Compositions including therapeutic NKD1-specific t cell receptors and methods of using the same to treat colorectal cancer

Abstract

The present disclosure provides compositions including T cell receptors (TCRs) that bind to NKD1 -derived peptides having the amino acid sequence SLLHTIYEV (SEQ ID NO: 1) or FLDTPIAKV (SEQ ID NO: 2), or its HLA-A2 bound form. Also disclosed herein are nucleic acids encoding the TCRs, vectors comprising the nucleic acids, and host cells (e.g., modified lymphocytes) comprising the TCRs, nucleic acids, or vectors, as well as methods for preparing and using said compositions to treat colorectal cancer.

Description

Atty. Dkt. No.: 115872-3406COMPOSITIONS INCLUDING THERAPEUTIC NKD1-SPECIFIC T CELL RECEPTORS AND METHODS OF USING THE SAME TO TREAT COLORECTAL CANCER CROSS-REFERENCE TO RELATED APPLICATION

[0001] T 'his application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 748,114, filed January 22, 2025, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present disclosure relates to compositions including T cell receptors (TCRs) that bind to NKD1 -derived peptides having the amino acid sequence SLLHTIYEV (SEQ ID NO: 1) or FLDTPIAKV (SEQ ID NO: 2), or its HLA-A2 bound form. Also disclosed herein are nucleic acids encoding the TCRs, vectors comprising the nucleic acids, and host cells comprising the TCRs, nucleic acids, or vectors. The present disclosure provides methods for preparing compositions comprising the TCRs, the nucleic acids, the vectors and / or the host cells (e.g., modified lymphocytes), and methods of using the compositions for treating colorectal cancer.GOVERNMENT SUPPORT

[0003] This invention was made with government support under CA023766, CA008748, and CA241894 awarded by the National Institutes of Health. The government has certain rights in the invention.”BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] Metastatic CRC (mCRC) is the 2ndleading cause of cancer death in the US with a 5-year survival rate of M o1. Despite its lethality, treatment options for mCRC are limited to a few regimens offering only up to 2 years of median survival2, emphasizing a critical need for more options. Chimeric antigen receptor (CAR) T cells are emerging as a powerful approach in cancer therapy3 5. However, there has been limited success in solid tumors, in part, due to the inability to identify cancer-specific targets. Extracellular target -1- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406proteins on the tumor are expressed in normal tissues making on-target off-tumor toxicity a significant risk, are not essential for cancer proliferation or survival, and many do not tend to harbor targetable mutations6 s. One method to increase the repertoire is by analyzing the immunopeptidome, which is the set of peptides derived from intracellular proteins presented in the context of human leukocyte antigen (HLA) class I molecules9,10.

[0006] However, T cell-based therapies developed from these endeavors have relied on peptide sequences found on tumor-associated stromal cells11, on infrequent HLA alleles12, on infrequent mutations susceptible to epitope editing and acquired resistance13,14, on patient-specific but not disease-specific neoantigens, on primary lesions without accounting for the evolving phenotypes of advanced tumors15,16, and have not been successful in the clinic. Furthermore, previous research on the immunopeptidome of CRC has been limited by the use of early-stage15, surgically resected heterogeneous tissues without HLA30or microsatellite status matching16and by excluding metastatic lesions. Murine models of cancer progression31and multi-site analysis of clinical samples19from advanced cancer patients revealed that tumors undergo a significant evolution and diversification of their gene expression profiles and associated immunopeptidomes as tumors progress, despite minimal genomic changes. As such, a common immunopeptidome signature in an HLA-controlled advanced microsatellite stable (MSS) mCRC, the target of most cancer therapeutics and the cause of most cancer related deaths, remains an unaddressed challenge.SUMMARY OF THE PRESENT TECHNOLOGY

[0007] In one aspect, the present disclosure provides a T cell receptor (TCR) construct comprising a TCR alpha chain variable region and a TCR beta chain variable region, wherein: (a) (i) the TCR alpha chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 4, a CDR2 amino acid sequence of SEQ ID NO: 5, and a CDR3 amino acid sequence of SEQ ID NO: 6 or having at least 80 % identity to SEQ ID NO: 6, and (ii) the TCR beta chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 8, a CDR2 amino acid sequence of SEQ ID NO: 9, and a CDR3 amino acid sequence of SEQ ID NO: 10, or having at least 80 % identity to SEQ ID NO: 10; or (b) (i) the TCR alpha chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14 or having at least 80 % identity to SEQ ID NO: 14, and (ii) the-2- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406TCR beta chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 16, a CDR2 amino acid sequence of SEQ ID NO: 17, and a CDR3 amino acid sequence of SEQ ID NO: 18, or having at least 80 % identity to SEQ ID NO: 18; or (c) (i) the TCR alpha chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 20, a CDR2 amino acid sequence of SEQ ID NO: 21, and a CDR3 amino acid sequence of SEQ ID NO: 22 or having at least 80 % identity to SEQ ID NO: 22, and (ii) the TCR beta chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 24, a CDR2 amino acid sequence of SEQ ID NO: 25, and a CDR3 amino acid sequence of SEQ ID NO: 26, or having at least 80 % identity to SEQ ID NO: 26; or (d) (i) the TCR alpha chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 28, a CDR2 amino acid sequence of SEQ ID NO: 29, and a CDR3 amino acid sequence of SEQ ID NO: 30 or having at least 80 % identity to SEQ ID NO: 30, and (ii) the TCR beta chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 32, a CDR2 amino acid sequence of SEQ ID NO: 33, and a CDR3 amino acid sequence of SEQ ID NO: 34, or having at least 80 % identity to SEQ ID NO: 34; or (e) (i) the TCR alpha chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 36, a CDR2 amino acid sequence of SEQ ID NO: 37, and a CDR3 amino acid sequence of SEQ ID NO: 38 or having at least 80 % identity to SEQ ID NO: 38, and (ii) the TCR beta chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 40, a CDR2 amino acid sequence of SEQ ID NO: 41, and a CDR3 amino acid sequence of SEQ ID NO: 42, or having at least 80 % identity to SEQ ID NO: 42. In certain embodiments, the NKD1 peptide is bound to a HLA-A2 molecule. The HLA-A2 molecule may be a HLA-A*02:01, HLA-A*02:02 or HLA-A*02:04 molecule. Additionally or alternatively, in certain embodiments, the TCR construct of the present technology comprises a TCR alpha chain variable region comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 7, and a TCR beta chain variable region comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 11. In other embodiments, the TCR construct of the present technology comprises a TCR alpha chain variable region comprising the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 15, and a TCR beta chain variable region comprising the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80 % identity to SEQ ID NO:-3- 4906-6039-7446.1Atty. Dkt. No.: 115872-340619. In other embodiments, the TCR construct of the present technology comprises a TCR alpha chain variable region comprising the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 23, and a TCR beta chain variable region comprising the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 27. In other embodiments, the TCR construct of the present technology comprises a TCR alpha chain variable region comprising the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 31, and a TCR beta chain variable region comprising the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 35. In other embodiments, the TCR construct of the present technology comprises a TCR alpha chain variable region comprising the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 39, and a TCR beta chain variable region comprising the amino acid sequence of SEQ ID NO: 43 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 43.

[0008] Additionally or alternatively, in certain embodiments, the TCR construct specifically binds to aNKDl peptide having the amino acid sequence SLLHTIYEV (SEQ ID NO: 1) or FLDTPIAKV (SEQ ID NO: 2). In some embodiments of the TCR construct described herein, one or more TCR alpha chain variable regions and one or more TCR beta chain variable regions are covalently linked to each other to form TCR heterodimers or multimers.

[0009] Additionally or alternatively, in some embodiments, the TCR construct further comprises an antibody or antigen binding fragment that specifically binds an additional target antigen, optionally wherein the additional target antigen is not NKD1. In some embodiments, the additional target antigen is selected from the group consisting of CD3, CD28, CD5, CD 16 or CD56. In some embodiments of the TCR construct, the antigen binding fragment is a scFv. Additionally or alternatively, in some embodiments, the scFv is a humanized anti-CD3 scFv.

[0010] In any of the preceding embodiments, the TCR construct is soluble or comprises a transmembrane domain. In some embodiments, the TCR construct is linked to at least one molecular marker or reporter via a linker.-4- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0011] In one aspect, the present disclosure provides a fusion protein comprising any and all embodiments of the TCR construct described herein operably linked to one or more polypeptides selected from among Fc receptors; Fc domains, including IgA, IgD, IgG, IgE, and IgM; cytokines, including IL-2 or IL- 15; toxins; enzymes; CD247 (CD3-zeta), CD28, CD137, CD134 domain, or combinations thereof. The TCR construct may be linked to the one or more polypeptides directly or via a linker.

[0012] In another aspect, the present disclosure provides a composition comprising any and all embodiments of the TCR construct disclosed herein and a pharmaceutically-acceptable carrier, wherein the TCR construct is optionally conjugated to an isotope, a dye, a drug, a chromagen, a contrast agent, a toxin, an enzymes, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA or any combination thereof. In another aspect, the present disclosure provides a composition comprising the fusion protein of the present technology and a pharmaceutically-acceptable carrier.

[0013] In one aspect, the present disclosure provides an engineered immune cell comprising any and all embodiments of the TCR construct disclosed herein and / or a nucleic acid encoding the TCR construct. In certain embodiments, the engineered immune cell is a lymphocyte (e.g., a T cell, a CD4+ T cell, a CD8+ T cell, a B cell, a tumor infiltrating lymphocyte, or a natural killer (NK) cell), or a myeloid cell (e.g., macrophage). The engineered immune cell may be derived from an autologous donor, an allogenic donor or an iPS engineered cell. Additionally or alternatively, in some embodiments, the engineered immune cell further comprises at least one co-stimulatory receptor or a TCR that binds to an additional target antigen that is not NKD1.

[0014] In one aspect, the present disclosure provides a nucleic acid encoding the TCR construct of the present technology or the fusion protein of the present technology. In some embodiments, the nucleic acid encoding the TCR construct or the fusion protein is operably linked to a promoter, such as a constitutive promoter or a conditional promoter. In another aspect, the present disclosure provides a vector comprising any and all embodiments of the nucleic acid described herein. The vector may be a viral vector (e.g., a retroviral vector or a lentiviral vector), a plasmid, an extracellular vesicle (EV) vector, or a nanoparticle. Also disclosed herein are host cells comprising any and all embodiments of the nucleic acids described herein or the vectors described herein. Also disclosed herein are kits comprising -5- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406any and all embodiments of the nucleic acids described herein or the vectors described herein and instructions for use.

[0015] In one aspect, the present disclosure provides a method for preparing immune cells for cancer therapy comprising isolating immune cells from a donor subject; and transducing the immune cells with any and all embodiments of the nucleic acids described herein or the vectors described herein. In another aspect, the present disclosure provides a method of treatment comprising isolating immune cells from a donor subject; transducing the immune cells with any and all embodiments of the nucleic acids described herein or the vectors described herein; and administering the transduced immune cells to a recipient subject. The donor subject and the recipient subject may be the same or different. In some embodiments, the immune cells isolated from the donor subject comprise one or more lymphocytes or myeloid cells. In certain embodiments, the one or more lymphocytes is a T cell, a B cell, or a natural killer (NK) cell. In some embodiments, the one or more myeloid cells is a macrophage or a monocyte.

[0016] In another aspect, the present disclosure provides a method for treating a NKD1-expressing cancer in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the TCR construct described herein, the fusion protein described herein, the composition described herein, or the engineered immune cell described herein, optionally wherein the cancer is a relapsed or refractory cancer. In yet another aspect, the present disclosure provides a method for treating a NKD1 -expressing cancer in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the vector disclosed herein, wherein the vector is configured to deliver the nucleic acid of the present technology to T cells in situ, optionally wherein the cancer is a relapsed or refractory cancer.

[0017] In one aspect, the present disclosure provides a method for treating of inhibiting tumor growth or metastasis in a subject with a NKD1 -expressing cancer comprising contacting a tumor cell with an effective amount of any and all embodiments of the TCR construct described herein, the fusion protein described herein, the composition described herein, or the engineered immune cell described herein. In another aspect, the present disclosure provides a method for treating of inhibiting tumor growth or metastasis in a subject with a NKD1 -expressing cancer comprising contacting a tumor cell with an effective amount of any and all embodiments of the vector disclosed herein, wherein the -6- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406vector is configured to deliver the nucleic acid of the present technology to T cells in situ, optionally wherein the cancer is a relapsed or refractory cancer.

[0018] In any of the preceding embodiments of the methods disclosed herein, the TCR construct, the fusion protein, the composition, the engineered immune cell or the vector is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratum orally.

