 1 depicts a bar graph showing normalized levels of IL2, IFNγ, and TNFα cytokines produced in GUCY2C-TAC T cells after co-culture with antigen-positive or antigen-negative target cells, as measured by flow cytometry. [Figure 14] 1 depicts a bar graph showing the percentage of target cells killed after co-culture with GUCY2C-TAC T cells. DETAILED DESCRIPTION OF THE INVENTION

[0019] Cancer is a major health challenge. According to the American Cancer Society, more than one million people are diagnosed with cancer each year in the United States. While patients with early-stage disease can be effectively treated with conventional therapies (surgery, radiation, and chemotherapy), patients with advanced disease have fewer options available, and those options are typically palliative in nature.

[0020] Active immunotherapy attempts to harness a patient's immune system to eliminate tumors, providing an option for patients who have failed conventional treatments. Typically, this treatment involves infusing large numbers of tumor-specific T cells into patients. Currently, most engineered T cell therapies, involving genetic modification of T cells, result in either (i) forced expression of a T cell receptor (TCR) or (ii) a chimeric antigen receptor (CAR) specific for an antigen target on the tumor. To date, the chimeric antigen receptors used to engineer T cells consist of (i) a targeting domain, usually a single-chain variable fragment (scFV), (ii) a transmembrane domain, and (iii) a cytoplasmic domain containing signaling elements from the T cell receptor and associated proteins. Such chimeric antigen receptors are also referred to as "T bodies" or "chimeric immune receptors (CIRs)," although most researchers now use the term "CAR." One advantage of the CAR approach is that any patient's immune cells can be targeted to any desired target in a major histocompatibility complex (MHC)-independent manner. This is attractive because MHC presentation is often defective in tumor cells.

[0021] CARs are thought of in modular terms, and scientists have spent considerable time investigating the impact of different cytoplasmic signaling domains on CAR function. Traditional CARs generally share two main components: (i) the CD3 zeta cytoplasmic domain, which contains immunotyrosine-based activation motifs (ITAMs) important for T cell activation, and (ii) costimulatory receptor components that trigger important survival pathways, such as the Akt pathway.

[0022] First-generation CARs employed a single signaling domain from either CD3ζ or FcεRIγ. Second-generation CARs combined the signaling domain of CD3ζ with the cytoplasmic domain of a costimulatory receptor from either the CD28 or TNFR family of receptors. Most CAR-engineered T cells currently in clinical trials employ second-generation CARs in which CD3ζ is linked to the cytoplasmic domain of either CD28 or CD137. These second-generation CARs have demonstrated antitumor activity in CD19-positive tumors. Third-generation CARs have combined multiple costimulatory domains, but there are concerns that third-generation CARs may lose antigen specificity.

[0023] While CAR-engineered T cells have shown considerable promise in clinical applications, they rely on synthetic methods to replace the native activation signal provided by the T cell receptor (TCR). Because this synthetic receptor does not deliver all of the signaling components that associate with the TCR (e.g., ITAMs on CD3γ, CD3δ, and CD3ε), it is unclear whether T cells are optimally activated by the CAR or how CAR activation impacts T cell differentiation (e.g., progression to memory). Furthermore, because the CAR signaling domain is decoupled from its natural regulatory partners by the very nature of the CAR's structure, there is an inherent risk that the CAR may cause low-level constitutive activation, which could result in off-target toxicity. Thus, the synthetic nature of prototypical CARs may subvert the canonical mechanisms that limit TCR activation, underpinning the severe toxicity often associated with therapeutic doses of conventional CAR T cells.

[0024] Given these limitations, it is preferable to redirect T cells to attack tumors via their native TCR. Alternative chimeric receptors, called T cell antigen coupler (TAC or TAC) receptors, have been developed using a different biological approach to direct T cells to attack tumors. While CARs are fully synthetic receptors that bring together components of the T cell receptor (TCR) signaling complex, TAC receptors redirect TCRs to tumor targets, recapitulating the native TCR signaling architecture. For example, in some embodiments, the TACs disclosed herein activate native major histocompatibility complex (MHC) signaling through the T cell receptor (TCR) while retaining MHC-agnostic targeting. Furthermore, the TACs disclosed herein recruit the T cell receptor (TCR) in combination with co-receptor stimulation. Furthermore, in some embodiments, the TACs disclosed herein exhibit enhanced activity and safety.

[0025] Specific Terms The term "antigen-binding domain" refers to any substance or molecule that directly or indirectly binds to a target (e.g., GUCY2C). Antigen-binding domains include antibodies or fragments thereof, peptides, peptidomimetics, proteins, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, or small molecules that bind to a target.

[0026] As used herein, unless otherwise specified, the term "antibody" is understood to mean an intact antibody (e.g., an intact monoclonal antibody), or a fragment thereof, such as an Fc fragment of an antibody (e.g., an Fc fragment of a monoclonal antibody), or an engineered, engineered, or chemically conjugated intact antibody, antigen-binding fragment, or antigen-binding fragment of an antibody (e.g., an antigen-binding fragment of a monoclonal antibody) comprising an Fc fragment. Generally, antibodies are multimeric proteins containing four polypeptide chains. Two of the polypeptide chains are called immunoglobulin heavy chains (H chains) and two of the polypeptide chains are called immunoglobulin light chains (L chains). The heavy and light chains of an immunoglobulin are connected by interchain disulfide bonds. The immunoglobulin heavy chains are connected by interchain disulfide bonds. The light chain contains one variable region (V L ) and one constant region (C L The heavy chain consists of one variable region (V H) and at least three constant regions (CH1, CH2, and CH3). The variable regions determine the binding specificity of the antibody. Each variable region contains three hypervariable regions known as complementarity-determining regions (CDRs) flanked by four relatively conserved regions known as framework regions (FRs). The extent of FRs and CDRs has been defined (Kabat, E.A. et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed., USDapartment of Health and Human Services, NIH Publication No. 91-3242; and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). CDRs can also be identified by alignment of amino acid sequences. Because FRs contain conserved amino acid sequences, CDR sequences can be identified by identifying non-conserved amino acid residues between variable regions with conserved FRs. The three CDRs, designated CDR1, CDR2, and CDR3, contribute to the binding specificity of the antibody. Naturally occurring antibodies are used as starting materials for engineered antibodies, such as chimeric antibodies and humanized antibodies. Examples of antibody-based antigen-binding fragments include Fab, Fab', (Fab')2, Fv, single-chain antibodies (e.g., scFv), minibodies, and diabodies. Examples of modified or engineered antibodies include chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies). An example of a chemically conjugated antibody is an antibody conjugated to a toxin moiety.

[0027] As used herein, the term "T cell" refers to a type of lymphocyte that plays a central role in cell-mediated immunity. T cells, also called T lymphocytes, are distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T cell receptor (TCR) on the cell surface. There are several subsets of T cells with different functions, including, but not limited to, T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, and natural killer T cells.

[0028] As used herein, the term "γδ T cell" or "gamma delta T cell" or "gd T cell" refers to any lymphocyte that has a γδ T cell receptor (TCR) on its surface, which contains one γ chain and one δ chain.

[0029] The term "T cell antigen coupler" or TAC is used interchangeably with "trifunctional T cell antigen coupler" or Tri-TAC and refers to an engineered nucleic acid construct or polypeptide that includes (a) an antigen-binding domain that binds to a target, (b) an antigen-binding domain that binds to a protein associated with the T cell receptor (TCR) complex, and (c) a T cell receptor signaling domain.

[0030] As used herein, the terms "polynucleotide" and / or "nucleic acid sequence" and / or "nucleic acid" refer to a sequence of nucleoside or nucleotide monomers, consisting of bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted sequences, including non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may contain naturally occurring bases, including adenine, guanine, cytosine, thymidine, and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza- and deaza-adenine, guanine, cytosine, thymidine, and uracil, as well as xanthine and hypoxanthine. The nucleic acids of the present disclosure may be isolated from biological organisms, formed by recombinant laboratory methods, or obtained by chemical synthesis or other known protocols for producing nucleic acids.

[0031] As used herein, the term "isolated polynucleotide" or "isolated nucleic acid sequence" refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences that naturally flank the nucleic acid from which it is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid). The term "nucleic acid" is intended to include DNA and RNA, and can be either double-stranded or single-stranded, and refer to either the sense or antisense strand. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences.

[0032] As used herein, the term "recombinant nucleic acid" or "engineered nucleic acid" refers to a nucleic acid or polynucleotide that does not occur in nature (e.g., is not naturally found in a biological organism). For example, a recombinant nucleic acid can be formed by laboratory methods of genetic recombination (such as molecular cloning) to create a sequence that would not otherwise be found in nature. Recombinant nucleic acids may also be produced by chemical synthesis or other known protocols for producing nucleic acids.

[0033] As used herein, the terms "peptide," "polypeptide," or "protein" refer to a chain of amino acids. As used herein, the term protein further refers to a larger molecule comprising one or more chains of amino acids, and in some embodiments, a protein fragment or domain or a full-length protein. Furthermore, as used herein, the term protein refers to either a linear chain of amino acids or a chain of amino acids that has been processed and folded into a functional protein. Protein structure is divided into four different levels: (1) primary structure—refers to the sequence of amino acids in the polypeptide chain; (2) secondary structure—refers to the regular, local substructures on the polypeptide backbone chain, such as α-helices and β-sheets; (3) tertiary structure—refers to the three-dimensional structure in the case of monomeric and multimeric protein molecules; and (4) quaternary structure—refers to the three-dimensional structure comprising the aggregation of two or more individual polypeptide chains that operate as a single functional unit. The use of peptide or polypeptide herein does not imply that the chain of amino acids is not a protein (i.e., a chain of amino acids with secondary, tertiary, or quaternary structure).

[0034] The term "isolated polypeptide" refers to a polypeptide that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or that is substantially free of chemical precursors or other chemicals when chemically synthesized.

[0035] As used herein, the term "vector" refers to a polynucleotide used to deliver a nucleic acid into a cell. In some embodiments, the vector is an expression vector that contains an expression control sequence (e.g., a promoter) operably linked to the nucleic acid to be expressed in the cell. Vectors known in the art include, but are not limited to, plasmids, phages, cosmids, and viruses.

[0036] As used herein, the term "tumor antigen" or "tumor-associated antigen" refers to an antigenic substance (e.g., presented by an MHC complex) produced by a tumor cell that elicits an immune response in a host. In some embodiments, the tumor antigen is on the surface of a tumor cell.

[0037] As used herein, the term "transmembrane and cytoplasmic domain" refers to a polypeptide comprising the transmembrane and cytoplasmic domains of a protein associated with the T cell receptor (TCR) complex. In some embodiments, such transmembrane and cytoplasmic domains may include, but are not limited to, protein domains that (a) associate with lipid rafts and / or (b) bind to Lck.

[0038] As used herein, "TCR co-receptor" refers to a molecule that helps the T cell receptor (TCR) communicate with antigen-presenting cells. Examples of TCR co-receptors include, but are not limited to, CD4, LAG3, and CD8.

[0039] As used herein, "TCR costimulator" or "costimulatory domain" refers to a molecule that enhances the response of T cells to antigens and can be considered a signal that leads to TCR activation. Examples of TCR costimulators include, but are not limited to, ICOS, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, lymphocyte fiction-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0040] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and, in some embodiments, refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some embodiments, the mammal is a human. None of these terms require the supervision of a medical professional.

[0041] As used herein, the terms "treatment," "treating," and the like, in some embodiments, refer to administering an agent or performing a procedure with the intent to obtain an effect. The effect can be preventative, in that it completely or partially prevents a disease or its symptoms, and / or therapeutic, in that it affects a partial or complete cure of the disease and / or symptoms of the disease. "Treatment," as used herein, can include the treatment of a disease or disorder (e.g., cancer) in a mammal, particularly a human, and includes (a) preventing the development of a disease or disease symptoms in a subject who may be predisposed to the disease but who has not yet been diagnosed with the disease (including, for example, diseases that may be associated with or caused by a primary disease), (b) inhibiting the disease, i.e., halting its development, and (c) palliating the disease, i.e., causing regression of the disease. Treating can refer to any indication of success in treating, ameliorating, or preventing cancer, including any objective or subjective parameter, such as relief, remission, reducing symptoms, or making disease symptoms more tolerable to patients, slowing the rate of deterioration or decline, or making the end point of deterioration less debilitating. Treating or ameliorating symptoms is based on one or more objective or subjective parameters, including the results of a physician's examination. Thus, the term "treating" includes administering a compound or agent of the present invention to prevent, delay, alleviate, stop, or inhibit the onset of symptoms or symptoms associated with a disease (e.g., cancer). The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject.