[0019] Additionally or alternatively, in some embodiments, the methods of the present technology further comprise administering an additional cancer therapy. Examples of the additional cancer therapy may be selected from among surgery, chemotherapy, radiation therapy, immunotherapy, monoclonal antibodies, anti-cancer nucleic acids or proteins, anticancer viruses or microorganisms, and any combinations thereof. In some embodiments, the methods of the present technology further comprise sequentially, separately, or simultaneously administering to the subject at least one therapeutic agent. Additionally or alternatively, in certain embodiments, the methods of the present technology further comprise administering a cytokine to the subject. Examples of cytokines include, but are not limited to, interferon a, interferon P, interferon y, complement C5a, IL-2, TNF alpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2. In any and all embodiments of the methods disclosed herein, the cancer or tumor is colorectal cancer. The colorectal cancer may be primary or metastatic.

[0020] Also disclosed herein are methods for imaging a NKD1 -expressing tumor in a subject comprising administering to the subject an effective amount of any and all embodiments of the TCR construct disclosed herein, the fusion protein disclosed herein, or the composition disclosed herein, wherein the TCR construct, the fusion protein, or the composition is conjugated to a detectable label.-7- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIGs. 1A-1E. Fetal gene signature marks metastatic disease. FIG. 1A.Patient derived organoid (PDO) generation. FIG. IB. Kernel density plot depicting the distribution of tumor cells from surgically resected lesions along the X-axis based on singlecell RNAseq expression of intestinal or fetal progenitor gene programs. Labels on left represent individual patient. FIG. 1C. Kaplan-Meier plot showing disease-free survival for patients in TCGA with high or low GSEA enrichment score (>1STD or <1STD of the mean, respectively) for the fetal progenitor signature. FIG. ID. UMAP of scRNA-seq of PDOs shows retention of distinct transcriptomic profiles. FIG. IE. Ternary plot of probabilities of cell state assignments per cell, calculates as Markov absorption probabilities from scRNAseq data. Probability distributions are shown for metastatic organoid lines grown in growth media and base media. ISC, intestinal stem cell signature; Fetal, fetal progenitor signature; Diff. Int., differentiated intestinal cell signature.

[0022] FIGs. 2A-2C. Quantitative analysis of peptide presentation from CRC-derived organoids. FIG. 2A. Workflow for organoid derivation and immunopeptidomic analysis. FIG. 2B. Surface HLA-A2 expression on organoids. FIG. 2C. Cumulative number of peptides detected and source genes per organoid source. Nml, normal epithelium; pri, primary tumor; met, metastatic lesion.

[0023] FIGs. 3A-3F. Target peptide selection. FIG. 3A. Target peptide selection workflow. FIG. 3B. Number of shared peptides with high-binding capacity to HLA-A2 in each organoid source, nml: normal, pri: primary tumor, met: metastasis. FIG. 3C. Selection of source genes with low expression in healthy tissue (top) and peptides not found in essential organs (IEDB, adrenal gland, aorta, bladder, bone marrow, brain, cerebellum, colon, esophagus, gall bladder, heart, kidney, liver, lung, lymph node, pancreas, skin, small intestine, spleen, tongue, trachea, thyroid, thymus, testis, prostate, ovary, uterus) (bottom). Black bracket: potential target genes / peptides; (#), number of peptides per associated gene.FIG. 3D. Matrix shows number of times NKD1 -derived peptides (row) were found among all tested organoid samples (columns). Each column corresponds to a patient-derived organoid line. FIG. 3E. Left axis, NKD1 transcript level in all samples in TCGA. Right axis, fraction of samples with >5.1 FPKM. FIG. 3F. Left axis, NKD1 transcript level per cell line. Right axis, fraction of cell lines with >5.1 TPM.-8- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0024] FIGs. 4A-4C. Target validation. FIG. 4A. Volcano plot shows differentially expressed genes in nml (left) vs. pri+met (right). Dot plot shows NKD1 expression level in tested organoids, p, primary lesion; br and li, brain and liver metastases. FIG. 4B. Bar graphs shows the relative western blot band intensity of NKD1 to GAPDH from each organoid line normalized to that of colorectal cancer cell line, SW480 (top). Li and di, liver and diaphragm lesions; p, primary tumor; n, normal epithelium. Western blot gel simultaneously developed for NKD1 and GAPDH protein (bottom). FIG. 4C. Fluorescence in situ hybridization (FISH) against NKD1 mRNA in FFPE slides from primary surgical specimens.

[0025] FIGs. 5A-5B. NKDl-specific CD8+ T cell expansion and identification. FIG. 5A. Workflow to identify NKDl-specific CD8-dependent TCR sequences. FIG. 5B.Flow cytometry plots show CD8+ T cells from 3 different donors that have been expanded with autologous monocyte derived dendritic cells (moDCs) for 2 weeks in the presence of NKD1-1 and NKD1-2 peptides, and double-stained with HLA-A2 dextramers loaded with respective peptides. Gates show sorted population for TCR 10X sequencing.

[0026] FIGs. 6A-6C. Functional characterization of NKD1 specific TCRs. FIG. 6A. Flow cytometry plots show CD8 T cells individually transduced with candidate TCRs double stained with corresponding target dextramers. FIG. 6B. Relative functional activity. Line graph shows % of TNFu+ T cells amongst transduced CD8 T cells after co-culture with T2 cells previously pulsed with titrating amounts of target peptide. WT1 derived peptide (RMFPNAPYL (SEQ ID NO: 3)) was used as negative control. E:T was 4:1. Left panels: TCRs #1 through #6, Right panels: TCRs #5, #6, #8 through #10. FIG. 6C. Line graph shows % of specific cytolytic activity against colorectal cancer cell line SW620 (GFP+) or non-specific melanoma cell line A375 (RFP+) after co-incubation with transduced CD8 T cells at a E:T of 4:1.

[0027] FIG. 7 shows MS immunopeptidome landscape in CRC.

[0028] FIG. 8 shows that immunopeptidome source proteins and locations are associated with house-keeping genes.

[0029] FIG. 9 shows that CRC-specific peptides are associated with the WNT pathway.

[0030] FIG. 10 shows that CRC-specific peptides associated with the WNT Pathway are not expressed in healthy adult tissue.-9- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0031] FIGs 11A-11F. NKD1 specific TCR efficacy and specificity. FIG. 11 A. Dot plots show specific cytotoxic activity of TCR#8 T cells against multiple patient derived organoids (PDOs) by relative luciferase activity after 3 days of co-culture at an E:T ratio of 4:1. FIG. 11B. Graph shows tumor size change over time in vivo. NSG mice were engrafted with 5 x 106SW620 cells at day 0.10 xlO6transduced T cells with either NKD1-specific TCR#8 or NY-ESO1 have been injected at days 4 and 7. Lines are averages of each group over time, p-value: 0.0051 at day 28. FIG. 11C. (bottom) Heatmap represents NKDl-specific TCR#8’s X-scan. Library of T2 cells expressing original target peptide sequence as well as peptides with substituted amino acids at each position with remainder amino acids were co-cultured for 3 days with either NKDl-specific TCR#8 T cells or nonspecific CMV-specific TCR at E:T ratio of 1:1. Color represents relative fold change reduction, (top) TCR#8 peptide recognition motif calculated from heatmap data. FIG. HD. Bar graph shows % of TNFa+ TCR#8 T cells after co-incubation with T2 cells at E:T ratio of 1 : 1 pulsed with predicted off-target peptides as defined by Scanprosite. FIG. HE.Volcano plot identifies additional potential off-target peptides. Library of T2 cells expressing original target peptide as well as 6,913 candidate off-target peptides, defined by BLOSUM80 similarity score with human proteome, were co-cultured for 3 days with either NKDl-specific TCR#8 T cells or non-specific CMV-specific TCR at E:T ratio of 1:1. Red dot: NKDl-specific peptide; Blue dot: single potential off-target. FIG. HF. Graph shows cytotoxic activity of T cells transduced with TCR#8 or CMV-specific TCR (control) against neuroblastoma cell line GIMEN overexpressing NKD1 or ZNF483, the parent protein of off-target peptide identified in FIG. HD.DETAILED DESCRIPTION

[0032] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0033] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel etal. eds. (2007) Current Protocols in Molecular Biology, the se ies Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University -10- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406Press); MacPherson et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. ( \999 Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).Definitions

[0034] As it would be understood, the section or subsection headings as used herein is for organizational purposes only and are not to be construed as limiting and / or separating the subject matter described.

[0035] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0036] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).-11- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0037] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another. “Administration” of a cell or vector or other agent and compositions containing same can be performed in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of animals, by the treating veterinarian. In some embodiments, administering or a grammatical variation thereof also refers to more than one doses with certain interval. In some embodiments, the interval is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year or longer. In some embodiments, one dose is repeated for once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration, inhalation, injection, and topical application. In some embodiments, the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer.

[0038] As used herein “adoptive cell therapeutic composition” refers to any composition comprising cells suitable for adoptive cell transfer. In some embodiments, the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of T-cells,-12- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells and peripheral blood mononuclear cells. In another embodiment, TILs, T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells or peripheral blood mononuclear cells form the adoptive cell therapeutic composition. In one embodiment, the adoptive cell therapeutic composition comprises T cells.

[0039] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, / .< ., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In some embodiments, amino acids forming a polypeptide are in the D form. In some embodiments, the amino acids forming a polypeptide are in the L form. In some embodiments, a first plurality of amino acids forming a polypeptide are in the D form, and a second plurality of amino acids are in the L form.

[0040] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.

[0041] As used herein, an “antigen” refers to a molecule to which an antibody (or antigen binding fragment thereof) can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen may be a polypeptide. An antigen may also be administered to an animal to generate an immune response in the animal.-13- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0042] As used herein, the term “biological sample” means sample material derived from living cells. Biological samples may include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject.Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or from cancers. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from non-diseased individuals, as controls or for basic research. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

[0043] As used herein, “CDRs” are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains or the complementarity determining region amino acid sequences of a TCR which are the hypervariable regions of the TCR alpha (a) and beta (P) chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th U. S. Department of Health and Human Services, National Institutes of Health (1987). The binding site of the TCR is comprised of six CDR loops, with each a and P chain contributing three loops, called CDR1, 2, and 3. The CDR1 and 2 loop sequences are constant for each type of chain and are therefore referred to as “germline derived,” whereas the CDR3 loops vary in an almost unlimited fashion and largely dictate the TCR specificity for peptide.

[0044] As used herein, the term “cell population” refers to a group of at least two cells expressing similar or different phenotypes. In non-limiting examples, a cell population can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at -14- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406least about 900, at least about 1000 cells, at least about 10,000 cells, at least about 100,000 cells, at least about 1 x 106cells, at least about 1 x 107cells, at least about 1 x 108cells, at least about 1 x 109cells, at least about 1 x IO10cells, at least about 1 x 1011cells, at least about 1 x 1012cells, or more cells expressing similar or different phenotypes.

[0045] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0046] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0047] As used herein, the term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.Conformational and non-conformational epitopes are distinguished in that the binding to the-15- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406former but not the latter is lost in the presence of denaturing solvents. In some embodiments, the epitope is a conformational epitope or a non-conformational epitope.

[0048] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, if required for proper expression and function.

[0049] As used herein, an "expression vector" includes vectors capable of expressing DNA that is operably linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[0050] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0051] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%-16- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by =HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity. Two sequences are deemed “unrelated” or “non-homologous” if they share less than 40% identity, or less than 25% identity, with each other.

[0052] As used herein, the terms “identical” or percent “identity”, when used in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (z.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein)), when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (e.g., NCBI web site). Such sequences are then said to be “substantially identical.” This term also refers to, or can be applied to, the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as those that have substitutions. In some embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or 50-100 amino acids or nucleotides in length.

[0053] As used herein, the term “immune cell” refers to any cell that plays a role in the immune response of a subject. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as-17- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes. As used herein, the term “engineered immune cell” refers to an immune cell that is genetically modified. As used herein, the term “native immune cell” refers to an immune cell that naturally occurs in the immune system.

[0054] As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.

[0055] The term “lymphocyte” refers to all immature, mature, undifferentiated, and differentiated white blood cell populations that are derived from lymphoid progenitors including tissue specific and specialized varieties, and encompasses, by way of non-limiting example, B cells, T cells, NKT cells, and NK cells. In some embodiments, lymphocytes include all B cell lineages including pre-B cells, progenitor B cells, early pro-B cells, late pro-B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, plasma B cells, memory B cells, B-l cells, B-2 cells, and anergic AN1 / T3 cell populations.