[0042] As used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, reference to "an antibody" in some embodiments includes multiple antibodies, etc.

[0043] As used herein, all numerical values ​​or ranges include whole integers within or encompassing such ranges, and fractions of values ​​or integers within or encompassing ranges, unless the context clearly dictates otherwise. Thus, for example, a reference to a range of 90-100% includes 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc. In another example, a reference to a range of 1 to 5,000 times includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5 times, etc., 2.1, 2.2, 2.3, 2.4, 2.5 times, etc.

[0044] As used herein, "about" a number refers to a range that is inclusive of the number, ranging from 10% below to 10% above the number. Ranges preceded by "about" refer to ranges that extend from 10% below the lower limit of the range to 10% above the upper limit of the range.

[0045] "Percent identity (%)" refers to the degree to which two sequences (nucleotides or amino acids) have the same residues at the same positions in the alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" refers to the % identity of an amino acid sequence to SEQ ID NO: Y, and states that X% of the residues in the amino acid sequence are identical to the residues in the sequence disclosed in SEQ ID NO: Y. Generally, a computer program is used for such calculations. Exemplary programs for comparing and aligning pairs of sequences include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and Gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984).

[0046] As used herein, the term "selective binding" refers to the higher affinity with which a molecule (e.g., a protein such as the target antigen-binding domain of TAC) binds to its target molecule (e.g., a target antigen such as GUCY2C) than to other molecules. Unless otherwise indicated, the terms "selective binding" and "specific binding" are used interchangeably herein.

[0047] As used herein, the term GUCY2C refers to the enzyme guanylate cyclase 2C. GUCY2C is a transmembrane protein that functions as a receptor for the endogenous peptides guanylin and uroguanylin and heat-stable Escherichia coli enterotoxin. The encoded protein activates the cystic fibrosis transmembrane conductance regulator. GUCY2C generates cGMP after activation by binding guanylin or uroguanylin and regulates intestinal homeostasis, tumorigenesis, and obesity. Cell surface expression of GUCY2C is found on the luminal surface of intestinal epithelium and in certain hypothalamic neurons. Overexpression of GUCY2C is found in tumors that develop from intestinal metaplasia, including cancers of the colon, esophagus, stomach, and pancreas. Overexpression is maintained in more than 95% of colorectal cancer metastases.

[0048] T cell antigen coupler (TAC) In certain embodiments, nucleic acids encoding a GUCY2C T cell-antigen coupler (TAC) polypeptide are disclosed herein. In some embodiments, the nucleic acid encoding the GUCY2C TAC comprises (a) a first polynucleotide encoding an antigen-binding domain that binds to GUCY2C, (b) a second polynucleotide encoding an antigen-binding domain that binds to the TCR complex, and (c) a third polynucleotide encoding a transmembrane domain and a cytoplasmic domain. In some embodiments, the nucleic acid comprises, in order (e.g., 5' to 3'), (a) a first polynucleotide, (b) a second polynucleotide, and (c) a third polynucleotide encoding the cytoplasmic domain and a transmembrane domain of a TCR co-receptor. In some embodiments, the nucleic acid encoding the GUCY2C TAC does not encode a costimulatory domain. In some embodiments, the nucleic acid encoding the GUCY2C-TAC does not encode a co-activation domain.

[0049] Further disclosed herein in certain embodiments is a GUCY2C T cell-antigen coupler (TAC) polypeptide. In some embodiments, the GUCY2C TAC polypeptide comprises (a) an antigen-binding domain that binds to GUCY2C, (b) an antigen-binding domain that binds to the TCR complex, and (c) a transmembrane domain and a cytoplasmic domain. In some embodiments, the GUCY2C TAC polypeptide comprises, in order (e.g., from the N-terminus to the C-terminus), (a) an antigen-binding domain that binds to GUCY2C, (b) an antigen-binding domain that binds to the TCR complex, and (c) a transmembrane domain and a cytoplasmic domain. In some embodiments, the GUCY2C TAC polypeptide does not comprise a costimulatory domain. In some embodiments, the GUCY2C TAC polypeptide does not comprise a co-activation domain.

[0050] Further disclosed herein, in certain embodiments, is an expression vector comprising a nucleic acid encoding a GUCY2C TAC polypeptide described herein.

[0051] Further disclosed herein, in certain embodiments, are T cells comprising a nucleic acid encoding a GUCY2C TAC polypeptide described herein, a T cell comprising an expression vector encoding a GUCY2C TAC polypeptide described herein, or a T cell comprising a GUCY2C TAC polypeptide described herein.

[0052] Further disclosed herein, in certain embodiments, is a method of treating cancer in an individual in need thereof, the method comprising administering to the individual T cells comprising a GUCY2C T cell-antigen coupler (TAC) polypeptide described herein.

[0053] TCR complex protein antigen-binding domain In certain embodiments, the GUCY2C TAC comprises an antigen-binding domain that binds to a protein associated with the TCR complex. A "TCR complex protein antigen-binding domain," also referred to as a "TCR complex antigen-binding domain," an "antigen-binding domain that binds to a TCR complex," or an "antigen-binding domain that binds to a protein associated with the TCR complex," refers to any substance or molecule that binds directly or indirectly to a protein associated with the TCR complex. In some embodiments, an antigen-binding domain that binds to a protein associated with the TCR complex selectively binds to a protein of the TCR. In some embodiments, an antigen-binding domain that binds to a protein associated with the TCR complex comprises a substance that specifically binds to a protein of the TCR.

[0054] In some embodiments, the TCR complex protein antigen-binding domain is selected from an antibody or fragment thereof, such as a single-chain antibody (e.g., a single-chain fragment variable antibody (scFv)), a single-domain antibody (e.g., a heavy-chain-only antibody (VHH), a shark heavy-chain-only antibody (VNAR)), a nanobody, a diabody, a minibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or an Fv fragment, that binds to a TCR protein. In some embodiments, the TCR complex protein antigen-binding domain is selected from an ankyrin repeat protein (DARPin), an affibody, an adnectin, an affilin, a filomer, a finomar, an affimer, a peptide aptamer, a lectin, a knottin, a centilin, an anticalin, a peptide, a peptidomimetic, a protein, a glycoprotein, or a proteoglycan, or a naturally occurring ligand for a TCR protein. In some embodiments, the TCR complex protein antigen-binding domain is a non-protein compound that binds to the TCR protein, including but not limited to a carbohydrate, lipid, nucleic acid, or small molecule. In some embodiments, the TCR complex protein antigen-binding domain is a designed ankyrin repeat (DARPin) targeted to the TCR protein. In some embodiments, the TCR complex protein antigen-binding domain is a single-chain variable fragment (scFv) targeted to the TCR protein. In some embodiments, the TCR complex protein antigen-binding domain is a nanobody targeted to the TCR protein.

[0055] TCR-associated proteins include, but are not limited to, TCR alpha (α) chain, TCR beta (β) chain, TCR gamma (γ) chain, TCR delta (δ) chain, CD3γ chain, CD3δ chain, and CD3ε chain. In some embodiments, the antigen-binding domain that binds to a protein associated with the TCR complex is an antibody against the TCR alpha (α) chain, TCR beta (β) chain, TCR gamma (γ) chain, TCR delta (δ) chain, CD3γ chain, CD3δ chain, and / or CD3ε chain. In some embodiments, the protein associated with the TCR complex is CD3. In some embodiments, the protein associated with the TCR complex is CD3ε. In some embodiments, the antigen-binding domain that binds to CD3 is an antibody, e.g., a single-chain antibody, e.g., a single-chain variable fragment (scFv). Examples of CD3 antibodies include, but are not limited to, UCHT1, OKT3, F6A, L2K, muromonab, otelixizumab, teplizumab, visilizumab, CD3-12, MEM-57, 4D10A6, CD3D, or TR66.

[0056] In some embodiments, the antigen binding domain that binds to the TCR complex is UCHT1, or a variant thereof. In some embodiments, the UCHT1 antigen binding domain is encoded by SEQ ID NO: 31. In some embodiments, the UCHT1 antigen binding domain comprises SEQ ID NO: 32. In some embodiments, the UCHT1 antigen binding domain is mutated. In some embodiments, the UCHT1 antigen binding domain comprises a Y to T mutation (Y182T) at the position corresponding to amino acid 182 of SEQ ID NO: 32. In some embodiments, the UCHT1(Y182T) antigen binding domain is encoded by SEQ ID NO: 43. In some embodiments, the UCHT1(Y182T) antigen binding domain comprises SEQ ID NO: 44. In some embodiments, the antigen binding domain that binds to the TCR complex is humanized UCHT1 (huUCHT1). In some embodiments, the huUCHT1 antigen binding domain is encoded by SEQ ID NO: 39. In some embodiments, the huUCHT1 antigen binding domain comprises SEQ ID NO: 40. In some embodiments, the huUCHT1 has a Y to T mutation (Y177T) at the position corresponding to amino acid 177 of SEQ ID NO: 40. In some embodiments, the huUCHT1(Y177T) antigen-binding domain is encoded by SEQ ID NO: 41. In some embodiments, the huUCHT1 antigen-binding domain comprises SEQ ID NO: 42.

[0057] In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 31 (UCHT1). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 31 (UCHT1). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 31 (UCHT1). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 31 (UCHT1). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 31 (UCHT1). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 31 (UCHT1). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 31 (UCHT1). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 31 (UCHT1). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO: 31 (UCHT1).In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 31 (UCHT1). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises the nucleotide sequence of SEQ ID NO: 31 (UCHT1).

[0058] In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises the amino acid sequence of SEQ ID NO: 32 (UCHT1).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1) (i.e., the antigen-binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence that includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each of which has 100% identity to the corresponding CDRs of the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 80% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 85% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 90% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 95% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 96% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 97% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 98% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 99% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1).

[0059] In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 43 (UCHT1(Y182T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 43 (UCHT1(Y182T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 43 (UCHT1(Y182T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 43 (UCHT1(Y182T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 43 (UCHT1(Y182T)). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 43 (UCHT1(Y182T)). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 43 (UCHT1(Y182T)). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 43 (UCHT1(Y182T)).In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO: 43 (UCHT1(Y182T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 43 (UCHT1(Y182T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises the nucleotide sequence of SEQ ID NO: 43 (UCHT1(Y182T)).

[0060] In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)).In some embodiments, the antigen-binding domain that binds to a protein associated with a TCR complex comprises the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)) (i.e., the antigen-binding domain that binds to a protein associated with a TCR complex comprises amino acid sequences that include CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each of which has 100% identity to the corresponding CDRs of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T))). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 80% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 85% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 90% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 95% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 96% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 97% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 98% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 99% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 44 (UCHT1(Y182T)).

[0061] In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 39 (huUCHT1). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 39 (huUCHT1). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 39 (huUCHT1). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 39 (huUCHT1). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 39 (huUCHT1). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 39 (huUCHT1). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 39 (huUCHT1). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 39 (huUCHT1).In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO: 39 (huUCHT1). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 39 (huUCHT1). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises the nucleotide sequence of SEQ ID NO: 39 (huUCHT1).

[0062] In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 40 (huUCHT1).In some embodiments, the antigen-binding domain that binds to a protein associated with a TCR complex comprises the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1) (i.e., the antigen-binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each of which has 100% identity to the corresponding CDRs of the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 80% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, the CDR sequences of the antigen binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1), and the non-CDR (e.g., framework) sequences of the antigen binding domain that binds to a protein associated with a TCR complex have at least 85% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, the CDR sequences of the antigen binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1), and the non-CDR (e.g., framework) sequences of the antigen binding domain that binds to a protein associated with a TCR complex have at least 90% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1).In some embodiments, the CDR sequences of the antigen binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1), and the non-CDR (e.g., framework) sequences of the antigen binding domain that binds to a protein associated with a TCR complex have at least 95% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, the CDR sequences of the antigen binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1), and the non-CDR (e.g., framework) sequences of the antigen binding domain that binds to a protein associated with a TCR complex have at least 96% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, the CDR sequences of the antigen binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1), and the non-CDR (e.g., framework) sequences of the antigen binding domain that binds to a protein associated with a TCR complex have at least 97% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1). In some embodiments, the CDR sequences of the antigen binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1), and the non-CDR (e.g., framework) sequences of the antigen binding domain that binds to a protein associated with a TCR complex have at least 98% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 99% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 40 (huUCHT1).

[0063] In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 41 (huUCHT1(Y177T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 41 (huUCHT1(Y177T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 41 (huUCHT1(Y177T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 41 (huUCHT1(Y177T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 41 (huUCHT1(Y177T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 41 (huUCHT1(Y177T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 41 (huUCHT1(Y177T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 41 (huUCHT1(Y177T)).In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO: 41 (huUCHT1(Y177T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 41 (huUCHT1(Y177T)). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises the nucleotide sequence of SEQ ID NO: 41 (huUCHT1(Y177T)).

[0064] In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, an antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)).In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, the CDR sequences of the antigen binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)) (i.e., the antigen binding domain that binds to a protein associated with a TCR complex comprises amino acid sequences comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each of which have 100% identity to the corresponding CDRs of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T))). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 80% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 85% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 90% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 95% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 96% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 97% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 98% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 99% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 42 (huUCHT1(Y177T)).

[0065] In some embodiments, the antigen-binding domain that binds to a protein associated with a TCR complex is OKT3. In some embodiments, the mouse OKT3 antigen-binding domain is encoded by SEQ ID NO: 33. In some embodiments, the OKT3 antigen-binding domain comprises SEQ ID NO: 34.

[0066] In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 33 (OKT3). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 33 (OKT3). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 33 (OKT3). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 33 (OKT3). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 33 (OKT3). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 33 (OKT3). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 33 (OKT3). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 33 (OKT3). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO: 33 (OKT3).In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 33 (OKT3). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises the nucleotide sequence of SEQ ID NO: 33 (OKT3).

[0067] In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises the amino acid sequence of SEQ ID NO: 34 (OKT3).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3) (i.e., the antigen-binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence that includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each of which has 100% identity to the corresponding CDRs of the amino acid sequence of SEQ ID NO: 34 (OKT3)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 80% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 85% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 90% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 95% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 96% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 97% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 98% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 99% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 34 (OKT3).

[0068] In some embodiments, the antigen-binding domain that binds to a protein associated with a TCR complex is F6A. In some embodiments, the mouse F6A antigen-binding domain is encoded by SEQ ID NO: 35. In some embodiments, the F6A antigen-binding domain comprises SEQ ID NO: 36.

[0069] In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 35 (F6A). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 35 (F6A). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 35 (F6A). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 35 (F6A). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 35 (F6A). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 35 (F6A). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 35 (F6A). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 35 (F6A). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO: 35 (F6A).In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 35 (F6A). In some embodiments, the polynucleotide encoding an antigen binding domain that binds to a protein associated with a TCR complex comprises the nucleotide sequence of SEQ ID NO: 35 (F6A).

[0070] In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises the amino acid sequence of SEQ ID NO: 36 (F6A).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 36 (F6A) (i.e., the antigen-binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each of which has 100% identity to the corresponding CDRs of the amino acid sequence of SEQ ID NO: 36 (F6A)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 36 (F6A), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 80% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 36 (F6A), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 85% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 36 (F6A), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 90% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 36 (F6A).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 36 (F6A), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 95% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 36 (F6A), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 96% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 36 (F6A), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 97% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 36 (F6A), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 98% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 36 (F6A). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 36 (F6A), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 99% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 36 (F6A).

[0071] In some embodiments, the antigen-binding domain that binds to a protein associated with a TCR complex is L2K. In some embodiments, the mouse L2K antigen-binding domain is encoded by SEQ ID NO: 37. In some embodiments, the L2K antigen-binding domain comprises SEQ ID NO: 38.

[0072] In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 37 (L2K). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 37 (L2K). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 37 (L2K). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 37 (L2K). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 85 (L2K). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 37 (L2K). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO: 85 (L2K). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO: 37 (L2K). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO: 85 (L2K).In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 37 (L2K). In some embodiments, the polynucleotide encoding an antigen-binding domain that binds to a protein associated with a TCR complex comprises the nucleotide sequence of SEQ ID NO: 37 (L2K).

[0073] In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the antigen binding domain that binds to a protein associated with a TCR complex comprises the amino acid sequence of SEQ ID NO: 38 (L2K).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 38 (L2K) (i.e., the antigen-binding domain that binds to a protein associated with a TCR complex comprises an amino acid sequence that includes CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, each of which has 100% identity to the corresponding CDRs of the amino acid sequence of SEQ ID NO: 38 (L2K)). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 38 (L2K), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 80% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 38 (L2K), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 85% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 38 (L2K), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 90% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 38 (L2K).In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 38 (L2K), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 95% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 38 (L2K), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 96% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 38 (L2K), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 97% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 38 (L2K), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 98% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 38 (L2K). In some embodiments, the CDR sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have 100% identity to the CDR sequences of the amino acid sequence of SEQ ID NO: 38 (L2K), and the non-CDR (e.g., framework) sequences of the antigen-binding domain that binds to a protein associated with a TCR complex have at least 99% sequence identity to the non-CDR (e.g., framework) sequences of the amino acid sequence of SEQ ID NO: 38 (L2K).

[0074] The amino acid and nucleotide sequences of exemplary antigen-binding domains that bind to proteins associated with the TCR complex are provided in Table 1.

[0075] [Table 1]

[0076] Transmembrane and cytoplasmic domains In some embodiments, the GUCY2C T cell antigen coupler polypeptide comprises a T cell receptor signaling domain polypeptide. In some embodiments, the GUCY2C T cell antigen coupler polypeptide comprises a transmembrane domain of a TCR signaling domain. In some embodiments, the GUCY2C T cell antigen coupler polypeptide comprises a cytoplasmic domain of a TCR signaling domain polypeptide. In some embodiments, the GUCY2C T cell antigen coupler polypeptide comprises a transmembrane domain and a cytoplasmic domain of a TCR signaling domain polypeptide.

[0077] In some embodiments, the T cell receptor signaling domain polypeptide comprises a TCR co-receptor domain. In some embodiments, the TCR signaling domain polypeptide comprises a transmembrane domain and / or a cytoplasmic domain of a TCR co-receptor. In some embodiments, the TCR co-receptor is CD4, CD8, LAG3, or a chimeric variation thereof.

[0078] In some embodiments, the TCR co-receptor is CD4. In some embodiments, the GUCY2C TAC comprises the transmembrane and cytoplasmic domains of the CD4 co-receptor. In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 45 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 45 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 45 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 45 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:45 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:45 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO:45 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO:45 (CD4 transmembrane and cytoplasmic domains).In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO:45 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:45 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises the nucleotide sequence of SEQ ID NO:45 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:46 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO:46 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 46 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 46 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 46 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 46 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 46 (CD4 transmembrane and cytoplasmic domains).In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 46 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 46 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 46 (CD4 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise the amino acid sequence of SEQ ID NO: 46 (CD4 transmembrane and cytoplasmic domains).

[0079] In some embodiments, the TCR co-receptor is CD8. In some embodiments, the TCR co-receptor is CD8α. In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 47 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 47 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 47 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 47 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:47 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:47 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO:47 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO:47 (CD8 transmembrane and cytoplasmic domains).In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO:47 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:47 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises the nucleotide sequence of SEQ ID NO:47 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:48 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO:48 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 48 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 48 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 48 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 48 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 48 (CD8 transmembrane and cytoplasmic domains).In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 48 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 48 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 48 (CD8 transmembrane and cytoplasmic domains). In some embodiments, the cytoplasmic and transmembrane domains comprise the amino acid sequence of SEQ ID NO: 48 (CD8 transmembrane and cytoplasmic domains).

[0080] In some embodiments, the TCR signaling domain polypeptide comprises a chimera of sequences or domains from co-receptors. In some embodiments, the TCR signaling domain polypeptide comprises a chimera of CD8α and CD8β, in which the CD8α arginine-rich region is replaced with the CD8β arginine-rich region (CD8α+R(β) chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domain comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 49 (CD8α+R(β) chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domain comprises a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 49 (CD8α+R(β) chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domain comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 49 (CD8α+R(β) chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO:49 (CD8α+R(β) chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:49 (CD8α+R(β) chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:49 (CD8α+R(β) chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO:49 (CD8α+R(β) chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO:49 (CD8α+R(β) chimera).In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO: 49 (CD8α+R(β) chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 49 (CD8α+R(β) chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises the nucleotide sequence of SEQ ID NO: 49 (CD8α+R(β) chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 50 (CD8α+R(β) chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 50 (CD8α+R(β) chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 50 (CD8α+R(β) chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 50 (CD8α+R(β) chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 50 (CD8α+R(β) chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 50 (CD8α+R(β) chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 50 (CD8α+R(β) chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 50 (CD8α+R(β) chimera).In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 50 (CD8α+R(β) chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 50 (CD8α+R(β) chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise the amino acid sequence of SEQ ID NO: 50 (CD8α+R(β) chimera).

[0081] In some embodiments, the TCR signaling domain polypeptide comprises a chimera of CD8α and CD8β, wherein a CD8α CXCP domain containing an Lck-binding motif is appended to the C-terminus of the CD8β cytoplasmic domain (CD8β+Lck chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domain comprises a nucleotide sequence having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 51 (CD8β+Lck chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domain comprises a nucleotide sequence having at least 75% sequence identity to the nucleotide sequence of SEQ ID NO: 51 (CD8β+Lck chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domain comprises a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 51 (CD8β+Lck chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domain comprises a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 51 (CD8β+Lck chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:51 (CD8β+Lck chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:51 (CD8β+Lck chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO:51 (CD8β+Lck chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO:51 (CD8β+Lck chimera).In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO: 51 (CD8β+Lck chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 51 (CD8β+Lck chimera). In some embodiments, the polynucleotide encoding the cytoplasmic and transmembrane domains comprises the nucleotide sequence of SEQ ID NO: 51 (CD8β+Lck chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 52 (CD8β+Lck chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 52 (CD8β+Lck chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 52 (CD8β+Lck chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 52 (CD8β+Lck chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 52 (CD8β+Lck chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 52 (CD8β+Lck chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 52 (CD8β+Lck chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 52 (CD8β+Lck chimera).In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 52 (CD8β+Lck chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 52 (CD8β+Lck chimera). In some embodiments, the cytoplasmic and transmembrane domains comprise the amino acid sequence of SEQ ID NO: 52 (CD8β+Lck chimera).

[0082] In some embodiments, the TCR signaling domain polypeptide comprises both the cytoplasmic and transmembrane domains of a TCR co-receptor protein, in some embodiments, the cytoplasmic and transmembrane domains are from the same co-receptor or from different co-receptors.

[0083] The amino acid and nucleotide sequences of exemplary transmembrane and cytoplasmic domains are provided in Table 2.

[0084] [Table 2]

[0085] Configurations, Linkers, and Connectors In some embodiments, the nucleic acids disclosed herein are arranged in the following order from the 5' end to the 3' end: (1) a first polynucleotide encoding an antigen-binding domain that binds to GUCY2C, (2) a second polynucleotide encoding an antigen-binding domain that binds to the TCR complex, and (3) a third polynucleotide encoding the transmembrane domain and cytoplasmic domain. In some embodiments, the nucleic acids disclosed herein are arranged in the following order from the 5' end to the 3' end: (1) a first polynucleotide encoding an antigen-binding domain that binds to GUCY2C, (2) a second polynucleotide encoding an antigen-binding domain that binds to the TCR complex, and (3) a third polynucleotide encoding the transmembrane domain and cytoplasmic domain. In some embodiments, the nucleic acids disclosed herein are arranged in the following order from the 3' end to the 5' end: (1) a first polynucleotide encoding an antigen-binding domain that binds to GUCY2C, (2) a second polynucleotide encoding an antigen-binding domain that binds to the TCR complex, and (3) a third polynucleotide encoding the transmembrane domain and cytoplasmic domain. In some embodiments, the nucleic acids described herein are arranged in the following order from the 5' end to the 3' end: (1) a first polynucleotide encoding an antigen-binding domain that binds to the TCR complex, (2) a second polynucleotide encoding an antigen-binding domain that binds to GUCY2C, and (3) a third polynucleotide encoding the transmembrane domain and cytoplasmic domain. In some embodiments, the nucleic acids described herein are arranged in the following order from the 5' end to the 3' end: (1) a first polynucleotide encoding an antigen-binding domain that binds to the TCR complex, (2) a second polynucleotide encoding an antigen-binding domain that binds to GUCY2C, and (3) a third polynucleotide encoding the transmembrane domain and cytoplasmic domain. In some embodiments, the nucleic acids described herein are arranged in the following order from the 3' end to the 5' end: (1) a first polynucleotide encoding an antigen-binding domain that binds to the TCR complex, (2) a second polynucleotide encoding an antigen-binding domain that binds to GUCY2C, and (3) a third polynucleotide encoding the transmembrane domain and cytoplasmic domain.