[0056] As used herein, "operably linked" with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a polypeptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide affects secretion of the fusion polypeptide. In some instances, the nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.-18- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0057] As used herein, the term “pharmaceutically-acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20thedition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0058] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single-and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and doublestranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0059] As used herein, the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.

[0060] As used herein, “prevention” or “preventing” of a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.-19- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0061] As used herein, the term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0062] As used herein, “regulatory sequence” of a nucleic acid molecule means a cisacting nucleotide sequence that influences expression, positively or negatively, of an operably linked gene. Regulatory regions include sequences of nucleotides that confer inducible ( / .< ., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression ( / .< ., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration, gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.

[0063] Particular examples of gene regulatory regions are promoters and enhancers. Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Polymerase II and III are examples of promoters. A polymerase II or “pol II” promoter catalyzes the transcription of DNA to synthesize precursors of mRNA, and most shRNA and microRNA. Examples of pol II promoters are known in the art and include without limitation, the phosphoglycerate kinase (“PGK”) promoter; EFl -alpha; CMV (minimal cytomegalovirus promoter); and LTRs from retroviral and lentiviral vectors. In some embodiments, the promoter is a constitutive promoter. As used herein, the term “constitutive promoter” refers to a promoter that allows for continual transcription of the coding sequence or gene under its control in all or most tissues of a subject at all or most developing stages. Non-limiting examples of the constitutive promoters include a CMV promoter, a simian virus 40 (SV40) promoter, a polyubiquitin C -20- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406(UBC) promoter, an EFl -alpha promoter, a PGK promoter and a CAG promoter. In some embodiments, the promoter is a conditional promoter, which allows for continual transcription of the coding sequence or gene under certain conditions. In further embodiments, the conditional promoter is an immune cell specific promoter, which allows for continual transcription of the coding sequence or gene in an immune cell. Non-limiting examples of the immune cell specific promoters include a promoter of a B29 gene promoter, a CD 14 gene promoter, a CD43 gene promoter, a CD45 gene promoter, a CD68 gene promoter, a IFN-P gene promoter, a WASP gene promoter, a T-cell receptor P-chain gene promoter, a V9 y (TRGV9) gene promoter, a V26 (TRDV2) gene promoter, and the like.

[0064] Enhancers are known to influence gene expression when positioned 5' or 3' of the gene, or when positioned in or a part of an exon or an intron. Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more.

[0065] Regulatory regions also include, but are not limited to, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.

[0066] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0067] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.-21- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0068] As used herein, “specifically binds” refers to a molecule (e.g., an antibody or antigen binding fragment thereof or TCR) which recognizes and binds another molecule (e.g., an antigen), but that does not substantially recognize and bind other molecules. The terms “specific binding,” “specifically binds to,” or is “specific for” a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide), as used herein, can be exhibited, for example, by a molecule having a KD for the molecule to which it binds to of about 104M, IO5M, 106M, 107M, 104M, 109M, 10l0M, 10 " M, or 10l2M. The term “specifically binds” may also refer to binding where a molecule binds to a particular polypeptide, or an epitope on a particular polypeptide, without substantially binding to any other polypeptide, or polypeptide epitope.

[0069] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0070] The term “T cell receptor” or “TCR” as used herein includes in all grammatical forms native TCRs as well as TCR variants, fragments and constructs. The term thus includes heterodimers comprising TCR alpha and beta chains as well as multimers and single chain constructs; optionally comprising further domains and / or moieties. In its native form, the TCR exists as a complex of several proteins on the surface of T cells. The T cell receptor is composed of two (separate) protein chains, which are produced from the independent T cell receptor alpha and beta (TCRa and TCRP) genes and are called alpha (a-) and beta (P-) chains. Each chain of the TCR possesses one N-terminal immunoglobulin-like (Ig)-variable (V) domain / region, one Ig-constant-like (C) domain / region, a transmembrane / cell membrane-spanning region anchoring the chain in the plasma membrane, and a short cytoplasmic tail at the C-terminal end.

[0071] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0072] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv)-22- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0073] It is also to be appreciated that the various modes of treatment or prevention of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.TCR Compositions of the Present Technology

[0074] The sequences of the CDR regions and the variable regions of the TCR alpha and beta chains of the TCRs described herein are shown in Table 1.-23- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406-24- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406-25- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0075] In one aspect, the present disclosure refers to a T cell receptor (TCR) that binds to a NKD1 peptide having the amino acid sequence SLLHTIYEV (SEQ ID NO: 1) or FLDTPIAKV (SEQ ID NO: 2), or its HLA-A2 bound form, wherein the TCR comprises: a CDR3 of the TCR alpha chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 30 or SEQ ID NO: 38, and / or a CDR3 of the TCR beta chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34 or SEQ ID NO: 42. Further envisaged are TCR sequence variants comprising a CDR3 alpha comprising or consisting of an amino acid sequence having at least 80 % identity to SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22, SEQ ID NO: 30 or SEQ ID NO: 38, preferably at least 85 % identity, more preferably 90 % or 95 % identity, and / or CDR3 beta comprising or consisting of an amino acid sequence having at least 80 % identity to SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO: 26, SEQ ID NO: 34 or SEQ ID NO: 42, preferably at least 85 % identity, more preferably 90 % or 95 % identity, provided that the TCR retains the advantageous capabilities of the TCR evaluated in the appended examples, i.e. is capable of binding to the antigenic target specified herein.-26- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0076] Antigen specificity is conferred by the variable regions of the alpha and beta chain. Both variable domains of the TCR alpha chain and beta chain comprise three hypervariable or complementarity determining regions (CDR1 alpha / beta, CDR2 alpha / beta and CDR3 alpha / beta) surrounded by framework (FR) regions. CDR3 is the prime determinant of antigen recognition and specificity (i.e. the ability to recognize and interact with a specific antigen), whereas CDR1 and CDR2 mainly interact with the MHC molecule presenting the antigenic peptide.

[0077] The TCR provided herein is capable of recognizing and specifically recognizing NKD1, in particular NKD1 in its MHC bound form as will be discussed somewhere else herein in detail. An antigenic peptide is said to be present in its “MHC bound form” when it forms a complex with an MHC molecule (which may be present on the surface of an antigen presenting cell such as a dendritic cell or a tumor cell, or it may be immobilized by for example coating to a bead or plate.).

[0078] Native TCRs recognize antigenic peptides bound to (“presented / displayed on”) the major histocompatibility complex (MHC) molecules at the surface of an antigen presenting cell. An antigenic peptide presented on a MHC molecule is also referred to as a “peptide: MHC complex” herein. There are two different classes of MHC molecules: MHC I and MHC II, which present peptides from different cell compartments. MHC class I molecules are expressed on the surface of all nucleated cells throughout the human body and display peptide or protein fragments from intracellular compartments to cytotoxic T cells. In humans, the MHC is also called the human leukocyte antigen (HLA). There are three major types of MHC class I: HLA- A, HLA-B and HLA-C. Once a TCR binds to its specific peptide: MHC complex, the T cell is activated and exerts biological effector functions.

[0079] The terms “binding to” and “recognizing” in all grammatical forms are used interchangeably herein. The antigenic target is particularly envisaged to be recognized by the TCRs disclosed herein when being bound by an MHC class I molecule, specifically an HLA-A molecule, preferably an HLA-A*02 molecule. In particular the antigenic target is recognized by the TCR of the present disclosure when presented by an HLA-molecule encoded by the HLA-A*02:01, HLA- A*02:02 or HLA-A*02:04 allele. Said MHC molecules, i.e. encoded by the HLA-A*02:01, HLA-A*02:02 and HLA-A*02:04 alleles, can be presented on the surface of a cell, for instance the surface of a tumor cell, or on a -27- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406(solid) carrier. In the context of the present disclosure, the NKD1 peptide is particularly envisaged to be recognized by the TCR disclosed herein when being bound by HLA-A2 which is a HLA-A*02:01, HLA-A*02:02, or HLA-A*02:04 encoded molecule. In a preferred embodiment of the present disclosure, the TCR can specifically recognized NKD1 peptide when bound by HLA-A*02:01, HLA-A*02:02 and HLA-A*02:04 encoded molecules, i.e. is capable of binding all three HLA-A*02 allele encoded molecules. That means, the TCR of the present disclosure is capable of binding to each of the molecules encoded by the HLA alleles HLA-A*02:01 , HLA-A*02:02 and HLA-A*02:04. It is however not envisaged that all HLA-A2 molecules are recognized at the same time in one patient, but are to be understood as alternatives.

[0080] As noted previously, CDR1 and CDR2 of the TCR alpha and beta chains are thought to be mainly involved in MHC recognition. There is a limited “pool” of CDR1 and CDR2 sequences known to be involved in HLA-A*02-restricted antigen recognition, and it is envisaged that the CDR3 domains of the present disclosure can in principle be combined with any of the CDR1 and CDR2 domains depicted in SEQ ID NOs: 4-5, 8-9, 12-13, 16-17, 20-21, 24-25, 28-29, 32-33, 36-37, or 40-41 provided that the TCR retains its ability to recognize its antigenic target, preferably in its HLA-A*02 (HLA-A*02:01, HLA-A*02:02 and HLA-A*02:04 ) bound form.

[0081] Useful examples of CDR1 and CDR2 domains include the CDR1 alpha comprising or consisting of the sequence as depicted in SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20, SEQ ID NO: 28, or SEQ ID NO: 36, the CDR2 alpha comprising or consisting of the sequence as depicted in SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 29, or SEQ ID NO: 37, the CDR1 beta comprising or consisting of the sequence as depicted in SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24, SEQ ID NO: 32 or SEQ ID NO: 40 and the CDR2 beta comprising or consisting of the sequence as depicted in SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 25, SEQ ID NO: 33 or SEQ ID NO: 41.

[0082] In accordance with the foregoing, the present disclosure inter alia provides a TCR comprising two polypeptide chains, each of which comprises a human variable region comprising at least one complementarity determining region (i.e. in particular CDR3, and preferably a CDR1 , and / or CDR2) of a TCR. A TCR with particular advantageous properties (as shown in the appended examples) comprises a first polypeptide chain comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4-28- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406(CDR1 alpha), a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 5 (CDR2 alpha), and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 6 (CDR3 alpha), and / or a second polypeptide chain comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 8 (CDR1 beta), a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 (CDR2 beta), and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 10 (CDR3 beta). In another embodiment, the TCR comprises a first polypeptide chain comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 12 (CDR1 alpha), a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 13 (CDR2 alpha), and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 14 (CDR3 alpha), and / or a second polypeptide chain comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 16 (CDR1 beta), a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 17 (CDR2 beta), and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 18 (CDR3 beta). In another embodiment, the TCR comprises a first polypeptide chain comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 20 (CDR1 alpha), a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 21 (CDR2 alpha), and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 22 (CDR3 alpha), and / or a second polypeptide chain comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 24 (CDR1 beta), a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 25 (CDR2 beta), and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 26 (CDR3 beta). In another embodiment, the TCR comprises a first polypeptide chain comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 28 (CDR1 alpha), a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 29 (CDR2 alpha), and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 30 (CDR3 alpha), and / or a second polypeptide chain comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 32 (CDR1 beta), a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 33 (CDR2 beta), and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 34 (CDR3 beta). In another embodiment, the TCR comprises a first polypeptide chain comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO:-29- 4906-6039-7446.1Atty. Dkt. No.: 115872-340636 (CDR1 alpha), a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 37 (CDR2 alpha), and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 38 (CDR3 alpha), and / or a second polypeptide chain comprising a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 40 (CDR1 beta), a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 41 (CDR2 beta), and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 42 (CDR3 beta).

[0083] The present disclosure further provides a TCR comprising a TCR alpha chain variable region comprising or consisting of an amino acid sequence as depicted in SEQ ID NO: 7 and / or a TCR beta chain variable region comprising or consisting of an amino acid sequence as depicted in SEQ ID NO: 11. In some embodiments, the present disclosure provides a TCR comprising a TCR alpha chain variable region comprising or consisting of an amino acid sequence as depicted in SEQ ID NO: 15 and / or a TCR beta chain variable region comprising or consisting of an amino acid sequence as depicted in SEQ ID NO: 19.

[0084] In some embodiments, the present disclosure provides a TCR comprising a TCR alpha chain variable region comprising or consisting of an amino acid sequence as depicted in SEQ ID NO: 23 and / or a TCR beta chain variable region comprising or consisting of an amino acid sequence as depicted in SEQ ID NO: 27. In some embodiments, the present disclosure provides a TCR comprising a TCR alpha chain variable region comprising or consisting of an amino acid sequence as depicted in SEQ ID NO: 31 and / or a TCR beta chain variable region comprising or consisting of an amino acid sequence as depicted in SEQ ID NO: 35. In some embodiments, the present disclosure provides a TCR comprising a TCR alpha chain variable region comprising or consisting of an amino acid sequence as depicted in SEQ ID NO: 39 and / or a TCR beta chain variable region comprising or consisting of an amino acid sequence as depicted in SEQ ID NO: 43.