[0086] In some embodiments, the GUCY2C TAC polypeptide disclosed herein comprises (1) an antigen-binding domain that binds to GUCY2C, (2) an antigen-binding domain that binds to the TCR complex, and (3) a transmembrane domain and a cytoplasmic domain, in the order of N-terminus to C-terminus. In some embodiments, the GUCY2C TAC polypeptide disclosed herein comprises (1) an antigen-binding domain that binds to GUCY2C, (2) an antigen-binding domain that binds to the TCR complex, and (3) a transmembrane domain and a cytoplasmic domain, in the order of C-terminus to N-terminus. In some embodiments, the GUCY2C TAC polypeptide described herein comprises (1) an antigen-binding domain that binds to the TCR complex, (2) an antigen-binding domain that binds to GUCY2C, and (3) a transmembrane domain and a cytoplasmic domain, in the order of N-terminus to C-terminus. In some embodiments, the GUCY2C TAC polypeptide described herein comprises (1) an antigen-binding domain that binds to the TCR complex, (2) an antigen-binding domain that binds to GUCY2C, and (3) a transmembrane domain and a cytoplasmic domain, in this order from C-terminus to N-terminus.

[0087] In some embodiments, the antigen-binding domain that binds to GUCY2C, the antigen-binding domain that binds to a TCR complex, and / or the transmembrane and cytoplasmic domains are directly fused. For example, the antigen-binding domain that binds to GUCY2C and the transmembrane and cytoplasmic domains are both fused to the antigen-binding domain that binds to a TCR complex. In some embodiments, the antigen-binding domain that binds to GUCY2C, the antigen-binding domain that binds to a TCR complex, and / or the transmembrane and cytoplasmic domains are linked by at least one linker. In some embodiments, the antigen-binding domain that binds to GUCY2C and the antigen-binding domain that binds to a TCR complex are directly fused and linked to the transmembrane and cytoplasmic domain by a linker. In some embodiments, the antigen-binding domain that binds to a TCR complex and the transmembrane and cytoplasmic domains are directly fused and linked to the antigen-binding domain that binds to GUCY2C by a linker.

[0088] In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises 1 to 40 amino acids. In some embodiments, the peptide linker comprises 1 to 30 amino acids. In some embodiments, the peptide linker comprises 1 to 15 amino acids. In some embodiments, the peptide linker comprises 1 to 10 amino acids. In some embodiments, the peptide linker comprises 1 to 6 amino acids. In some embodiments, the peptide linker comprises 30 to 40 amino acids. In some embodiments, the peptide linker comprises 32 to 36 amino acids. In some embodiments, the peptide linker comprises 5 to 30 amino acids. In some embodiments, the peptide linker comprises 5 amino acids. In some embodiments, the peptide linker comprises 10 amino acids. In some embodiments, the peptide linker comprises 15 amino acids. In some embodiments, the peptide linker comprises 20 amino acids. In some embodiments, the peptide linker comprises 25 amino acids. In some embodiments, the peptide linker comprises 30 amino acids. In some embodiments, the peptide linker comprises a glycine and / or serine rich linker.

[0089] In some embodiments, at least one linker comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 26 ((G4S)4-based linker), SEQ ID NO: 28 (G4S-based linker), SEQ ID NO: 6 (linker 1), SEQ ID NO: 8 (linker 2), SEQ ID NO: 10 (CD4-based linker), SEQ ID NO: 12 (short helix connector), SEQ ID NO: 14 (long helix connector), SEQ ID NO: 16 (large domain connector), SEQ ID NO: 22 (Whitlow), or SEQ ID NO: 24 (G4S3 linker). In some embodiments, at least one linker comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO: 26 ((G4S)4-based linker), SEQ ID NO: 28 (G4S-based linker), SEQ ID NO: 6 (linker 1), SEQ ID NO: 8 (linker 2), SEQ ID NO: 10 (CD4-based linker), SEQ ID NO: 12 (short helix connector), SEQ ID NO: 14 (long helix connector), SEQ ID NO: 16 (large domain connector), SEQ ID NO: 22 (Whitlow), or SEQ ID NO: 24 (G4S3 linker). In some embodiments, at least one linker comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 26 ((G4S)4-based linker), SEQ ID NO: 28 (G4S-based linker), SEQ ID NO: 6 (linker 1), SEQ ID NO: 8 (linker 2), SEQ ID NO: 10 (CD4-based linker), SEQ ID NO: 12 (short helix connector), SEQ ID NO: 14 (long helix connector), SEQ ID NO: 16 (large domain connector), SEQ ID NO: 22 (Whitlow), or SEQ ID NO: 24 (G4S3 linker). In some embodiments, at least one linker comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 26 ((G4S)4-based linker), SEQ ID NO: 28 (G4S-based linker), SEQ ID NO: 6 (linker 1), SEQ ID NO: 8 (linker 2), SEQ ID NO: 10 (CD4-based linker), SEQ ID NO: 12 (short helix connector), SEQ ID NO: 14 (long helix connector), SEQ ID NO: 16 (large domain connector), SEQ ID NO: 22 (Whitlow), or SEQ ID NO: 24 (G4S3 linker).In some embodiments, at least one linker comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO: 26 ((G4S)4-based linker), SEQ ID NO: 28 (G4S-based linker), SEQ ID NO: 6 (linker 1), SEQ ID NO: 8 (linker 2), SEQ ID NO: 10 (CD4-based linker), SEQ ID NO: 12 (short helix connector), SEQ ID NO: 14 (long helix connector), SEQ ID NO: 16 (large domain connector), SEQ ID NO: 22 (Whitlow), or SEQ ID NO: 24 (G4S3 linker). In some embodiments, at least one linker comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO: 26 ((G4S)4-based linker), SEQ ID NO: 28 (G4S-based linker), SEQ ID NO: 6 (linker 1), SEQ ID NO: 8 (linker 2), SEQ ID NO: 10 (CD4-based linker), SEQ ID NO: 12 (short helix connector), SEQ ID NO: 14 (long helix connector), SEQ ID NO: 16 (large domain connector), SEQ ID NO: 22 (Whitlow), or SEQ ID NO: 24 (G4S3 linker). In some embodiments, at least one linker comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO: 26 ((G4S)4-based linker), SEQ ID NO: 28 (G4S-based linker), SEQ ID NO: 6 (linker 1), SEQ ID NO: 8 (linker 2), SEQ ID NO: 10 (CD4-based linker), SEQ ID NO: 12 (short helix connector), SEQ ID NO: 14 (long helix connector), SEQ ID NO: 16 (large domain connector), SEQ ID NO: 22 (Whitlow), or SEQ ID NO: 24 (G4S3 linker). In some embodiments, at least one linker comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 26 ((G4S)4-based linker), SEQ ID NO: 28 (G4S-based linker), SEQ ID NO: 6 (linker 1), SEQ ID NO: 8 (linker 2), SEQ ID NO: 10 (CD4-based linker), SEQ ID NO: 12 (short helix connector), SEQ ID NO: 14 (long helix connector), SEQ ID NO: 16 (large domain connector), SEQ ID NO: 22 (Whitlow), or SEQ ID NO: 24 (G4S3 linker).In some embodiments, at least one linker comprises the amino acid sequence of SEQ ID NO: 26 ((G4S)4-based linker), SEQ ID NO: 28 (G4S-based linker), SEQ ID NO: 6 (linker 1), SEQ ID NO: 8 (linker 2), SEQ ID NO: 10 (CD4-based linker), SEQ ID NO: 12 (short helix connector), SEQ ID NO: 14 (long helix connector), SEQ ID NO: 16 (large domain connector), SEQ ID NO: 22 (Whitlow), or SEQ ID NO: 24 (G4S3 linker).

[0090] In some embodiments, at least one linker comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO:25 ((G4S)4-based linker), SEQ ID NO:27 (G4S-based linker), SEQ ID NO:5 (linker 1), SEQ ID NO:7 (linker 2), SEQ ID NO:9 (CD4-based linker), SEQ ID NO:11 (short helix connector), SEQ ID NO:13 (long helix connector), SEQ ID NO:15 (large domain connector), SEQ ID NO:21 (Whitlow), or SEQ ID NO:23 (G4S3 linker). In some embodiments, at least one linker comprises a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO:25 ((G4S)4-based linker), SEQ ID NO:27 (G4S-based linker), SEQ ID NO:5 (linker 1), SEQ ID NO:7 (linker 2), SEQ ID NO:9 (CD4-based linker), SEQ ID NO:11 (short helix connector), SEQ ID NO:13 (long helix connector), SEQ ID NO:15 (large domain connector), SEQ ID NO:21 (Whitlow), or SEQ ID NO:23 (G4S3 linker). In some embodiments, at least one linker comprises a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:25 ((G4S)4-based linker), SEQ ID NO:27 (G4S-based linker), SEQ ID NO:5 (linker 1), SEQ ID NO:7 (linker 2), SEQ ID NO:9 (CD4-based linker), SEQ ID NO:11 (short helix connector), SEQ ID NO:13 (long helix connector), SEQ ID NO:15 (large domain connector), SEQ ID NO:21 (Whitlow), or SEQ ID NO:23 (G4S3 linker). In some embodiments, at least one linker comprises a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO:25 ((G4S)4-based linker), SEQ ID NO:27 (G4S-based linker), SEQ ID NO:5 (linker 1), SEQ ID NO:7 (linker 2), SEQ ID NO:9 (CD4-based linker), SEQ ID NO:11 (short helix connector), SEQ ID NO:13 (long helix connector), SEQ ID NO:15 (large domain connector), SEQ ID NO:21 (Whitlow), or SEQ ID NO:23 (G4S3 linker).In some embodiments, at least one linker comprises a nucleotide sequence having at least 96% identity to the nucleotide sequence of SEQ ID NO:25 ((G4S)4-based linker), SEQ ID NO:27 (G4S-based linker), SEQ ID NO:5 (linker 1), SEQ ID NO:7 (linker 2), SEQ ID NO:9 (CD4-based linker), SEQ ID NO:11 (short helix connector), SEQ ID NO:13 (long helix connector), SEQ ID NO:15 (large domain connector), SEQ ID NO:21 (Whitlow), or SEQ ID NO:23 (G4S3 linker). In some embodiments, at least one linker comprises a nucleotide sequence having at least 97% identity to the nucleotide sequence of SEQ ID NO:25 ((G4S)4-based linker), SEQ ID NO:27 (G4S-based linker), SEQ ID NO:5 (linker 1), SEQ ID NO:7 (linker 2), SEQ ID NO:9 (CD4-based linker), SEQ ID NO:11 (short helix connector), SEQ ID NO:13 (long helix connector), SEQ ID NO:15 (large domain connector), SEQ ID NO:21 (Whitlow), or SEQ ID NO:23 (G4S3 linker). In some embodiments, at least one linker comprises a nucleotide sequence having at least 98% identity to the nucleotide sequence of SEQ ID NO:25 ((G4S)4-based linker), SEQ ID NO:27 (G4S-based linker), SEQ ID NO:5 (linker 1), SEQ ID NO:7 (linker 2), SEQ ID NO:9 (CD4-based linker), SEQ ID NO:11 (short helix connector), SEQ ID NO:13 (long helix connector), SEQ ID NO:15 (large domain connector), SEQ ID NO:21 (Whitlow), or SEQ ID NO:23 (G4S3 linker). In some embodiments, at least one linker comprises a nucleotide sequence having at least 99% identity to the nucleotide sequence of SEQ ID NO:25 ((G4S)4-based linker), SEQ ID NO:27 (G4S-based linker), SEQ ID NO:5 (linker 1), SEQ ID NO:7 (linker 2), SEQ ID NO:9 (CD4-based linker), SEQ ID NO:11 (short helix connector), SEQ ID NO:13 (long helix connector), SEQ ID NO:15 (large domain connector), SEQ ID NO:21 (Whitlow), or SEQ ID NO:23 (G4S3 linker).In some embodiments, at least one linker comprises the nucleotide sequence of SEQ ID NO:25 ((G4S)4-based linker), SEQ ID NO:27 (G4S-based linker), SEQ ID NO:5 (linker 1), SEQ ID NO:7 (linker 2), SEQ ID NO:9 (CD4-based linker), SEQ ID NO:11 (short helix connector), SEQ ID NO:13 (long helix connector), SEQ ID NO:15 (large domain connector), SEQ ID NO:21 (Whitlow), or SEQ ID NO:23 (G4S3 linker).

[0091] In some embodiments, the peptide linker connecting the antigen-binding domain that binds GUCY2C to the antigen-binding domain that binds the TCR complex (e.g., UCHT1) is known as a connector to distinguish this protein domain from other linkers within the TAC. The connector can be any size. In some embodiments, the connector between the antigen-binding domain that binds the TCR complex and the antigen-binding domain that binds GUCY2C is a short helix comprising SEQ ID NO: 12. In some embodiments, the connector between the antigen-binding domain that binds the TCR complex and the antigen-binding domain that binds GUCY2C is a short helix encoded by SEQ ID NO: 11. In some embodiments, the connector between the antigen-binding domain that binds the TCR complex and the antigen-binding domain that binds GUCY2C is a long helix comprising SEQ ID NO: 14. In some embodiments, the connector between the antigen-binding domain that binds the TCR complex and the antigen-binding domain that binds GUCY2C is a long helix encoded by SEQ ID NO: 13. In some embodiments, the connector between the antigen-binding domain that binds the TCR complex and the antigen-binding domain that binds GUCY2C is a large domain comprising SEQ ID NO: 16. In some embodiments, the connector between the antigen binding domain that binds to the TCR complex and the antigen binding domain that binds to GUCY2C is the large domain encoded by SEQ ID NO:15.

[0092] In some embodiments, a nucleic acid or TAC disclosed herein comprises a leader sequence. In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:1 (muIgG leader), SEQ ID NO:17 (huIgG leader), SEQ ID NO:19 (huCD8a leader), or SEQ ID NO:29 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 85% sequence identity to the nucleotide sequence of SEQ ID NO:1 (muIgG leader), SEQ ID NO:17 (huIgG leader), SEQ ID NO:19 (huCD8a leader), or SEQ ID NO:29 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:1 (muIgG leader), SEQ ID NO:17 (huIgG leader), SEQ ID NO:19 (huCD8a leader), or SEQ ID NO:29 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:1 (muIgG leader), SEQ ID NO:17 (huIgG leader), SEQ ID NO:19 (huCD8a leader), or SEQ ID NO:29 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 96% sequence identity to the nucleotide sequence of SEQ ID NO:1 (muIgG leader), SEQ ID NO:17 (huIgG leader), SEQ ID NO:19 (huCD8a leader), or SEQ ID NO:29 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 97% sequence identity to the nucleotide sequence of SEQ ID NO:1 (muIgG leader), SEQ ID NO:17 (huIgG leader), SEQ ID NO:19 (huCD8a leader), or SEQ ID NO:29 (huCD8a leader).In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO:1 (muIgG leader), SEQ ID NO:17 (huIgG leader), SEQ ID NO:19 (huCD8a leader), or SEQ ID NO:29 (huCD8a leader). In some embodiments, the leader sequence is encoded by a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:1 (muIgG leader), SEQ ID NO:17 (huIgG leader), SEQ ID NO:19 (huCD8a leader), or SEQ ID NO:29 (huCD8a leader). In some embodiments, the leader sequence comprises the nucleotide sequence of SEQ ID NO:1 (muIgG leader), SEQ ID NO:17 (huIgG leader), SEQ ID NO:19 (huCD8a leader), or SEQ ID NO:29 (huCD8a leader).

[0093] In some embodiments, a nucleic acid or TAC disclosed herein comprises a leader sequence. In some embodiments, the leader sequence comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:2 (muIgG leader), SEQ ID NO:18 (huIgG leader), SEQ ID NO:20 (huCD8a leader), or SEQ ID NO:30 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:2 (muIgG leader), SEQ ID NO:18 (huIgG leader), SEQ ID NO:20 (huCD8a leader), or SEQ ID NO:30 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2 (muIgG leader), SEQ ID NO:18 (huIgG leader), SEQ ID NO:20 (huCD8a leader), or SEQ ID NO:30 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2 (muIgG leader), SEQ ID NO:18 (huIgG leader), SEQ ID NO:20 (huCD8a leader), or SEQ ID NO:30 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:2 (muIgG leader), SEQ ID NO:18 (huIgG leader), SEQ ID NO:20 (huCD8a leader), or SEQ ID NO:30 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:2 (muIgG leader), SEQ ID NO:18 (huIgG leader), SEQ ID NO:20 (huCD8a leader), or SEQ ID NO:30 (huCD8a leader). In some embodiments, the leader sequence comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2 (muIgG leader), SEQ ID NO: 18 (huIgG leader), SEQ ID NO: 20 (huCD8a leader), or SEQ ID NO: 30 (huCD8a leader).In some embodiments, the leader sequence comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 (muIgG leader), SEQ ID NO: 18 (huIgG leader), SEQ ID NO: 20 (huCD8a leader), or SEQ ID NO: 30 (huCD8a leader). In some embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 2 (muIgG leader), SEQ ID NO: 18 (huIgG leader), SEQ ID NO: 20 (huCD8a leader), or SEQ ID NO: 30 (huCD8a leader).

[0094] In some embodiments, the GUCY2C T cell antigen coupler polypeptide comprises a tag, e.g., a Myc tag. In some embodiments, the tag comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:4 (Myc tag). In some embodiments, the tag comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID NO:4 (Myc tag). In some embodiments, the tag comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:4 (Myc tag). In some embodiments, the tag comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:4 (Myc tag). In some embodiments, the tag comprises an amino acid sequence having at least 96% identity to the amino acid sequence of SEQ ID NO:4 (Myc tag). In some embodiments, the tag comprises an amino acid sequence having at least 97% identity to the amino acid sequence of SEQ ID NO:4 (Myc tag). In some embodiments, the tag comprises an amino acid sequence having at least 98% identity to the amino acid sequence of SEQ ID NO:4 (Myc tag). In some embodiments, the tag comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 4 (Myc tag). In some embodiments, the tag comprises the amino acid sequence of SEQ ID NO: 4 (Myc tag).

[0095] In some embodiments, the tag comprises a nucleotide sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO:3 (Myc tag). In some embodiments, the tag comprises a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO:3 (Myc tag). In some embodiments, the tag comprises a nucleotide sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO:3 (Myc tag). In some embodiments, the tag comprises a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO:3 (Myc tag). In some embodiments, the tag comprises a nucleotide sequence having at least 96% identity to the nucleotide sequence of SEQ ID NO:3 (Myc tag). In some embodiments, the tag comprises a nucleotide sequence having at least 97% identity to the nucleotide sequence of SEQ ID NO:3 (Myc tag). In some embodiments, the tag comprises a nucleotide sequence having at least 98% identity to the nucleotide sequence of SEQ ID NO:3 (Myc tag). In some embodiments, the tag comprises a nucleotide sequence having at least 99% identity to the nucleotide sequence of SEQ ID NO:3 (Myc tag). In some embodiments, the tag comprises the nucleotide sequence of SEQ ID NO: 3 (Myc tag).

[0096] Exemplary linker, connector, tag, and leader sequences are provided in Table 3.

[0097] [Table 3]

[0098] GUCY2C antigen-binding domain In certain embodiments, the GUCY2C TAC polypeptide comprises a GUCY2C antigen-binding domain. In some embodiments, the GUCY2C antigen-binding domain selectively binds to GUCY2C. In some embodiments, the GUCY2C antigen-binding domain binds to GUCY2C on a target cell. In some embodiments, the target cell is a cell associated with a disease state, including, but not limited to, cancer. In some embodiments, the target cell is a tumor cell.

[0099] In some embodiments, the GUCY2C antigen-binding domain is an antibody or a fragment thereof. In some embodiments, the GUCY2C antigen-binding domain is selected from a single-chain antibody (e.g., a single-chain fragment variable antibody (scFv)), a single-domain antibody (e.g., a heavy-chain-only antibody (VHH), a shark heavy-chain-only antibody (VNAR)), a nanobody, a diabody, a minibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or an Fv fragment that binds to GUCY2C. In some embodiments, the GUCY2C antigen-binding domain is a nanobody. In some embodiments, the GUCY2C antigen-binding domain is selected from an amino acid sequence according to any one of SEQ ID NOs: 53-71.

[0100] In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 53 to 71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 53 to 71.

[0101] In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 53-71. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 53-71.

[0102] In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 53-62, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 72-74, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 53-62, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 72-74, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 53-62, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 72-74, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 53-62, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 72-74, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of any one of SEQ ID NOs: 53-62, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 72-74, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of any one of SEQ ID NOs: 53-62, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 72-74, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of any one of SEQ ID NOs: 53-62, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 72-74, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 53-62, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 72-74, respectively.

[0103] In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 63-71, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75-77, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 63-71, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75-77, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 90% identical to the amino acid sequence of any one of SEQ ID NOs: 63-71, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75-77, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 63-71, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75-77, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 96% identical to the amino acid sequence of any one of SEQ ID NOs: 63-71, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75-77, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 97% identical to the amino acid sequence of any one of SEQ ID NOs: 63-71, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75-77, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 98% identical to the amino acid sequence of any one of SEQ ID NOs: 63-71, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75-77, respectively. In some embodiments, the GUCY2C antigen-binding domain comprises an amino acid sequence at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 63-71, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75-77, respectively.

[0104] The amino acid sequences of exemplary GUCY2C antigen-binding domains are provided in Table 4.

[0105] [Table 4]

[0106] In some embodiments, the GUCY2C antigen-binding domain is a nanobody and comprises (a) a VHH CDR1 having amino acids selected from the group consisting of SEQ ID NOs: 72-75, (b) a VHH CDR2 having amino acids selected from the group consisting of SEQ ID NOs: 73-76, and (c) a VHH CDR3 having amino acids selected from the group consisting of SEQ ID NOs: 74-77. In some embodiments, the GUCY2C antigen-binding domain is a nanobody and comprises a CDR1 having the amino acid sequence of SEQ ID NO: 72, a CDR2 having the amino acid sequence of SEQ ID NO: 73, and a CDR2 having the amino acid sequence of SEQ ID NO: 74. In some embodiments, the GUCY2C antigen-binding domain is a nanobody and comprises a CDR1 having the amino acid sequence of SEQ ID NO: 75, a CDR2 having the amino acid sequence of SEQ ID NO: 76, and a CDR2 having the amino acid sequence of SEQ ID NO: 77.

[0107] The CDRs of exemplary GUCY2C antigen-binding domains are provided in Table 5.

[0108] [Table 5]

[0109] Specific TAC In certain embodiments, disclosed herein is a GUCY2C TAC protein comprising an amino acid sequence having the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153.

[0110] In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153.In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 96% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153.In some embodiments, the GUCY2C TAC protein comprises the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153.

[0111] In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 85% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 96% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively.In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 97% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 98% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises the amino acid sequence of any one of SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively.

[0112] In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 137, 139, 141, 143, 145, 147, 149, 151, and 153, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 85% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 137, 139, 141, 143, 145, 147, 149, 151, and 153, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 137, 139, 141, 143, 145, 147, 149, 151, and 153, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 137, 139, 141, 143, 145, 147, 149, 151, and 153, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 96% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 137, 139, 141, 143, 145, 147, 149, 151, and 153, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively.In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 97% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 137, 139, 141, 143, 145, 147, 149, 151, and 153, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 98% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 137, 139, 141, 143, 145, 147, 149, 151, and 153, and comprises the CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 75 to 77, respectively. In some embodiments, the GUCY2C TAC protein comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 137, 139, 141, 143, 145, 147, 149, 151, and 153, and comprises the CDR1, CDR2, and CDR3 sequences, respectively, of SEQ ID NOs: 75 to 77. In some embodiments, the GUCY2C TAC protein comprises the amino acid sequence of any one of SEQ ID NOs: 99, 101, 103, 105, 107, 109, 111, 113, 115, 137, 139, 141, 143, 145, 147, 149, 151, and 153, and comprises the CDR1, CDR2, and CDR3 sequences, respectively, of SEQ ID NOs: 75 to 77.