[0085] TCR sequence variants comprising alpha chain variable regions comprising an amino acid sequence having at least 80% identity, more preferably at least 85 % identity, more preferably 90 % or 95 % to SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 31 or SEQ ID NO: 39 and / or a TCR beta chain variable region comprising or consisting of an amino acid sequence having at least 80 % identity, more preferably at least 85% identity, more preferably 90 % or 95 % to SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO: 27, SEQ ID NO: 35 or SEQ ID NO: 43 are also envisaged herein; provided that the -30- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406TCR retains the advantageous capabilities of the TCR evaluated in the appended examples, i.e. is capable of binding to the antigenic target specified herein.

[0086] The TCR of the present disclosure may further comprise an alpha chain constant region and / or a TCR beta chain constant region. The constant region can be a human constant region or derived from another species, yielding a “chimeric” TCR. For instance, human alpha and / or beta chains can be replaced by their murine counterparts (“murinization”) which has been found to enhance surface expression of human TCRs by supporting preferential pairing of the TCR alpha and beta chains, and a more stable association with the CD3 co-receptor. Suitable constant regions of the alpha chain and / or beta chain can be human, or murine. Instead of replacing complete human constant regions by their murine counterparts, it is also possible to exchange only some amino acids in the human constant regions for the corresponding amino acids of the murine constant region (“minimal murinization”), as further explained in the section “TCR sequence variants” herein. Further it is envisaged by the present disclosure that constant and variable regions can be combined in way suitable for the purpose. In this scenario the constant and variable regions may be derived from human, mouse or achieved by the process of minimal murinization as described above.

[0087] As noted previously, the term “TCR” encompasses TCR variants, which include TCR sequence variants, fragments and constructs. All TCR variants are envisaged to be functional variants of the TCR disclosed herein. The term “functional variant” as used herein refers to a TCR, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent NKD1 -specific TCR, its variable regions or its antigenbinding regions and shares its biological activity, i.e. its ability to specifically bind to the antigenic target for which the parent NKD1 -specific TCR of the present disclosure has antigenic specificity to a similar, the same or even a higher extent as the TCR disclosed herein and evaluated in the appended examples. Also encompassed by the present disclosure are TCR sequence variants.

[0088] The term “TCR variants” includes “sequence variants” of the TCR disclosed herein, i.e. variants substantially comprising the amino acid sequence of the TCR as described herein (also referred to as the “parent” NKD1 -specific TCR) but containing at least one amino acid modification (i.e. a substitution, deletion, or insertion) as compared to the “parent” NKD1 -specific TCR amino acid sequence, provided that the variant preferably -31- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406retains the antigenic specificity of the “parent” NKDl-specific TCR disclosed herein. TCR sequence variants of the present disclosure are typically prepared by introducing appropriate nucleotide changes into the nucleic acids encoding the “parent” NKDl-specific TCR, or by peptide synthesis. Generally, the aforementioned amino acid modifications may be introduced into, or present in, the variable region or the constant region of the TCR, and may serve to modulate properties like binding strength and specificity, post-translational processing (e.g. glycosylation), thermodynamic stability, solubility, surface expression or TCR assembly.

[0089] As set out previously, amino acid modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequences of the parent NKDl-specific TCR. Exemplary insertional variants of a TCR of the present disclosure include fusion products of said TCR and an enzyme or another functional polypeptide. Exemplary substitutional variants of a TCR of the present disclosure are those including amino acid substitutions in variable regions or CDRs of the alpha and / or beta chain, the framework region or the constant region. Particularly envisaged herein are conservative amino acid substitutions. Conservative amino acid substitutions are known in the art, and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid that has the same chemical or physical properties. For instance, the conservative amino acid substitution can be in an acidic amino acid substituted for another acidic amino acid (e.g. Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g. Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, Vai, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc. that may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0090] Accordingly, the amino acid sequences of SEQ ID NOs: 7, 11, 15, 19, 23, 27, 31, 35, 39, or 43 can for instance serve as “subject sequence” or “reference sequence”, while the amino acid sequence of a CDR3 different therefrom can serve as “query sequence”.-32- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0091] Cysteine Modification. The addition of a disulfide bond in the constant region has been reported to foster correct pairing of the TCR alpha and beta chains (Kuball J et al., Blood. 2007 Mar. 15; 109(6):2331-8.). Thus, the addition of one or more cysteine bonds in the constant region is also envisaged herein.

[0092] Constructs and Fragments. The term “TCR” as used herein further comprises TCR constructs. The term “construct” includes proteins or polypeptides comprising at least one antigen binding domain of the TCR of the present technology, but do not necessarily share the basic structure of a native TCR (i.e. variable domains incorporated into a TCR alpha chain and a TCR beta chain forming a heterodimer). TCR constructs and fragments are typically obtained by routine methods of genetic engineering and are often artificially constructed to comprise additional functional protein or polypeptide domains. In accordance with the foregoing, TCR constructs and fragments of the present disclosure are envisaged to comprise at least one CDR3 alpha and / or at least one CDR3 beta as disclosed elsewhere herein. Further envisaged herein are constructs and fragments comprising at least one CDR1 alpha, CDR2 alpha, CDR1 beta, CDR2 beta, alpha chain variable region, beta chain variable region, alpha chain and / or beta chain, or combinations thereof, optionally in combination with further protein domains or moieties as exemplified herein. The TCR constructs and fragments provided herein are envisaged to be capable of specifically binding to the same antigenic target as the TCR described herein and evaluated in the appended Examples.

[0093] Multimers. The TCR construct of the present disclosure encompasses heterodimers and multimers in which at least one TCR alpha chain variable region or TCR alpha chain and at least one TCR beta chain variable region are covalently linked to each other to form TCR heterodimers or multimers. A “multimer” as used in the present disclosure describes a molecule of diverse subunits or functional entities while a heterodimer comprises only two functional entities. In its simplest form a multivalent TCR construct according to the present disclosure comprises a multimer of two or three or four or more TCRs associated (e.g. covalently or otherwise linked) with one another, preferably via a linker molecule. In this context “covalently linked” means a chemical bond between two molecules, sharing electron pairs describing a stable balance between atom bonds.

[0094] Suitable linker to a spherical body, preferably a uniform bead, more preferably a polystyrene bead, most preferably a bio-compatible polystyrene bead. Such TCR constructs can also be comprised of a TCR of the present technology and a bead having a pre-defined -33- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406fluorescence dye incorporated into the bead. Suitable linker molecules include, but are not limited to, multivalent attachment molecules such as avidin, streptavidin, neutravidin and extravidin, each of which has four binding sites for biotin. Thus, biotinylated TCRs can be formed into multimers having a plurality of TCR binding sites. The number of TCRs in the multimer will depend upon the quantity of TCR in relation to the quantity of linker molecule used to make the multimers, and also on the presence or absence of any other biotinylated molecules. Exemplary multimers are dimeric, trimeric, tetrameric or pentameric or higher-order multimer TCR constructs. Multimers of the present disclosure may also comprise further functional entities such as labels or drugs or (solid) carriers.

[0095] Fusion Proteins. A TCR heterodimer or multimer also relates to fusion proteins or polypeptides comprising at least one TCR alpha chain, TCR alpha chain variable region or CDR3 alpha and / or at least one TCR beta chain, TCR beta chain variable region or CDR3 beta; and further one or more fusion component(s). It may be at least one TCR alpha chain as defined herein and / or at least one TCR beta chain as defined herein and / or an antibody or a single chain antibody fragment (scFv) which is directed against an antigen or epitope on the surface of lymphocytes, and also the TCR alpha chain(s) and TCR beta chain(s) are linked to each other and fused, optionally via a linker, to said antibody or scFv. Useful components include Fc receptors; Fc domains (derived from IgA, IgD, IgG, IgE, and IgM); cytokines (such as IL-2 or IL- 15); toxins; antibodies or antigen-binding fragments thereof (such as anti-CD3, anti-CD28, anti-CD5, anti-CD16 or anti-CD56 antibodies or antigen-binding fragments thereof); CD247 (CD3-zeta), CD28, CD137, CD134 domains; or any combinations thereof.

[0096] Exemplary antibody fragments that can be used as fusion components include fragments of full-length antibodies, such as (s)dAb, Fv, Fd, Fab, Fab', F(ab')2 or “r IgG” (“half antibody”); modified antibody fragments such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, minibodies, multibodies such as triabodies or tetrabodies, and single domain antibodies such as nanobodies or single variable domain antibodies comprising only one variable domain, which might be VHH, VH or VL.

[0097] TCR constructs of the present disclosure may be fused to one or more antibody or antibody fragments, yielding monovalent, bivalent and polyvalent / multivalent constructs and thus monospecific constructs, specifically binding to only one target antigen as well as -34- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406bispecific and polyspecific / multispecific constructs, which specifically bind more than one target antigens, e.g. two, three or more, through distinct antigen binding sites.

[0098] Optionally, a linker may be introduced between the one or more of the domains or regions of the TCR construct of the present disclosure, i.e. between the TCR alpha chain CDR3, TCR alpha chain variable region, and / or a TCR alpha chain, the TCR beta chain CDR3, TCR beta chain variable region, and / or a TCR beta chain, and / or the one or more fusion component(s) described herein. Linkers are known in the art and have been reviewed, inter alia, by Chen et al. , Adv Drug Deliv Rev. 2013 Oct. 15; 65(10): 1357-1369. In general, linkers include flexible, cleavable and rigid linkers and will be selected depending on the type of construct and intended use / application. For example, for therapeutic application, non-immunogenic, flexible linkers are often preferred in order to ensure a certain degree of flexibility or interaction between the domains while reducing the risk of adverse immunogenic reactions. Such linkers are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids and include “GS” linkers consisting of stretches of Gly and Ser residues.

[0099] Particularly useful TCR constructs envisaged in accordance with the present disclosure are those comprising at least one TCR alpha chain, TCR alpha chain variable region or CDR3 alpha as defined herein, at least one TCR beta chain, TCR beta chain variable region or CDR3 beta as defined herein, optionally linked to each other and fused, optionally via a liker, to at least one antibody or an antibody fragment (such as a single chain antibody fragment (scFv)) directed against an antigen or epitope on the surface of lymphocytes. Useful antigenic targets recognized by the antibody or antibody fragment (e.g. scFv) include CD3, CD28, CD5, CD16, and CD56. Said construct can in general have any structure as long the “TCR portion” (i.e. TCR alpha and beta chain or variable regions or CDR3s thereof) retains its ability to recognize the antigenic target defined herein, and the “antibody portion” binds to the desired surface antigen or epitope, thereby recruiting and targeting the respective lymphocyte to the target cell. Such constructs may advantageously serve as “adapters” joining an antigen presenting cell displaying the antigenic target (such as a tumor cell) and a lymphocyte (such as a cytotoxic T cell or NK cell) together. An example of such a fusion protein is a construct engineered according to the principle of a bispecific T cell engager (BiTE®) consisting of two single-chain variable fragments (scFvs) of different antibodies, on a single peptide chain of about 55 kilodaltons (kDa).-35- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406Accordingly, a TCR construct of the present disclosure may comprise at least one TCR antigen binding domain as described herein (for instance a TCR variable alpha and variable beta chain fused to each other) linked to a scFv (or other binding domain) of the desired binding specificity, e.g. CD3 or CD56. The scFv (or other binding domain) binds to T cells such as via the CD3 receptor or to CD56 for NK cell activation, and the other to a tumor cell via an antigenic target specifically expressed on the tumor cell. Also envisaged herein are tribodies comprising at least one TCR antigen binding domain as described herein, an scFv (or other binding domain) and a further domain e.g. for targeting the construct to a site of action within the body (e.g. an Fc domain).

[0100] Soluble Forms. The TCR of the present disclosure can be provided in soluble form. Soluble TCRs are useful as diagnostic tools, and carriers or “adapters” that specifically target therapeutic agents or effector cells to, for instance, a cancer cell expressing the antigenic target recognized by the soluble TCR. Soluble TCRs (sTCRs) will typically be fragments or constructs comprising TCR alpha and / or beta chains, or variable regions or CDRs thereof and optionally stabilized via disulfide bonds or covalently linked via a suitable linker molecule, e.g. as described above in the context of TCR constructs of the present disclosure. They will typically not comprise e.g. a transmembrane region. In some circumstances amino acid modifications in the polypeptide sequence may be introduced in order to enhance solubility of the molecules, and / or correct folding and pairing of the alpha and beta chains (if desired), in particular when produced in a recombinant host that does not provide for the aforementioned features. When using E. coll as production host cells for instance folding and pairing of the TCR alpha and beta chains is typically accomplished in vitro. A TCR according to the present disclosure may therefore for instance comprise additional cysteine residues, as described elsewhere herein.