[0113] In some embodiments, the GUCY2C TAC protein is encoded by a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152. In some embodiments, the GUCY2C TAC protein is encoded by a nucleic acid sequence having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152. In some embodiments, the GUCY2C TAC protein is encoded by a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152. In some embodiments, the GUCY2C TAC protein is encoded by a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152.In some embodiments, the GUCY2C TAC protein is encoded by a nucleic acid sequence having at least 96% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152. In some embodiments, the GUCY2C TAC protein is encoded by a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152. In some embodiments, the GUCY2C TAC protein is encoded by a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152. In some embodiments, the GUCY2C TAC protein is encoded by a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152.In some embodiments, the GUCY2C TAC protein is encoded by the nucleic acid sequence of SEQ ID NO: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152.

[0114] The amino acid and nucleic acid sequences of an exemplary GUCY2C TAC are provided in Table 6.

[0115] [Table 6-1]

[0116] [Table 6-2]

[0117] Vector constructs In certain embodiments, disclosed herein is a vector comprising the GUCY2C TAC nucleic acid sequence disclosed herein. In some embodiments, the vector further comprises a promoter. In some embodiments, the promoter is functional in mammalian cells. Promoters, which are regions of DNA that initiate transcription of a particular nucleic acid sequence, are well known in the art. A "promoter functional in mammalian cells" refers to a promoter that drives the expression of an associated nucleic acid sequence in mammalian cells. A promoter that drives the expression of a nucleic acid sequence is said to be "operably linked" to the nucleic acid sequence.

[0118] A variety of delivery vectors and expression vehicles are used to introduce the nucleic acids described herein into cells.

[0119] In certain embodiments, (a) a first polynucleotide encoding an antigen-binding domain that binds to GUCY2C; (b) a second polynucleotide encoding an antigen-binding domain that binds to a protein associated with the TCR complex; and (c) a third polynucleotide encoding a T cell receptor signaling domain; and (d) a promoter that is functional in mammalian cells; Disclosed herein is a vector comprising:

[0120] In some embodiments, the first polynucleotide and the third polynucleotide are fused to the second polynucleotide, and the coding sequence is operably linked to a promoter. In some embodiments, the second polynucleotide and the third polynucleotide are fused to the first polynucleotide, and the coding sequence is operably linked to a promoter. In some embodiments, the vector is designed for expression in mammalian cells. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector.

[0121] In some embodiments, useful vectors include vectors derived from retroviruses, lentiviruses, murine stem cell viruses (MSCV), poxviruses, adenoviruses, and adeno-associated viruses. Other useful delivery vectors include vectors derived from herpes simplex viruses, transposons, vaccinia viruses, human papillomaviruses, simian immunodeficiency viruses, HTLVs, human foamy viruses, and variants thereof. Additional useful vectors include vectors derived from spumaviruses, mammalian type B retroviruses, mammalian type C retroviruses, avian type C retroviruses, mammalian type D retroviruses, and HTLV / BLV retroviruses. An example of a lentiviral vector useful in the disclosed compositions and methods is the pCCL4 vector.

[0122] Pharmaceutical Composition In certain embodiments, disclosed herein are pharmaceutical compositions comprising engineered T cells (transduced with and / or expressing a GUCY2C TAC polypeptide) disclosed herein and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, but are not limited to, buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), or preservatives. In some embodiments, the engineered T cells are formulated for intravenous administration.

[0123] The pharmaceutical composition is administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration are determined by factors such as the patient's symptoms and the type and severity of the patient's disease, but the appropriate dosage is determined by clinical trials. When an "immunologically effective amount," an "antitumor effective amount," a "tumor-inhibiting effective amount," or a "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered will be determined by a physician taking into account individual differences in age, body weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject).

[0124] In some embodiments, the engineered T cells and / or pharmaceutical compositions described herein are administered in a 10 1 ~10 15 cells / weight (kg), 10 4 ~10 9 Cells / kg body weight, optionally 10 5 ~10 8 cells / weight (kg), 10 6 ~10 7 cells / kg body weight, or 10 5 ~10 6 In some embodiments, the engineered T cells and / or pharmaceutical compositions described herein are administered at a dose of 10 cells / kg body weight, including all integer values ​​within those ranges. 1In some embodiments, the modified T cells and / or pharmaceutical compositions described herein are administered at doses exceeding 10 cells / kg body weight. 15 It is administered at a dose of less than cells / kg body weight.

[0125] In some embodiments, the engineered T cells and / or pharmaceutical compositions described herein are administered in an amount of 0.5×10 6 cells, 2 x 10 6 cells, 4 x 10 6 cells, 5 x 10 6 cells, 1.2×10 7 cells, 2 x 10 7 cells, 5 x 10 7 cells, 2 x 10 8 cells, 5 x 10 8 cells, 2 x 10 9 Cells, 0.5~2000×10 6 cells, 0.5~2×10 6 cells, 0.5~2×10 7 cells, 0.5~2×10 8 cells, or 0.5–2 × 10 9 The dose is administered to the cells (including all integer values ​​within those ranges).

[0126] Also disclosed herein are pharmaceutical compositions comprising engineered / modified T cells and unmodified T cells, or pharmaceutical compositions comprising different populations of engineered / modified T cells with or without unmodified T cells. One of skill in the art will understand that a therapeutic amount of engineered / modified T cells need not be essentially homogeneous. In some embodiments, engineered / modified T cells, upon activation, are capable of activating unmodified T cells within the same pharmaceutical composition / cell population. In some embodiments, engineered / modified T cells are capable of activating unmodified T cells only when activated in response to binding of an antigen to a TAC (e.g., GUCY2C) expressed by the engineered / modified T cells.

[0127] In some embodiments, the T cell composition is administered multiple times at these dosages. In some embodiments, the dosage is administered single or multiple times, for example, daily, weekly, biweekly, or monthly, hourly, or upon recurrence, relapse, or progression of the cancer being treated. The cells, in some embodiments, are administered by injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0128] In some embodiments, the pharmaceutical composition is substantially free, e.g., free at detectable levels, of contaminants selected from the group consisting of, e.g., endotoxin, mycoplasma, replication-competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, bacteria, fungi, mycoplasma, IL-2, and IL-7.

[0129] In some embodiments, the modified / engineered T cells and / or pharmaceutical compositions are administered by methods including, but not limited to, aerosol inhalation, injection, infusion, ingestion, transfusion, implantation, or transplantation. The modified / engineered T cells and / or pharmaceutical compositions may be administered to a subject intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenous (iv) injection, intravenous (iv) infusion, or intraperitoneally. The modified / engineered T cells and / or pharmaceutical compositions thereof may be administered to a patient by intradermal or subcutaneous injection. The modified / engineered T cells and / or pharmaceutical compositions thereof may be administered by iv injection. The modified / engineered T cells and / or pharmaceutical compositions thereof may be injected directly into a tumor, lymph node, or site of infection.

[0130] Pharmaceutical compositions can be prepared by known methods for preparing pharmaceutically acceptable compositions to be administered to subjects, such that an effective amount of T cells is combined in a mixture with a pharmaceutically acceptable carrier.Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, 20th Edition, Mack Publishing Company, Easton, Pa., USA, 2000).Based on this, compositions can include, but are not limited to, a solution of a substance combined with one or more pharmaceutically acceptable carriers or diluents, and can be included in a buffer solution with an appropriate pH and isotonicity with physiological fluids.

[0131] Suitable pharmaceutically acceptable carriers include essentially chemically inert and non-toxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline, glycerol solution, N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), dioleylphosphotidylethanolamine (DOPE), and liposomes. In some embodiments, such compositions contain a therapeutically effective amount of the compound together with an appropriate amount of carrier to provide a form for direct administration to a patient.

[0132] Pharmaceutical compositions include, but are not limited to, lyophilized powders or aqueous or non-aqueous sterile injection solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats, and solutes that make the composition substantially compatible with the tissues or blood of the intended recipient. Other components that may be present in such compositions include, for example, water, surfactants (e.g., Tween), alcohol, polyol, glycerin, and vegetable oil. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions.

[0133] The pharmaceutical compositions disclosed herein can be formulated into various forms and administered by several different means. Pharmaceutical formulations can be administered orally, rectally, or parenterally, optionally containing conventionally acceptable carriers, adjuvants, and vehicles. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, or intrasternal injection and infusion techniques. Administration includes injection or infusion, including intraarterial, intracardiac, intraventricular, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural, and subcutaneous), inhalation, transdermal, transmucosal, sublingual, buccal, and topical (including epidermal, cutaneous, enema, eye drops, ear drops, intranasal, and vaginal) administration. In some exemplary embodiments, the route of administration is by injection, such as intramuscular, intravenous, subcutaneous, or intraperitoneal injection.

[0134] Liquid formulations include oral, intravenous, intranasal, ophthalmic, otic, aerosol, etc. In certain embodiments, a combination of different formulations is administered. In certain embodiments, the composition is formulated for a sustained release profile.

[0135] Treatment and Use In certain embodiments, disclosed herein are methods of using the engineered T cells disclosed herein in the treatment of a GUCY2C-expressing cancer in an individual in need thereof.

[0136] In some embodiments, the antigen-binding domain of a TAC polypeptide disclosed herein that binds to GUCY2C binds to GUCY2C on tumor cells. In some embodiments, the antigen-binding domain of a TAC polypeptide disclosed herein that binds to GUCY2C selectively binds to GUCY2C on tumor cells.

[0137] In certain embodiments, disclosed herein are methods of treating a cancer that expresses GUCY2C in an individual in need thereof, the method comprising administering to the individual an engineered T cell disclosed herein or a pharmaceutical composition comprising an engineered T cell disclosed herein.

[0138] Further disclosed herein is the use of the engineered T cells disclosed herein in the preparation of a medicament for treating a GUCY2C-expressing cancer in an individual in need thereof. In certain embodiments, additionally disclosed herein is the use of the engineered T cells disclosed herein or the pharmaceutical composition disclosed herein for treating a GUCY2C-expressing cancer in an individual in need thereof.

[0139] In some embodiments, the engineered T cells disclosed herein are part of a combination therapy. In some embodiments, the efficacy of the therapies disclosed herein is assessed multiple times. In some embodiments, patients are stratified based on their response to the treatments disclosed herein. In some embodiments, the efficacy of the treatment determines participation in a clinical trial.

[0140] In some embodiments, the engineered T cells disclosed herein are administered in combination with lymphocyte-depleting therapy or to a subject who has undergone lymphocyte-depleting therapy. Examples of lymphocyte-depleting therapy include non-myeloablative lymphocyte-depleting chemotherapy, myeloablative lymphocyte-depleting chemotherapy, fludarabine, cyclophosphamide, corticosteroids, alemtuzumab, total body irradiation (TBI), and any combination thereof.

[0141] Cancers that can be treated with the engineered T cells disclosed herein include any form of neoplastic disease. In some embodiments, the cancer is colon cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, or pancreatic cancer. In some embodiments, the cancer is a primary cancer. In some embodiments, the cancer is a metastatic cancer.

[0142] In some embodiments, the cancer is primary colorectal cancer, primary gastric cancer, primary gastroesophageal junction cancer, primary esophageal cancer, or primary pancreatic cancer. In some embodiments, the cancer is metastatic colorectal cancer, metastatic gastric cancer, metastatic gastroesophageal junction cancer, metastatic esophageal cancer, or metastatic pancreatic cancer. In some embodiments, the colorectal cancer is colon adenocarcinoma. In some embodiments, the colorectal cancer is cecal adenocarcinoma. In some embodiments, the gastric cancer is gastric adenocarcinoma (e.g., gastric tubular adenocarcinoma). In some embodiments, the cancer is leukemia.

[0143] In some embodiments, the cancer to be treated is primary colorectal cancer. Colorectal cancer affects both men and women and accounts for 9.2% of all cancer deaths. The lack of response to targeted therapy, such as anti-EGFR antibodies, is associated with mutations in KRAS and BRAF oncogenes. In addition, immunotherapy, such as immune checkpoint inhibitors, has failed to demonstrate significant survival benefits in most patients with colorectal cancer due to low tumor mutation burden and reduced density of immune infiltration. Guanylyl cyclase C (GUCY2C) is overexpressed in more than 90% of colorectal cancers at all stages.