[0101] Besides additional cysteine bridges, other useful modifications include, for instance, the addition of leucine zippers and / or ribosomal skipping sequences, e.g. sequence 2A from picorna virus as described in Walseng et al., (2015), PLoS ONE 10(4): eOl 19559 to increase folding, expression and / or pairing of the TCR alpha and / or beta chains.

[0102] Modifications. The TCR of the present disclosure may further comprise one or more modifications as described herein. The modifications described below will typically be covalent modifications and can be accomplished using standard techniques known in the-36- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406art. In some circumstances, amino acid modifications in the TCRs may be required in order to facilitate the introduction of said modifications.

[0103] Molecular Markers. The TCR, in particular (soluble) TCR, of the present disclosure can be labelled with at least one molecular marker. Useful molecular markers are known in the art and can be coupled to the TCR or TCR variant using routine methods, optionally via linkers of various lengths.

[0104] In general, different marker fall into a variety of classes, depending on the assay in which they are to be detected - the following examples include, but are not limited to: isotopic marker, which may be radioactive or heavy isotopes, such as radioisotopes or radionuclides (e.g.3H,l4‘15N,35S,89Zr,90Y, "Tc,U1ln,125I,131I); magnetic marker (e.g. magnetic particles); redox active moieties; optical dyes (including, but not limited to, chromophores, phosphors and fluorophores) such as fluorescent groups (e.g. FITC, rhodamine, lanthanide phosphors), chemiluminescent groups, and fluorophores which can be either “small molecule” fluorophores or proteinaceous fluorophores; enzymatic groups (e.g. horseradish peroxidase, P-galactosidase, luciferase, alkaline phosphatase; biotinylated groups; or predetermined polypeptide epitopes recognized by a secondary reporter (e.g. leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.). Labelling with molecular markers is particularly envisaged when the TCR, TCR variants or especially soluble TCR constructs (such as those comprising at least one TCR alpha and / or TCR beta chain as described herein) are intended for diagnostic use.

[0105] Functional Moieties. The TCR, in particular soluble TCR, of the present disclosure can be modified by attaching further functional moieties, e.g. for reducing immunogenicity, increasing hydrodynamic size (size in solution) solubility and / or stability (e.g. by enhanced protection to proteolytic degradation) and / or extending serum half-life.

[0106] Exemplary functional moieties for use in accordance with the present disclosure include peptides or protein domains binding to other proteins in the human body (such as serum albumin, the immunoglobulin Fc region or the neonatal Fc receptor (FcRn), polypeptide chains of varying length (e.g. XTEN technology or PASylation®), non-proteinaceous polymers, including, but not limited to, various polyols such as polyethylene glycol (PEGylation), polypropylene glycol, polyoxyalkylenes, or copolymers of-37- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406polyethylene glycol and polypropylene glycol, or of carbohydrates, such as hydroxyethyl starch (e.g. HESylation®) or polysialic acid (e.g. PolyXen® technology).

[0107] Other useful functional moieties include “suicide” or “safety switches” that can be used to shut off effector host cells carrying a TCR disclosed herein in a patient's body. An example is the inducible Caspase 9 (iCasp9) “safety switch” described by Gargett and Brown Front Pharmacol. 2014; 5: 235. Briefly, effector host cells are modified by well-known methods to express a Caspase 9 domain whose dimerization depends on a small molecule dimerizer drug such as AP1903 / C1P, and results in rapid induction of apoptosis in the modified effector cells. The system is for instance described in EP2173869 (A2).Examples for other “suicide” “safety switches” are known in the art, e.g. Herpes Simplex Virus thymidine kinase (HSV-TK), expression of CD20 and subsequent depletion using anti-CD20 antibody or myc tags (Kieback et al., Proc Natl Acad Sci USA. 2008 Jan.15;105(2):623-8). The TCR of the present technology can also be modified by introducing an inducible so called “on-switch” (as for example described in WO2019175209A1), wherein the modified alpha and beta chains of the TCR only dimerize upon interaction with a small dimerizer drug subsequently resulting in a functional TCR which is only expressed on the cell surface in the presence of the dimerizer drug.

[0108] Glycosylation. TCRs with an altered glycosylation pattern are also envisaged herein. As known in the art, glycosylation patterns can depend on the amino acid sequence (e.g. the presence or absence of particular glycosylation amino acid residues, discussed below) and / or the host cell or organism in which the protein is produced. Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. Addition of N-linked glycosylation sites to the binding molecule is conveniently accomplished by altering the amino acid sequence such that it contains one or more tri-peptide sequences selected from asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline). O-linked glycosylation sites may be introduced by the addition of or substitution by, one or more serine or threonine residues to the starting sequence.

[0109] Another means of glycosylation of TCRs is by chemical or enzymatic coupling of glycosides to the protein. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or -38- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine.

[0110] Similarly, deglycosylation (i.e. removal of carbohydrate moieties present on the binding molecule) may be accomplished chemically, e.g. by exposing the TCRs to trifluoromethanesulfonic acid, or enzymatically by employing endo- and exo-glycosidases.

[0111] Conjugates. It is also conceivable to add a small molecule such as a drug to the TCR, in particular to the soluble TCR of the present disclosure. Linkage can be achieved via covalent bonds, or non-covalent interactions such as through electrostatic forces. Various linkers, known in the art, can be employed in order to form the drug conjugates.

[0112] Tags. The TCR, in particular soluble TCR, of the disclosure can be modified to introduce additional domains which aid in identification, tracking, purification and / or isolation of the respective molecule (tags). Non-limiting examples of such tags comprise peptide motives known as Myc-tag, HAT -tag, HA- tag, TAP- tag, GST-tag, chitin binding domain (CBD-tag), maltose binding protein (MBP- tag), Flag-tag, Strep-tag and variants thereof, His-tag, CD20, Her2 / neu tags, myc-tag, FLAG-tag, T7-tag, HA(hemagglutinin)-tag, or GFP-tags.

[0113] Epitope tags are useful examples of tags that can be incorporated into the TCR of the disclosure. Epitope tags are short stretches of amino acids that allow for binding of a specific antibody and therefore enable identification and tracking of the binding and movement of soluble TCRs or host cells within the patient's body or cultivated (host) cells. Detection of the epitope tag, and hence, the tagged TCR, can be achieved using a number of different techniques. Examples of such techniques include: immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (“Western”), and affinity chromatography. The epitope tags can for instance have a length of 6 to 15 amino acids, in particular 9 to 11 amino acids. It is also possible to include more than one epitope tag in the TCR of the present disclosure.

[0114] Tags can further be employed for stimulation and expansion of host cells carrying an TCR of the present technology by cultivating the cells in the presence of binding molecules specific for said tag.-39- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406Engineered Immune Cells

[0115] The presently disclosed subject matter also provides methods of using such cells for the treatment of a tumor. The engineered immune cells of the presently disclosed subject matter can be cells of the lymphoid lineage or myeloid lineage. Examples of myeloid cells include but are not limited to, mast cells, monocytes, macrophages, dendritic cells, eosinophils, neutrophils, basophils. The lymphoid lineage, comprising B, T, and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of immune cells of the lymphoid lineage include T cells, Natural Killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., those from which lymphoid cells can be differentiated). T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cells, including, but not limited to, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells)), and two types of effector memory T cells: e.g, TEM cells and TEMRA cells, Regulatory T cells (also known as suppressor T cells), Natural killer T cells, Mucosal associated invariant T cells, and 76 T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells.

[0116] Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation in order to perform their cytotoxic effect on target cells.

[0117] The engineered immune cells of the presently disclosed subject matter can express a TCR that specifically binds to a NKD1 antigen: :MHC complex, for the treatment of cancer such as colorectal cancer. Such engineered immune cells can be administered to a subject e.g., a human subject) in need thereof for the treatment of a NKD1 -expressing cancer such as colorectal cancer. In some embodiments, the immune cell is a lymphocyte, such as a T cell, a B cell or a natural killer (NK) cell. In certain embodiments, the engineered immune cell is a T cell. The T cell can be a CD4+T cell or a CD8+T cell. In certain embodiments, the T cell is a CD4+T cell. In certain embodiments, the T cell is a CD8+T cell.-40- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0118] The engineered immune cells of the present disclosure can further include at least one recombinant or exogenous co-stimulatory ligand. For example, the engineered immune cells of the present disclosure can be further transduced with at least one costimulatory ligand, such that the engineered immune cells co-expresses or is induced to coexpress the NKD1 -specific TCR and the at least one co-stimulatory ligand. The interaction between the NKD1 -specific TCR and the at least one co-stimulatory ligand provides a non-antigen-specific signal important for full activation of an immune cell (e.g., T cell). Co-stimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily, and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase reaction. Its primary role is in the regulation of immune cells. Members of TNF superfamily share a number of common features. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. TNF superfamily members include, without limitation, nerve growth factor (NGF), CD40L (CD40L) / CD 154, CD137L / 4-1BBL, TNF-a, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFP) / lymphotoxin-alpha (LT-a), lymphotoxin-beta (LT-P), CD257 / B cell-activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-induced TNF Receptor ligand (GITRL), TNF-related apoptosis-inducing ligand (TRAIL), and LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. These proteins share structural features with immunoglobulins — they possess an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both ligands for CD28, or PD-L1 / (B7-H1) that are ligands for PD-1. In certain embodiments, the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof. In certain embodiments, the engineered immune cell comprises one recombinant co-stimulatory ligand (e.g., 4-1BBL). In certain embodiments, the engineered immune cell comprises two recombinant co-stimulatory ligands (e.g., 4-1BBL and CD80). CARs comprising at least one co-stimulatory ligand are described in U.S. Patent No. 8,389,282, which is incorporated by reference in its entirety.4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0119] Furthermore, the engineered immune cells of the present disclosure can further comprise at least one exogenous cytokine. For example, a presently disclosed engineered immune cell can be further transduced with at least one cytokine, such that the engineered immune cell secretes the at least one cytokine as well as expresses the NKD1-specific TCR disclosed herein. In certain embodiments, the at least one cytokine is selected from the group consisting of IL-2, IL- 3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, IL-18, and IL-21.

[0120] The engineered immune cells can be generated from peripheral donor lymphocytes, e.g., those disclosed in Sadelain, M., etal., Nat Rev Cancer 3 :35-45 (2003) (disclosing peripheral donor lymphocytes genetically modified to express CARs), in Morgan, R.A. etal., Science 314: 126-129 (2006) (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex comprising the a and P heterodimer), in Panelli etal., J Immunol 164:495-504 (2000); Panelli et al., J Immunol 164:4382-4392 (2000) (disclosing lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs) in tumor biopsies), and in Dupont et al., Cancer Res 65:5417-5427 (2005); Papanicolaou et al., Blood 102:2498-2505 (2003) (disclosing selectively inv / Yro-expanded antigen-specific peripheral blood leukocytes employing artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells). The engineered immune cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0121] In certain embodiments, the engineered immune cells of the present disclosure (e.g., T cells) express from about 1 to about 5, from about 1 to about 4, from about 2 to about 5, from about 2 to about 4, from about 3 to about 5, from about 3 to about 4, from about 4 to about 5, from about 1 to about 2, from about 2 to about 3, from about 3 to about 4, or from about 4 to about 5 vector copy numbers per cell of a presently disclosed NKD1 -specific TCR.

[0122] For example, the higher the NKDl -specific TCR expression level in an engineered immune cell, the greater cytotoxicity and cytokine production the engineered immune cell exhibits. An engineered immune cell (e.g., T cell) having a high NKD1-specific TCR expression level can induce antigen-specific cytokine production or secretion and / or exhibit cytotoxicity to a tissue or a cell having a low expression level of a target antigen: :MHC complex, e.g., about 2,000 or less, about 1,000 or less, about 900 or less, -42- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406about 800 or less, about 700 or less, about 600 or less, about 500 or less, about 400 or less, about 300 or less, about 200 or less, about 100 or less of target antigen: :MHC complexes / cell. Additionally or alternatively, the cytotoxicity and cytokine production of a presently disclosed engineered immune cell (e.g., T cell) are proportional to the expression level of target antigen: :MHC complexes in a target tissue or a target cell. For example, the higher the expression level of target antigen: :MHC complexes in the target, the greater cytotoxicity and cytokine production the engineered immune cell exhibits.

[0123] The unpurified source of immune cells may be any source known in the art, such as the bone marrow, fetal, neonate or adult or other hematopoietic cell source, e.g., fetal liver, peripheral blood or umbilical cord blood. Various techniques can be employed to separate the cells. For instance, negative selection methods can remove non-immune cells initially. Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and / or stages of differentiation for both positive and negative selections.

[0124] A large proportion of terminally differentiated cells can be initially removed by a relatively crude separation. For example, magnetic bead separations can be used initially to remove large numbers of irrelevant cells. Suitably, at least about 80%, usually at least 70% of the total hematopoietic cells will be removed prior to cell isolation.

[0125] Procedures for separation include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that modify cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents joined to or used in conjunction with a mAb, including, but not limited to, complement and cytotoxins; and panning with antibody attached to a solid matrix, e.g., plate, chip, elutriation or any other convenient technique.

[0126] Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g, a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels.

[0127] The cells can be selected against dead cells, by employing dyes associated with dead cells such as propidium iodide (PI). Usually, the cells are collected in a medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable (e.g., sterile), isotonic medium.-43- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0128] In some embodiments, the engineered immune cells comprise one or more additional modifications. For example, in some embodiments, the engineered immune cells comprise and express (are transduced to express) an antigen recognizing receptor that binds to a second antigen that is different than the first target antigen: :MHC complex. The inclusion of an antigen recognizing receptor in addition to a presently disclosed NKD1-specific TCR on the engineered immune cell can increase the avidity of the NKD1 -specific TCR (or the engineered immune cell comprising the same) on a target cell, especially, when the NKD1 -specific TCR is one that has a low binding affinity to a particular target antigen: :MHC complex, e.g., a Ka of about 2 x 10'8M or more, about 5 x 10'8M or more, about 8 x 10'8M or more, about 9 x 10'8M or more, about 1 x 10'7M or more, about 2 x 10'7M or more, or about 5 x 10'7M or more.

[0129] In certain embodiments, the antigen recognizing receptor is a chimeric costimulatory receptor (CCR). CCR is described in Krause, et al., J. Exp. Med. 188(4): 619-626(1998), and US20020018783, the contents of which are incorporated by reference in their entireties. CCRs mimic co-stimulatory signals, but unlike, CARs, do not provide a T-cell activation signal, e.g., CCRs lack a CD3(^ polypeptide. CCRs provide co-stimulation, e.g., a CD28-like signal, in the absence of the natural co-stimulatory ligand on the antigen-presenting cell. A combinatorial antigen recognition, i.e., use of a CCR in combination with a NKD1 -specific TCR, can augment T-cell reactivity against the dual-antigen expressing cells, thereby improving selective targeting. Kloss et al., describe a strategy that integrates combinatorial antigen recognition, split signaling, and, critically, balanced strength of T-cell activation and costimulation to generate T cells that eliminate target cells that express a combination of antigens while sparing cells that express each antigen individually (Kloss et al., Nature Biotechnology 31 (1): 71 -75 (2013)). In certain embodiments, the CCR comprises (a) an extracellular antigen-binding domain that binds to an antigen different than the NKD1 target antigen: :MHC complex, (b) a transmembrane domain, and (c) a co-stimulatory signaling region that comprises at least one co-stimulatory molecule, including, but not limited to, CD28, 4-1BB, 0X40, ICOS, PD-1, CTLA-4, LAG-3, 2B4, and BTLA. In certain embodiments, the co-stimulatory signaling region of the CCR comprises one co-stimulatory signaling molecule. In certain embodiments, the one co-stimulatory signaling molecule is CD28. In certain embodiments, the one co-stimulatory signaling molecule is 4-1BB. In certain embodiments, the co-stimulatory signaling region of the CCR comprises -44- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406two co-stimulatory signaling molecules. In certain embodiments, the two co-stimulatory signaling molecules are CD28 and 4- IBB. A second antigen is selected so that expression of both the first target antigen: :MHC complex and the second antigen is restricted to the targeted cells (e.g., cancerous cells). In some embodiments, the second antigen is CD 19, CD22, CD20, CD21, CD23, CD72 and ROR1. Similar to a CAR, the extracellular antigenbinding domain of the CCR can be an scFv, a Fab, a F(ab)2; or a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the CCR comprises an scFv that binds to a B cell antigen (e.g., CD19, CD22, CD20, CD21, CD23, CD72 and ROR1), transmembrane domain comprising a CD28 polypeptide, and a co-stimulatory signaling region comprising two co-stimulatory signaling molecules that are CD28 and 4-1BB.Polynucleotides., Polypeptides and Analogs

[0130] Also included in the presently disclosed subject matter are polypeptides encoding the NKD1 -specific TCR variable domains described herein, CD3(^, CD8, CD28, etc. or fragments thereof, and polynucleotides encoding the same, that are modified in ways that enhance their biological activity when expressed in an engineered immune cell. The presently disclosed subject matter provides methods for optimizing an amino acid sequence or a nucleic acid sequence by producing an alteration in the sequence. Such alterations may comprise certain mutations, deletions, insertions, or post-translational modifications. The presently disclosed subject matter further comprises analogs of any naturally-occurring polypeptide of the presently disclosed subject matter. Analogs can differ from a naturally-occurring polypeptide of the presently disclosed subject matter by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the presently disclosed subject matter can generally exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identity or homology with all or part of a naturally-occurring amino acid sequence of the presently disclosed subject matter. The length of sequence comparison is at least about 5, about 10, about 15, about 20, about 25, about 50, about 75, about 100 or more amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'3and e'100indicating a closely related sequence. Modifications comprise in vivo and in vitro chemical derivatization of polypeptides, e.g, acetylation, carboxylation, phosphorylation, or-45- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides of the presently disclosed subject matter by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethyl sulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., beta (P) or gamma (y) amino acids.

[0131] In addition to full-length polypeptides, the presently disclosed subject matter also provides fragments of any one of the polypeptides or peptide domains of the presently disclosed subject matter. A fragment can be at least about 5, about 10, about 13, or about 15 amino acids. In some embodiments, a fragment is at least about 20 contiguous amino acids, at least about 30 contiguous amino acids, or at least about 50 contiguous amino acids. In some embodiments, a fragment is at least about 60 to about 80, about 100, about 200, about 300 or more contiguous amino acids. Fragments of the presently disclosed subject matter can be generated by methods known to those of ordinary skill in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

[0132] Non-protein analogs have a chemical structure designed to mimic the functional activity of a protein of the present technology. Such analogs are administered according to methods of the presently disclosed subject matter. Such analogs may exceed the physiological activity of the original polypeptide. Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs increase the antineoplastic activity of the original polypeptide when expressed in an engineered immune cell. These chemical modifications include, but are not limited to, substituting alternative R groups and varying the degree of saturation at specific carbon atoms of a reference polypeptide. The protein analogs can be relatively resistant to in vivo degradation, resulting in a more-46- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.

[0133] In accordance with the presently disclosed subject matter, the polynucleotides encoding the NKD1 -specific TCR variable domains described herein, CD3(^, CD8, and CD28 can be modified by codon optimization. Codon optimization can alter both naturally occurring and recombinant gene sequences to achieve the highest possible levels of productivity in any given expression system. Factors that are involved in different stages of protein expression include codon adaptability, mRNA structure, and various cis- elements in transcription and translation. Any suitable codon optimization methods or technologies that are known to ones skilled in the art can be used to modify the polynucleotides of the presently disclosed subject matter, including, but not limited to, OptimumGene™, Encor optimization, and Blue Heron.Vectors

[0134] Many expression vectors are available and known to those of skill in the art and can be used for expression of polypeptides provided herein. The choice of expression vector will be influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector in the cells.

[0135] Vectors also can contain additional nucleotide sequences operably linked to the ligated nucleic acid molecule, such as, for example, an epitope tag such as for localization, e.g., a hexa-his tag or a myc tag, hemagglutinin tag or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and / or membrane association.

[0136] Expression of NKD1 -specific TCRs or antigen binding fragments thereof can be controlled by any promoter / enhancer known in the art. Suitable bacterial promoters are well known in the art and described herein below. Other suitable promoters for mammalian cells, yeast cells and insect cells are well known in the art and some are exemplified below. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application and is within the level of skill of the skilled artisan. Promoters -47- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406which can be used include but are not limited to eukaryotic expression vectors containing the SV40 early promoter (Bernoist and Chambon, Nature 290:304-310(1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797(1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Set. USA I '. 1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)); prokaryotic expression vectors such as the P-lactamase promoter (Jay etal., Proc. Natl. Acad. Sci. USA 75:5543 (1981)) or the tac promoter (DeBoer etal., Proc. Natl. Acad. Sci. USA 50:21-25(1983)); see also "Useful Proteins from Recombinant Bacteria": in Scientific American 242:79-94 (1980)); plant expression vectors containing the nopaline synthetase promoter (Herrera- Estrella et al., Nature 505:209-213(1984)) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., Nucleic Acids Res. 9:2871(1981)), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., Nature 510: 1 15-120(1984)); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift etal., Cell 55:639-646 (1984); Ornitz etal., Cold Spring Harbor Symp. Quant. Biol. 50:399-409(1986); MacDonald, Hepatology 7:425-515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan etal., Nature 515: 115-122 (1985)), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl etal., Cell 55:647-658 (1984); Adams et al., Nature 515:533-538 (1985); Alexander et al., Mol. Cell Biol. 7: 1436-1444 (1987)), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 15:485-495 (1986)), albumin gene control region which is active in liver (Pinckert et al., Genes andDevel. 1:268-276 (1987)), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., Mol. Cell. Biol. 5: 1639-403 (1985)); Hammer et al., Science 255:53-58 (1987)), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al., Genes and Devel. 7:161-171 (1987)), beta globin gene control region which is active in myeloid cells (Magram et al., Nature 515:338-340 (1985)); Kollias et al., Cell 5:89-94 (1986)), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al., Cell 15:703-712 (1987)), myosin light chain-2 gene control-48- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406region which is active in skeletal muscle (Shani, Nature 514:283-286 (1985)), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason etal., Science 254: 1372- 1378 (1986)).

[0137] In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of a NKD1 -specific TCR, or antigen binding fragment thereof, in host cells. A typical expression cassette contains a promoter operably linked to the nucleic acid sequence encoding the polypeptide chains of interest and signals required for efficient polyadenylation of the transcript, ribosome binding sites and translation termination.Additional elements of the cassette can include enhancers. In addition, the cassette typically contains a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes.

[0138] Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a nucleic acid sequence encoding a germline TCR chain under the direction of the polyhedron promoter or other strong baculovirus promoter.

[0139] Any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a nucleic acid encoding any of the polypeptides provided herein. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified.Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesized nucleic acids encoding restriction endonuclease recognition sequences.

[0140] Exemplary plasmid vectors useful to produce the polypeptides provided herein contain a strong promoter, such as the HCMV immediate early enhancer / promoter or the MHC class I promoter, an intron to enhance processing of the transcript, such as the HCMV-49- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406immediate early gene intron A, and a polyadenylation (poly A) signal, such as the late SV40 poly A signal.

[0141] Genetic modification of engineered immune cells (e.g., T cells, NK cells) can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA or RNA construct. The vector can be a retroviral vector (e.g., gamma retroviral), which is employed for the introduction of the DNA or RNA construct into the host cell genome. For example, a polynucleotide encoding the NKD1 -specific TCR can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from an alternative internal promoter.

[0142] Non-viral vectors or RNA may be used as well. Random chromosomal integration, or targeted integration (e.g., using a nuclease, transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs), and / or clustered regularly interspaced short palindromic repeats (CRISPRs), or transgene expression (e.g., using a natural or chemically modified RNA) can be used.

[0143] For initial genetic modification of the cells to provide NKDl-specific TCR expressing cells, a retroviral vector is generally employed for transduction, however any other suitable viral vector or non-viral delivery system can be used. For subsequent genetic modification of the cells to provide cells comprising an antigen presenting complex comprising at least two co-stimulatory ligands, retroviral gene transfer (transduction) likewise proves effective. Combinations of retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller, et al., Mol. Cell. Biol. 5:431-437 (1985)); PA317 (Miller, et al., Mol. Cell. Biol. 6:2895-2902 (1986)); and CRIP (Danos, et al. Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988)). Non -amphotropic particles are suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art.