[0144] In some embodiments, the cancer to be treated is a primary cancer, such as primary gastroesophageal junction cancer or gastric tubular adenocarcinoma. Gastric cancer is the sixth most common cancer in the world and the second leading cause of cancer-related death worldwide. In most parts of the world, gastric cancer forms in the main part of the stomach (stomach body). In the United States, gastric cancer is more likely to affect the area where the esophagus faces the stomach, i.e., gastroesophageal junction cancer. Many gastric cancers develop from intestinal metaplasia, which causes more than 50% of gastric cancer and gastroesophageal junction cancer, characterized by ectopic overexpression of GUCY2C.

[0145] In some embodiments, the cancer to be treated is primary pancreatic cancer.Pancreatic cancer has the highest mortality rate of all major cancers.When all stages are combined, the 5-year relative survival rate is 10%.For people diagnosed with localized disease, the 5-year survival rate is only 39%.Most pancreatic cancers develop from intestinal metaplasia, which leads to more than 50% of pancreatic cancers being characterized by overexpression of GUCY2C. [Example]

[0146] The following examples are given for the purpose of illustrating various embodiments of the present invention and are not intended to limit the present invention in any way. The examples, together with the methods described herein, are representative of presently preferred embodiments and are exemplary, not limiting the scope of the present invention. Modifications thereof and other uses encompassed within the spirit of the invention as defined by the claims will occur to those skilled in the art.

[0147] Example 1: Production of TAC T cells targeting GUCY2C using different binder variants T cells were engineered using lentiviral vectors to express various GUCY2C-TAC receptors. The TAC receptors comprise a TAC scaffold (a myc-tagged extracellular CD3-binding domain fused to the CD4 transmembrane and cytoplasmic domains) combined with different anti-GUCY2C single-domain antibodies (nanobodies). GUCY2C-TAC variants using these different nanobodies are listed in Table 7. These include the parent G22 nanobody as well as humanized variants of both the G22 (G22H1-G22H10) and G23 (G23H1-G23H9) nanobodies.

[0148] [Table 7]

[0149] Example 2: Phenotype of GUCY2C-TAC T cells Using the TAC receptor bearing both the nonhumanized and humanized anti-GUCY2C nanobodies listed in Table 7, GUCY2C-TAC T cells were produced and phenotyped using flow cytometry. Cells were stained for TAC expression by targeting the Myc-Tag within the TAC receptor (Figure 1A). Double staining of anti-Myc with the transduction marker mStrawberry (mStraw) is shown, demonstrating transduction efficiencies ranging from 34% to 74%. TAC surface expression was quantified by measuring the mean fluorescence intensity (MFI) of TAC-positive T cells (Figure 1B). The data show that all selected GUCY2C-TAC receptors were transduced into T cells and expressed on their surface. Surface expression of the TAC constructs bearing the humanized nanobodies was comparable to or superior to that of the TAC receptor bearing the parent G22 nanobody.

[0150] Example 3: In vitro cytotoxicity of GUCY2C-TAC T cells T cell subsets were engineered to express non-humanized and humanized GUCY2C-TAC receptors as listed in Table 7. GUCY2C-TAC T cells were injected at 1 x 10 ET ratios of 1:5, 1:7.5, and 1:10. 4 NALM6 GUCY2C / eGFP Target cells were co-cultured with the TAC constructs and monitored using a live-cell imaging reader. Images were taken every 8 hours for 5 consecutive days. Tumor cells expressed GFP, indicating their presence. The observed GFP surface area was calculated for each time point and plotted against time (Figure 2). The data show that T cells engineered with the GUCY2C-TAC variants G22, G22H1, G22H5, G22H8, and G23H4, except for G23H9, were able to kill target cells. With the exception of G23H4 and G23H9, the cytotoxicity of the TAC constructs bearing humanized nanobodies is comparable to that of the TAC receptor characterized by the parent G22 nanobody.

[0151] Example 4: In vitro expansion of GUCY2C-TAC T cells T cells engineered with various non-humanized and humanized GUCY2C-TAC receptors included in Table 7 were tested for their proliferative capacity in response to antigen stimulation. To this end, GUCY2C-TAC T cells were cultured in NALM6 GUCY2C or N87 GUCY2C They were co-cultured with either of the target cells at an E:T ratio of 1:3 (Figure 3). GUCY2C N87 is a leukemia cell line engineered to overexpress GUCY2C and naturally expresses CD19. GUCY2C is a gastric carcinoma cell line engineered to overexpress GUCY2C and naturally expresses HER2. GUCY2C-TAC T cells were evaluated via a CTV (cell tracing violet) proliferation assay, briefly described below. Target cells were first inactivated using mitomycin C and then co-cultured with T cells pre-loaded with CTV dye. After 4 days of co-culture, T cells were analyzed by flow cytometry. As cells proliferate, they lose CTV expression, which can be converted to a mitotic index, calculated using FCS Express software and normalized by subtracting the T cell-only control. The positive control was NALM6. GUCY2C CD19-TAC (SEQ ID NO: 159) T cells in co-culture with target cells and N87 GUCY2C These included HER2-TAC (SEQ ID NO: 157) T cells in co-culture with target cells. The majority of GUCY2C-engineered T cell products expanded to a magnitude comparable to or exceeding that of the positive control TAC T cells.

[0152] Example 5: In vitro cytotoxicity of GUCY2C-TAC T cells in a repeated killing assay T cells were engineered with various non-humanized and humanized GUCY2C-TAC receptors as listed in Table 7. GUCY2C-TAC T cells were injected at an E:T ratio of 3:1 with 5x10 4 NALM6 GUCY2C / eGFP After 3-4 days of co-culture, GUCY2C-TAC T cells were collected from all wells and counted. These isolated T cells were then transferred to fresh NALM6 cells.GUCY2C / eGFP The TAC T cells were then co-cultured again with target cells. This process was repeated over multiple consecutive rounds using the same TAC T cells to mimic a chronic stimulation environment. The area of ​​GFP-expressing cells was calculated at the beginning and end of each round to indicate the number of viable tumor cells. A total of nine rounds were completed. CD19-TAC T cells were used as a positive control. Negative controls included wells with target cells alone, as well as wells with target cells co-cultured with non-transduced T cells (NTD). Overall findings are summarized below.

[0153] Cytotoxicity (FIG. 4A): All tested constructs were able to induce tumor cell killing over 9 rounds for a total of 32 days.

[0154] Long-term persistence and proliferation (Figure 4B): Repeated killing assays show that although the level of proliferation decreased over time, the cells remained functional as they continued to effectively kill tumor cells in successive rounds.

[0155] TAC T cell phenotype and cell composition (Figure 4C): GUCY2C-TAC T cells were phenotyped using flow cytometry at the beginning of the experiment and after rounds 1, 5, and 9. The percentages of CD4+ and CD8+ cells were graphed, demonstrating that GUCY2C-TAC T cells became enriched among CD8+ T cells in the coculture. The proportion of TCRαβ+ cells expressing the transduction marker mStrawberry (mStraw) was also examined. This analysis shows that the mStraw+ fraction of T cells engineered with various humanized GUCY2C-TAC receptors initially increased until round 5 and then decreased until round 9, with mStraw-negative cells generally being more abundant at the end of the assay. GUCY2C-TAC T cells were also stained for the activation / exhaustion marker, CD69. Specifically, the proportion of CD8+ CD69+ cells increased over time. GUCY2C-TAC T cells were also stained for a combination of exhaustion markers by first gating on CD8+ T cells. CD8+ CD39+ T cells were then examined for LAG3 and PD-1 expression. The data show that the combination of LAG3 / PD-1 / CD39 exhaustion markers was not significantly increased in CD8+ T cells.

[0156] The recapitulation of cytotoxicity and related observations of T cells expressing TAC constructs bearing humanized nanobodies was comparable to that obtained with TAC receptors featuring the parent G22 nanobody.

[0157] Example 6: In vivo activity of GUCY2C-TAC T cells against NALM6 leukemia tumor cells expressing GUCY2C NALM6 leukemia tumor cells constitutively expressing (i) a truncated GUCY2C protein containing the extracellular and transmembrane domains and (ii) luciferase (eLuc) were injected into NSG mice via the tail vein and allowed to establish for 12 days before treatment (Figure 5A). Treatment consisted of a subtherapeutic dose of 1 million TAC T cells (both nonhumanized and humanized GUCY2C-TAC variants, CD19-TAC, or untransduced cells matching the total number of T cells equivalent to 1 million TAC T cells) given as a single bolus injection. Tumor burden was represented by the intensity of the bioluminescent signal obtained by weekly in vivo imaging. The data demonstrate that the G22, G22H8, G22H1, and G22H5 GUCY2C-TAC T cell products were able to control tumors and induce sustained tumor regression.

[0158] NALM6 treated with either (1) GUCY2C-TAC T cells (Table 7), (2) CD19-TAC T cells (control), (3) non-transduced T cells (NTD), or (4) left untreated (NT) (negative control). eLuc / GUCY2C Survival curves of tumor-bearing mice. As shown in Figure 5B, the results confirm that the G22, G22H8, G22H1, and G22H5 GUCY2C-TAC T cell products induced improved survival beyond the efficacy achieved with the CD19-TAC control. G22H8 demonstrates more potent antitumor properties by inducing tumor regression and animal survival compared to the parental non-humanized G22 TAC variant.

[0159] Example 7: In vivo activity of GUCY2C-TAC T cells against GUCY2C-expressing N87 gastric cancer cells N87 gastric tumor cells constitutively expressing GUCY2C were injected subcutaneously into the hind flank of NSG mice to form solid tumor xenografts. Tumors were allowed to grow for 9 days before treatment. Tumor-bearing mice were treated with either nonhumanized or humanized GUCY2C-TAC variants, or untransduced TAC T cells (NTD), engineered TAC T cells, or TAC T cells engineered at either 1 million or 6 million, matching the total number of T cells to an equivalent of 6 million TAC T cells. Controls included a non-treated group (NT) and a 6 million HER2-TAC T positive control. All cells were injected into animals via the tail vein as a single dose. Tumor burden was measured by caliper measurement twice weekly. As shown in Figure 6, the data demonstrate that all GUCY2C-TAC T products were able to provide some degree of tumor control and durable tumor regression, with G22H8 demonstrating more potent tumor regression compared to the parental non-humanized G22TAC variant.

[0160] Example 8: Binding specificity of the G22H8 nanobody The binding specificity of G22H8 was analyzed using a membrane protein screen. The G22H8 nanobody was expressed as a GFP fusion protein, purified, and tested for its ability to bind to GUCY2C expressed in a human HEK cell line (Figure 7A). Target cells engineered with either an empty vector control or a GUCY2C expression plasmid were incubated with various concentrations of the fusion protein, and then fluorescence was measured. Concentration-dependent binding of the recombinant G22H8-GFP protein to the HEK-GUCY2C-expressing cell line was observed, confirming the ability of the G22H8 nanobody to bind to the GUCY2C antigen. Binding specificity was established using membrane proteome array technology, which consists of over 6,000 human membrane proteins and represents approximately 94% of all single-pass, multi-pass, and glycosylphosphatidylinositol (GPI)-anchored human proteins. In this study, G22H8-GFP was found to selectively bind to GUCY2C but not to other proteins (Figure 7B).

[0161] Example 9: GUCY2C expression levels in colon cancer cells GUCY2C expression was evaluated in cancer cells naturally and ectopically expressing GUCY2C. Expression was assessed by flow cytometry (Figure 8) and mRNA analysis by ddPCR (Figure 9). Cell lines endogenously expressing GUCY2C included SW1463 (adenocarcinoma; colon; Dukes' C type), T84 (carcinoma; colon), LS1034 (cecal adenocarcinoma), and H508 (cecal adenocarcinoma). Cell lines engineered to overexpress GUCY2C included HCT116 (colon carcinoma) and N87 (gastric tubular adenocarcinoma). Data showed varying levels of antigen expression, with SW1463 and H508 exhibiting lower GUCY2C levels compared to the engineered cell lines and T84 or LS1034. No GUCY2C expression was observed in HCT116 and SW480 cells.

[0162] Example 10: In vitro early activation assay of GUCY2C-TAC T cells A subset of T cells was engineered to express the humanized GUCY2C-TAC receptor as listed in Table 7. GUCY2C-TAC T cells were co-cultured with various target cells, both endogenous and engineered. After 4 hours of co-culture, cells were stained for the CD69 early activation marker.