[0144] Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al., Blood 80: 1418-1422(1992), or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and poly cations, e.g, by the method of Xu, et al. , Exp. Hemat.22:223-230 (1994); and Hughes, eta!., J. Clin. Invest. 89: 1817 (1992).-50- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406

[0145] Transducing viral vectors can be used to express a co-stimulatory ligand and / or secretes a cytokine (e.g., 4-1 BBL and / or IL- 12) in an engineered immune cell. In some embodiments, the chosen vector exhibits high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430 (1997); Kido et al., Current Eye Research 15:833-844 (1996); Bloomer etal., Journal of Virology 71 :6641-6649, 1997; Naldini et al, Science 272:263 267 (1996); and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319, (1997)). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, (1990); Friedman, Science 244: 1275-1281 (1989); Eglitis etal., BioTechniques 6:608-614, (1988); Tolstoshev etal., Current Opinion in Biotechnology 1:55-61(1990); Sharp, The Lancet 337 : 1277-1278 (1991); Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322 (1987); Anderson, Science 226:401-409 (1984); Moen, Blood Cells 17:407-416 (1991); Miller et al., Biotechnology 7:980-990 (1989); Le Gal La Salle et al., Science 259:988-990 (1993); and Johnson, Chest 107:77S-83S (1995)). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg etal., N. Engl. J. Med 323:370 (1990); Anderson etal., U.S. Pat. No. 5,399,346).

[0146] In certain non-limiting embodiments, the vector expressing a presently disclosed NKD1 -specific TCR is a retroviral vector, e.g., an oncoretroviral vector. In some instances, the retroviral vector is a SFG retroviral vector or murine stem cell virus (MSCV) retroviral vector. In certain non-limiting embodiments, the vector expressing a presently disclosed NKD1 -specific TCR is a lentiviral vector or a transposon vector.

[0147] Non-viral approaches can also be employed for the expression of a protein in a cell. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Nat'l. Acad. Sci. U.S.A.84:7413, (1987); Ono etal., Neuroscience Letters 17:259 (1990); Brigham etal., Am. J. Med. Sci. 298:278, (1989); Staubinger et al., Methods in Enzymology 101 :512 (1983)), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263 : 14621 (1988); Wu et al., Journal of Biological Chemistry 264: 16985 (1989)), or by microinjection under surgical conditions (Wolff et al., Science 247: 1465 (1990)). Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE-51- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue or are injected systemically. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g., Zinc finger nucleases, meganucleases, or TALE nucleases). Transient expression may be obtained by RNA electroporation.

[0148] cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element or intron (e.g., the elongation factor la enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0149] The resulting cells can be grown under conditions similar to those for unmodified cells, whereby the modified cells can be expanded and used for a variety of purposes.

[0150] Targeting vectors can be used to integrate a polynucleotide into the host cell's chromosome by methods known in the art, such as described by J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012). Briefly, suitable means include homologous recombination or use of a hybrid recombinase that specifically targets sequences at the integration sites. Targeting vectors are typically circular and linearized before used for homologous recombination. As an alternative, the foreign polynucleotides may be DNA fragments joined by fusion PCR or synthetically constructed DNA fragments which are then recombined into the host cell. It is also possible to use heterologous recombination which results in random or non-targeted integration.-52- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406Administration

[0151] Engineered immune cells expressing the NKD1 -specific TCRs of the presently disclosed subject matter can be provided systemically or directly to a subject for treating cancer, such as colorectal cancer. In certain embodiments, engineered immune cells are directly injected into an organ of interest. Additionally or alternatively, the engineered immune cells are provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature) or into the tissue of interest.Expansion and differentiation agents can be provided prior to, during or after administration of cells and compositions to increase production of T cells in vitro or in vivo.

[0152] Engineered immune cells of the presently disclosed subject matter can be administered in any physiologically acceptable vehicle, systemically or regionally, normally intravascularly, intraperitoneally, intrathecally, or intrapleurally, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). In certain embodiments, at least 1 x 105cells can be administered, eventually reaching 1 x 1010or more. In certain embodiments, at least 1 x 106cells can be administered. A cell population comprising engineered immune cells can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of engineered immune cells in a cell population using various well-known methods, such as fluorescence activated cell sorting (FACS). The ranges of purity in cell populations comprising engineered immune cells can be from about 50% to about 55%, from about 55% to about 60%, about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%; from about 85% to about 90%, from about 90% to about 95%, or from about 95 to about 100%. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage). The engineered immune cells can be introduced by injection, catheter, or the like. If desired, factors can also be included, including, but not limited to, interleukins, e.g., IL-2, IL-3, IL 6, IL-11, IL-7, IL-12, IL-15, IL-21, as well as the other interleukins, the colony stimulating factors, such as G-, M- and GM-CSF, interferons, e.g, y- interferon.

[0153] In certain embodiments, compositions of the presently disclosed subject matter comprise pharmaceutical compositions comprising engineered immune cells expressing a NKD1 -specific TCR with a pharmaceutically acceptable carrier. Administration can be -53- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406autologous or non-autologous. For example, engineered immune cells expressing a NKD1-specific TCR and compositions comprising the same can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived T cells of the presently disclosed subject matter or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a pharmaceutical composition of the presently disclosed subject matter (e.g., a pharmaceutical composition comprising engineered immune cells expressing a NKD1 -specific TCR, it can be formulated in a unit dosage injectable form (solution, suspension, emulsion).Formulations

[0154] Engineered immune cells expressing a NKD1 -specific TCR and compositions comprising the same can be conveniently provided as sterile liquid preparations, e.g, isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0155] Sterile injectable solutions can be prepared by incorporating the compositions of the presently disclosed subject matter, e.g, a composition comprising engineered immune cells, in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON' S PHARMACEUTICAL SCIENCE”, 17th edition,-54- 4906-6039-7446.1Atty. Dkt. No.: 115872-34061985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0156] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the presently disclosed subject matter, however, any vehicle, diluent, or additive used would have to be compatible with the engineered immune cells of the presently disclosed subject matter.

[0157] The compositions can be isotonic, / .< ., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of the presently disclosed subject matter may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is suitable particularly for buffers containing sodium ions.

[0158] Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

[0159] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the engineered immune cells as described in the presently disclosed subject matter. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems-55- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0160] One consideration concerning the therapeutic use of the engineered immune cells of the presently disclosed subject matter is the quantity of cells necessary to achieve an optimal effect. The quantity of cells to be administered will vary for the subject being treated. In certain embodiments, from about 102to about 1012, from about 103to about 1011, from about 104to about 1010, from about 105to about 109, or from about 106to about 108engineered immune cells of the presently disclosed subject matter are administered to a subject. More effective cells may be administered in even smaller numbers. In some embodiments, at least about 1 x 108, about 2 x 108, about 3 x 108, about 4 x 108, about 5 x 108, about 1 x 109, about 5 x 109, about 1 x 1010, about 5 x 1010, about 1 x 1011, about 5 x 1011, about 1 x 1012or more engineered immune cells of the presently disclosed subject matter are administered to a human subject. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. Generally, engineered immune cells are administered at doses that are nontoxic or tolerable to the patient.

[0161] The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and / or carrier in compositions to be administered in methods of the presently disclosed subject matter. Typically, any additives (in addition to the active cell(s) and / or agent(s)) are present in an amount of from about 0.001% to about 50% by weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as from about 0.0001 wt % to about 5 wt %, from about 0.0001 wt% to about 1 wt %, from about 0.0001 wt% to about 0.05 wt%, from about 0.001 wt% to about 20 wt %, from about 0.01 wt% to about 10 wt %, or from about 0.05 wt% to about 5 wt %. For any composition to be administered to an animal or human, and for any particular method of administration, toxicity should be determined, such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure -56- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation.Uses of the Engineered Immune Cells of the Present Technology

[0162] The present disclosure also provides a diagnostic composition comprising, as one or more diagnostic agent(s), the TCR, nucleic acid, the vector and / or the host cell as described herein. Typically, said diagnostic agent will comprise means for detecting its binding to a NKD1 -expressing tumor, or imaging of a NKD1 -expressing tumor, for instance a label as described in the context of the TCRs described herein. As regards the host cell, it is for instance conceivable to use modified host cells comprising a dye or a contrast agent that is released (instead of cytotoxic granules) upon antigen recognition. The detectable label can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and imaging. In general, almost any label useful in such methods can be applied to the present technology. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Labels useful in the practice of the present technology include magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels(e.g.,3H,14C,35S,125I,121I,13 JI,112In, "mTc), other imaging agents such as microbubbles (for ultrasound imaging),18F,UC,150, (for Positron emission tomography), "mTc (for Single photon emission tomography), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, and the like) beads. Patents that describe the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each incorporated herein by reference in their entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6thEd., Molecular Probes, Inc., Eugene OR.).

[0163] The present disclosure envisages the use of the diagnostic agents described in the foregoing for detecting, imaging, diagnosing and / or prognosing cancer in a subject which can be accomplished in vivo or in vitro.

[0164] Thus the present disclosure provides a diagnostic composition for use in detecting, diagnosing cancer in a subject in vivo, said composition comprising, as a diagnostic agent, the TCR, the nucleic acid, the vector and / or the host cell described herein.-57- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406The method will typically comprise (a) administering said diagnostic agent to the subject and (b) detecting binding of said diagnostic agent to NKD1 -expressing tumor cells.

[0165] Moreover, the present disclosure provides a method of detecting, diagnosing and / or prognosing cancer in a subject in vitro. In accordance the present disclosure also provides a method of detecting the presence of a cancer in a subject, comprising the steps of (a) providing a sample of a subject, said sample comprising one or more cells; (b) contacting said sample with the TCR, host cell and / or the pharmaceutical composition disclosed herein; thereby forming a complex, and (c) detecting the complex. Said complex is envisaged to be indicative for binding of the diagnostic agent to NKD1 and is indicative of the presence of a (cancer) cell expressing NKD1.

[0166] In both methods binding of the diagnostic agent to its antigenic target is detectable by using routine methods known in the art and will inter alia depend on the specific diagnostic agent used. Suitable labels that can be coupled to the diagnostic agent of the present disclosure are exemplified in the section relating to labeled TCR constructs.

[0167] Further it is envisaged by the present disclosure to comprise the use of a TCR, nucleic acid or vector as described herein for the generation of modified lymphocytes. As described somewhere else herein preferred lymphocytes include but not limited to cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells, natural killer (NK) cells, natural killer T (NKT) cells, gamma / delta- T cells.

[0168] For treatment, the amount of the engineered immune cells provided herein administered is an amount effective in producing the desired effect, for example, treatment or amelioration of the effects of cancer such as colorectal cancer, or one or more symptoms thereof. An effective amount can be provided in one or a series of administrations of the engineered immune cells provided herein. An effective amount can be provided in a bolus or by continuous perfusion. For adoptive immunotherapy using antigen-specific T cells, while cell doses in the range of about 106to about 1010are typically infused, lower doses of the engineered immune cells may be administered, e.g., about 104to about 108.

[0169] Upon administration of the engineered immune cells into the subject, the engineered immune cells are induced that are specifically directed against a NKD1 : :MHC complex. The engineered immune cells of the presently disclosed subject matter can be administered by any methods known in the art, including, but not limited to, pleural -58- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406administration, intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, intraperitoneal administration, and direct administration to the thymus. In certain embodiments, the engineered immune cells and the compositions comprising the same are intravenously administered to the subject in need. Methods for administering cells for adoptive cell therapies, including, for example, donor lymphocyte infusion and engineered immune cell therapies, and regimens for administration are known in the art and can be employed for administration of the engineered immune cells provided herein.

[0170] The presently disclosed subject matter provides various methods of using the engineered immune cells (e.g., T cells) provided herein, expressing any and all embodiments of the NKD1 -specific TCR described herein.

[0171] For example, the presently disclosed subject matter provides methods of reducing tumor burden in a subject. In one non-limiting example, the method of reducing tumor burden comprises administering an effective amount of the presently disclosed engineered immune cells to the subject and administering a suitable antibody targeted to the tumor, thereby inducing tumor cell death in the subject. In some embodiments, the engineered immune cells and the antibody are administered at different times. For example, in some embodiments, the engineered immune cells are administered and then the antibody is administered. In some embodiments, the antibody is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 30 hours, 26 hours, 48 hours or more than 48 hours after the administration of the engineered immune cells of the present technology.

[0172] The presently disclosed subject matter provides various methods of using the engineered immune cells (e.g., T cells) provided herein, expressing a NKD1 -specific TCR disclosed herein. For example, the presently disclosed subject matter provides methods of reducing tumor burden in a subject. In one non-limiting example, the method of reducing tumor burden comprises administering an effective amount of the presently disclosed engineered immune cells to the subject, thereby inducing tumor cell death in the subject.