[0163] An exemplary flow plot is shown (FIG. 10), and normalized results for various GUCYC-TAC constructs are shown as a bar graph (FIG. 11). The data demonstrate that GUCY2C-TAC T cells are activated when co-cultured with antigen-positive target cells. No reactivity was observed in co-culture with GUCY2C-negative cells such as HCT116 and N87. The observed activity was generally comparable to the HER2-TAC T reference cells for all GUCY2C-TAC candidates tested. T cell activation was significantly higher with SW1463 expressing low levels and engineered N87 expressing high levels. GUCY2CThis was similar across a wide range of antigen surface expression levels presented by the cells. Cancer cells did not result in activation of non-transduced T cells (NTD), further demonstrating that the activity observed in TAC T cells was antigen-dependent.

[0164] Example 11: In vitro cytokine production assay of GUCY2C-TAC T cells A subset of T cells was engineered to express the humanized GUCY2C-TAC receptor as listed in Table 7. GUCY2C-TAC T cells were co-cultured with various target cells, both endogenous and engineered. After 4 hours of co-culture, cells were permeabilized and stained for cytokines.

[0165] An exemplary flow plot is shown (Figure 12), and normalized results for various GUCY2C-TAC constructs are presented as a bar graph (Figure 13). The data demonstrate that GUCY2C-TAC T cells produced increased levels of cytokines when cocultured with antigen-positive target cells. No reactivity was observed in the absence of GUCY2C antigen. GUCY2C-TAC T cells were able to engage and activate target cells expressing endogenous GUCY2C antigen at various levels. Cytokine production across all GUCY2C-TAC variants was comparable. When comparing GUCY2C-TAC T cell activation to HER2-TAC in engineered cell lines, GUCY2C-TAC T cells were similar or superior to the HER2-TAC reference. Cancer cells did not result in activation of non-transduced T cells (NTD).

[0166] Example 12: In vitro cytotoxicity of GUCY2C-TAC T cells A subset of T cells was engineered to express the humanized GUCY2C-TAC receptor as listed in Table 7. GUCY2C-TAC T cells were co-cultured with various target cells, both endogenous and engineered. T cells were co-cultured with various target cells for 5 days. Target cells were engineered with GFP, and loss of GFP was used to assess cytotoxicity.

[0167] The data demonstrate that all GUCY2C-TAC candidates were able to induce cytotoxicity comparable to the HER2-TAC reference (Figure 14). Notably, GUCY2C-TAC T cells were able to induce cytotoxicity in LS1034 target cells that endogenously express the GUCY2C antigen.

[0168] [Table 8-1]

[0169] [Table 8-2]

[0170] [Table 8-3]

[0171] [Table 8-4]

[0172] [Table 8-5]

[0173] [Table 8-6]

[0174] [Table 8-7]

[0175] [Table 8-8]

[0176] Table 8-9

[0177] Table 8-10

[0178] Table 8-11

[0179] Table 8-12

[0180] Table 8-13

[0181] Table 8-14

[0182] Table 8-15

[0183] Table 8-16

[0184] Table 8-17

[0185] Table 8-18

[0186] Table 8-19

[0187] Table 8-20

[0188] Table 8-21

[0189] Table 8-22

[0190] Table 8-23

[0191] Table 8-24

[0192] Table 8-25

[0193] Table 8-26

Claims

1. a guanylate cyclase 2C (GUCY2C) T-cell antigen coupler (GUCY2C-TAC) protein, (a) a first polypeptide, (i) a CDR1 having the amino acid sequence of SEQ ID NO: 72, a CDR2 having the amino acid sequence of SEQ ID NO: 73, and a CDR3 having the amino acid sequence of SEQ ID NO: 74; or (ii) a CDR1 having the amino acid sequence of SEQ ID NO: 75, a CDR2 having the amino acid sequence of SEQ ID NO: 76, and a CDR3 having the amino acid sequence of SEQ ID NO: 77 a first polypeptide comprising a GUCY2C binding domain comprising: (b) a second polypeptide comprising an antigen-binding domain that binds a protein associated with the TCR complex; and (c) a third polypeptide comprising a TCR co-receptor cytoplasmic domain and a transmembrane domain; and Including, The GUCY2C-TAC protein, wherein the first polypeptide, the second polypeptide, and the third polypeptide are directly fused to each other or linked by at least one linker.

2. The GUCY2C-TAC protein of claim 1, wherein the GUCY2C binding domain comprises a CDR1 having the amino acid sequence of SEQ ID NO: 72, a CDR2 having the amino acid sequence of SEQ ID NO: 73, and a CDR3 having the amino acid sequence of SEQ ID NO:

74.

3. The GUCY2C-TAC protein according to claim 1 or 2, wherein the GUCY2C binding domain is a nanobody.

4. The GUCY2C-TAC protein of any one of claims 1 to 3, wherein the GUCY2C-binding domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 62.

5. The GUCY2C-TAC protein according to any one of claims 1 to 4, wherein the GUCY2C-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53 to 62.

6. The GUCY2C-TAC protein of claim 1, wherein the GUCY2C binding domain comprises a CDR1 having the amino acid sequence of SEQ ID NO: 75, a CDR2 having the amino acid sequence of SEQ ID NO: 76, and a CDR3 having the amino acid sequence of SEQ ID NO:

77.

7. The GUCY2C-TAC protein of claim 6, wherein the GUCY2C binding domain is a nanobody.

8. 8. The GUCY2C-TAC protein of claim 6 or 7, wherein the GUCY2C-binding domain comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-71.

9. The GUCY2C-TAC protein according to any one of claims 6 to 8, wherein the GUCY2C-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63 to 71.

10. The GUCY2C-TAC protein according to any one of claims 1 to 9, wherein the first polypeptide, the second polypeptide, and the third polypeptide are arranged in order from the N-terminus to the C-terminus.

11. The GUCY2C-TAC protein according to any one of claims 1 to 10, wherein the protein associated with the TCR complex is a CD3 protein.

12. The GUCY2C-TAC protein according to claim 11, wherein the CD3 protein is a CD3γ protein, a CD3δ protein, and / or a CD3ε protein.

13. The GUCY2C-TAC protein according to claim 12, wherein the CD3 protein is a CD3ε protein.

14. The GUCY2C-TAC protein according to any one of claims 1 to 13, wherein the antigen-binding domain that binds the protein associated with the TCR complex is derived from an antibody selected from UCHT1 OKT3, F6A, and L2K.

15. The GUCY2C-TAC protein of claim 14, wherein the antigen-binding domain that binds the protein associated with the TCR complex is a UCHT1 antigen-binding domain.

16. The GUCY2C-TAC protein according to claim 15, wherein the UCHT1 antigen-binding domain is a UCHT1 scFV.

17. The GUCY2C-TAC protein according to any one of claims 14 to 16, wherein the UCHT1 antigen-binding domain comprises a humanized variant of UCHT1 (huUCHT1).

18. 18. The GUCY2C-TAC protein of claim 17, wherein the UCHT1 antigen-binding domain comprises a humanized variant of UCHT1 (huUCHT1(Y177T)), which comprises a Y to T mutation at the position corresponding to amino acid 177 of SEQ ID NO:

40.

19. 19. The GUCY2C-TAC protein of any one of claims 1 to 18, wherein the antigen-binding domain that binds the protein associated with the TCR complex comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32 (UCHT1), SEQ ID NO: 44 (UCHT1(Y182T)), SEQ ID NO: 40 (huUCHT1), or SEQ ID NO: 42 (huUCHT1(Y177T)).

20. 19. The GUCY2C-TAC protein of any one of claims 1 to 18, wherein the CDR sequence of the antigen-binding domain that binds the protein associated with the TCR complex has 100% identity with the CDR sequence of the amino acid sequence of SEQ ID NO: 32 (UCHT1), SEQ ID NO: 44 (UCHT1(Y182T)), SEQ ID NO: 40 (huUCHT1), or SEQ ID NO: 42 (huUCHT1(Y177T)).

21. 19. The GUCY2C-TAC protein according to any one of claims 1 to 18, wherein the CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have 100% identity with the CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), SEQ ID NO: 44 (UCHT1(Y182T)), SEQ ID NO: 40 (huUCHT1), or SEQ ID NO: 42 (huUCHT1(Y177T)), and the non-CDR sequences of the antigen-binding domain that binds the protein associated with the TCR complex have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the non-CDR sequences of the amino acid sequence of SEQ ID NO: 32 (UCHT1), SEQ ID NO: 44 (UCHT1(Y182T)), SEQ ID NO: 40 (huUCHT1), or SEQ ID NO: 42 (huUCHT1(Y177T)).

22. The GUCY2C-TAC protein according to any one of claims 1 to 21, wherein the transmembrane domain is a CD4 transmembrane domain and the cytoplasmic domain is a CD4 cytoplasmic domain.

23. 23. The GUCY2C-TAC protein of claim 22, wherein the transmembrane domain and the cytoplasmic domain comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 46 (CD4 transmembrane domain and cytoplasmic domain).

24. The GUCY2C-TAC protein according to any one of claims 1 to 23, wherein the transmembrane domain is a CD8 transmembrane domain and the cytoplasmic domain is a CD8 cytoplasmic domain.

25. 25. The GUCY2C-TAC protein of any one of claims 1 to 24, wherein the component encoded by (a) and the component encoded by (c) are fused to the component encoded by (b).

26. 25. The GUCY2C-TAC protein according to any one of claims 1 to 24, wherein the component encoded by (b) and the component encoded by (c) are fused to the component encoded by (a).

27. 27. The GUCY2C-TAC protein of any one of claims 1 to 26, wherein at least one linker connects the component encoded by (a) to the component encoded by (b).

28. 28. The GUCY2C-TAC protein of claim 27, wherein the at least one linker is a glycine and / or serine-rich linker, a large protein domain, a long helix structure, or a short helix structure.

29. The GUCY2C-TAC protein of claim 27 or 28, wherein the at least one linker comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26 ((G4S)4-based linker), SEQ ID NO: 28 (G4S-based linker), SEQ ID NO: 14 (CD4-based linker), SEQ ID NO: 12 (short helix connector), SEQ ID NO: 14 (long helix connector), SEQ ID NO: 16 (large domain connector), or SEQ ID NO: 24 ((G4S)3-flexible linker).

30. 30. The GUCY2C-TAC protein of any one of claims 1 to 29, which does not contain a costimulatory domain.

31. 31. The GUCY2C-TAC protein according to any one of claims 1 to 30, which does not contain an activation domain.

32. 32. The GUCY2C-TAC protein of any one of claims 1 to 31, further comprising a leader sequence.

33. 33. The GUCY2C-TAC protein of claim 32, wherein the leader sequence comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2 (muIgG leader), SEQ ID NO: 18 (huIgG leader), SEQ ID NO: 20 (huCD8a-1 leader), or SEQ ID NO: 30 (huCD8a-2 leader).

34. A GUCY2C TAC protein comprising an amino acid sequence having at least 80% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153.

35. A GUCY2C TAC protein comprising an amino acid sequence according to any one of the amino acid sequences set forth in SEQ ID NOs: 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, and 153.

36. A GUCY2C TAC protein comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

131. A GUCY2C TAC protein comprising an amino acid sequence according to the amino acid sequence of SEQ ID NO:

131.

37. A nucleic acid sequence encoding a GUCY2C TAC protein according to any one of claims 1 to 36.

38. 38. The nucleic acid sequence of claim 37, wherein the nucleic acid comprises a sequence having at least 80% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152.

39. 39. The nucleic acid sequence of claim 37 or 38, wherein the nucleic acid sequence comprises the nucleic acid sequence of any one of SEQ ID NOs: 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, and 152.

40. A T cell expressing the GUCY2C-TAC protein of any one of claims 1 to 36.

41. A T cell comprising a nucleic acid sequence according to any one of claims 37 to 39.

42. 42. A pharmaceutical composition comprising the T cells of claim 40 or 41 and a pharmaceutically acceptable excipient.

43. 43. A method of treating a GUCY2C-expressing cancer in an individual in need thereof, comprising administering to said individual the pharmaceutical composition of claim 42.

44. 44. The method of claim 43, wherein the cancer is a solid cancer.

45. 45. The method of claim 43 or 44, wherein the cancer is colon cancer, gastric cancer, gastroesophageal junction cancer, esophageal cancer, or pancreatic cancer.

46. 45. The method of claim 43 or 44, wherein the cancer is primary colorectal cancer, primary gastric cancer, primary gastroesophageal junction cancer, primary esophageal cancer, or primary pancreatic cancer.

47. 45. The method of claim 43 or 44, wherein the cancer is metastatic colorectal cancer, metastatic gastric cancer, metastatic gastroesophageal junction cancer, metastatic esophageal cancer, or metastatic pancreatic cancer.

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