[0173] The presently disclosed engineered immune cells can reduce the number of tumor cells, reduce tumor size, and / or eradicate the tumor in the subject. In certain embodiments, the method of reducing tumor burden comprises administering an effective amount of engineered immune cells to the subject, thereby inducing tumor cell death in the -59- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406subject. In some embodiments, the cancer is a relapsed or refractory cancer. In some embodiments, the cancer is resistant to one or more cancer therapies, e.g., one or more chemotherapeutic drugs.

[0174] The presently disclosed subject matter also provides methods of increasing or lengthening survival of a subject with cancer (e.g., a tumor). In one non-limiting example, the method of increasing or lengthening survival of a subject with cancer (e.g., a tumor) comprises administering an effective amount of the presently disclosed engineered immune cell to the subject, thereby increasing or lengthening survival of the subject. The presently disclosed subject matter further provides methods for treating or preventing cancer (e.g., a tumor) in a subject, comprising administering the presently disclosed engineered immune cells to the subject. Also provided herein are methods for treating of inhibiting tumor growth or metastasis in a subject comprising contacting a tumor cell with an effective amount of any of the engineered immune cells provided herein. Cancers whose growth may be inhibited using the engineered immune cells of the presently disclosed subject matter include cancers typically responsive to immunotherapy. In some embodiments, the cancer is metastatic colorectal cancer.Combination Therapy

[0175] The compositions of the present technology may be employed in conjunction with other therapeutic agents useful in the treatment of cancers, such as colorectal cancer. For example, engineered immune cells expressing a NKDl-specific TCR disclosed herein may be separately, sequentially or simultaneously administered with at least one additional cancer therapy.

[0176] In some embodiments, the additional cancer therapy is selected from among surgery, a chemotherapy, a radiation therapy, an immunotherapy, a monoclonal antibody, an anti-cancer nucleic acid, an anti-cancer protein, an anti-cancer virus or microorganism, a cytokine, or any combination thereof.

[0177] Radiation therapy includes, but is not limited to, exposure to radiation, e.g., ionizing radiation, UV radiation, as known in the art. Exemplary dosages include, but are not limited to, a dose of ionizing radiation at a range from at least about 2 Gy to not more than about 10 Gy or a dose of ultraviolet radiation at a range from at least about 5 J / m2to not more than about 50 J / m2, usually about 10 J / m2.

[0178] The methods of the present technology may further comprise sequentially,-60- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406separately, or simultaneously administering to the subject at least one chemotherapeutic agent, optionally selected from the group consisting of 5 -fluorouracil (5-FU), leucovorin, capecitabine, irinotecan, oxaliplatin, adagrasib, ziv-Aflibercept, fruquintinib, regorafenib, tucatinib, trifluridine and tipiracil.

[0179] In some embodiments, the methods further comprise sequentially, separately, or simultaneously administering an immunotherapy to the subject. In some embodiments, the immunotherapy regulates immune checkpoints. In further embodiments, the immunotherapy comprises, or consists essentially of, or yet further consists of Bevacizumab, Cetuximab, Ramucirumab, Panitumumab, an immune checkpoint inhibitor, such as a Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) inhibitor, or a Programmed Cell Death 1 (PD-1) inhibitor, or a Programmed Death Ligand 1 (PD-L1) inhibitor. In yet further embodiments, the immune checkpoint inhibitor comprises, or consists essentially of, or yet further consists of an antibody or an equivalent thereof recognizing and binding to an immune checkpoint protein, such as an antibody or an equivalent thereof recognizing and binding to CTLA4 (for example, Yervoy (ipilimumab), CP-675,206 (tremelimumab), AK104 (cadonilimab), or AGEN1884 (zalifrelimab)), or an antibody or an equivalent thereof recognizing and binding to PD-1 (for example, Keytruda (pembrolizumab), Opdivo (nivolumab), Libtayo (cemiplimab), Tyvyt (sintilimab), BGB-A317 (tislelizumab), JS001 (toripalimab), SHR1210 (camrelizumab), GB226 (geptanolimab), JS001 (toripalimab), AB122 (zimberelimab), AK105 (penpulimab), HLX10 (serplulimab), BCD-100 (prolgolimab), AGEN2034 (balstilimab), MGA012 (retifanlimab), AK104 (cadonilimab), HX008 (pucotenlimab), PF-06801591 (sasanlimab), JNJ-63723283 (cetrelimab), MGD013 (tebotelimab), CT-011 (pidilizumab), or Jemperli (dostarlimab)), or an antibody or an equivalent thereof recognizing and binding to PD-L1 (for example, Tecentriq (atezolizumab), Imfinzi (durvalumab), Bavencio (avelumab), CS1001 (sugemalimab), orKN035 (envafolimab)).

[0180] In some embodiments, the methods further comprise sequentially, separately, or simultaneously administering a cytokine to the subject. In some embodiments, the cytokine is administered prior to, during, or subsequent to administration of the one or more engineered immune cells described herein. In some embodiments, the cytokine is selected from the group consisting of interferon a, interferon P, interferon y, complement C5a, IL-2, TNFa, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1,-61- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.Kits of the Present Technology

[0181] The presently disclosed subject matter provides kits for the treatment or prevention of a disease, such as colorectal cancer. In certain embodiments, the kit comprises a therapeutic or prophylactic composition containing an effective amount of a nucleic acid encoding a NKD1 -specific TCR of the present technology or an engineered immune cell expressing the NKD1 -specific TCR. In particular embodiments, the engineered immune cell further expresses at least one co-stimulatory ligand.

[0182] In one aspect, the kits of the present technology comprise a therapeutic or prophylactic composition including an effective amount of any of the engineered immune cells disclosed herein in unit dosage form. In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0183] If desired, the nucleic acid encoding a NKD1 -specific TCR of the present technology can be provided together with instructions for manufacturing engineered immune cells and administering the engineered immune cells to a subject having or at risk of developing cancer, such as colorectal cancer. The instructions will generally include information about the use of the composition for the treatment or prevention of cancer such as colorectal cancer.

[0184] In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of cancer such as colorectal cancer, or symptoms thereof; precautions; warnings; indications; counter-indications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly -62- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0185] The at least one engineered immune cell of the present technology may be provided in the form of a prefilled syringe or autoinjection pen containing a sterile, liquid formulation or lyophilized preparation (e.g., Kivitz etal., Clin. Ther. 28:1619-29 (2006)).

[0186] A device capable of delivering the kit components through an administrative route may be included. Examples of such devices include syringes (for parenteral administration).

[0187] The kit components may be packaged together or separated into two or more containers. In some embodiments, the containers may be vials that contain sterile, lyophilized formulations of engineered immune cell composition that are suitable for reconstitution. A kit may also contain one or more buffers suitable for reconstitution and / or dilution of other reagents. Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers.

[0188] Also provided herein are kits for use in the manufacture of an engineered immune cell that expresses a NKDl-specific TCR disclosed herein. In certain embodiments, the kit comprises a vector comprising a nucleic acid encoding an engineered NKDl-specific TCR of the present technology.EXAMPLES

[0189] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.Example 1: CRC undergoes a highly conserved fetal-like regenerative program throughout disease continuum

[0190] Using multimodal analysis of resected surgical specimens from normal colon, primary tumor and metastatic lesions, in conjunction with their respective patient derived organoid (PDO) models, it has been recently demonstrated that metastasis stem cells (MetSCs) enter a highly conserved fetal-like regenerative state that is enriched in developmental WNT signaling associated molecules (FIGs. 1A-1E), including NKD I19. This developmental reprogramming is required and enriched in metastatic lesions compared to primary tumor, is common across patients (FIG. IB) despite divergent somatic mutation profile, is crucial for cancer dissemination, and is associated with worse prognosis (FIG.-63- 4906-6039-7446.1Atty. Dkt. No.: 115872-34061C), signifying a potential for a disease-specific therapeutic vulnerability that could be shared across patients. Importantly, this is also successfully recapitulated and induced in our PDO model (FIGs. ID, IE), underscoring the significant utility of our platform for identifying and investigating novel therapeutic strategies. Prior attempts targeting canonical WNT pathways with small molecules have been marked by on-target off-tumor toxicity20, limiting its widespread applicability in patients. Therefore, intercepting a tumor-specific, reactivated developmental program associated immunopeptidome could offer a novel avenue to targeting WNT-dependent phenotypic states while circumventing associated toxicities.Example 2: NKDl-derived peptides are specific targets for mCRC

[0191] NKD1 is a WNT-associated oncofetal protein target specifically expressed in mCRC. NKD1 is involved in both canonical25(i.e. beta-catenin dependent) and non-canonical WNT-signaling pathways (e.g. WNT-PCP, WNT-Ca2+, WNT-RTK)26’27’29. NKD1 plays a crucial role in embryogenesis, including segmentation, presomitic mesoderm formation29, and gastrointestinal organogenesis19, through WNT-signaling mediated TCF / LEF1 activation35. It is downregulated in adult healthy tissue but is reactivated in malignant and pre-malignant colorectal lesions. NKD1 exhibits a low mutational burden (<l-3%) in MSS CRC40and is less methylated than in MSI high CRCs41. Its expression is high in colorectal adenomas42and further enriched in metastatic lesions compared to primary tumors19, and its knockdown leads to decreased proliferation43, confirming proliferative dependence on NKD1.

[0192] Healthy donors harbor oncofetal peptide specific T cell receptors (TCRs) in the periphery. Oncofetal antigens (OF A) and cancer germline antigens (CGA) are two major sources of targets for T cell-based therapies. OF As are usually transiently expressed during fetal development and are reactivated in cancer cells44. CGAs are genes exclusively expressed in germ cells and trophoblasts but become aberrantly re-expressed in cancer cells45. OF As and CGAs are particularly attractive as targets because they reflect the biology of cancer cells as they re-emerge from the process of de-differentiation into an embryonic / stem cell-like phenotype46,47, but also because they retain their immunogenicity due to their temporal (i.e. prior to thymic development) and anatomical (i.e. exclusive expression in germ cells) segregation from T cell tolerance. PHOX2B48, WT122, IRS223, MAGE-A349, MAGE-A150, NY-ESO-151are a few examples of immunogenic OFAs / CGAs -64- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406from which TCRs have been successfully isolated from patients for T cell based therapies. Consistent with these examples, we isolated multiple NKD1 specific TCRs from healthy peripheral blood mononuclear cells.

[0193] Patients with CRC were screened for the presence of preserved mismatch repair (pMMR) proteins or microsatellite stable (MSS) status, lacking germline mutations (representing 80-85% of all mCRC patients) and carrying the HLA-A2 associated alleles, which include *02:01, *02:04, *02:05 and *02:07. The proprietary MSK-Integrated Mutation Profiling of Actionable Cancer Targets (IMPACT)®, a next-generation sequencing (NGS) test available to MSKCC patients, captures >500 common somatic mutations and HLA alleles. Surgical specimens and associated organoids were annotated with biologic and clinical data, including somatic mutation profile, tumor mutational burden, MSS status, number of metastatic lesions, gender, and surgical and cancer-treatment history. We have accumulated a total of 11 from normal epithelium, 8 from primary tumor, 8 from metastatic lesions (FIG.2A).

[0194] We first confirmed surface HLA-A2 expression by flow cytometry. Mitigating concerns of HLA downregulation, our tumor PDOs retain elevated levels ofHLA-A2 expression on the cell surface (FIG. 2B) comparable to that of the normal epithelia. We then expanded PDOs to more than 40 million cells and transitioned them to base media (i.e. without growth factors) for 7 days to induce a fetal WNT program (FIG. IE). We sequentially pulled down peptide:MHC (pMHC) complexes using the BB7.2 clone antibody (anti -HL A- A2), followed by the W6 / 32 clone antibody (anti-HLA-A / B / C) to maximize capture. We then eluted bound peptides using Sepharose beads and tC18 columns, sequentially as previously published24. After solid phase extraction, samples were run through the Thermo OrbiTrap mass spectrometer using data independent acquisition (DIA) method. Spectra data was deconvoluted and peptide hits were searched with Spectronaut® using the sample-specific reference proteome library, created with the ProteomeGenerator2 script. Using UniProt and sample specific mRNA data as our reference library, we were able to remarkably identify a total of 38,600 peptides corresponding to 7,690 source genes (FIG. 2C)

[0195] In order to screen for target peptides from the immunopeptidome repertoire derived above, we selected putative tumor associated antigens that 1) are specific to organoids from metastatic and / or primary tumors but missing in normal colon, 2) are absent -65- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406in healthy immunopeptidome databases such as the Human HLA Atlas DB and the Immune Epitope DB (IEDB), 3) are from source proteins either missing in relevant healthy tissue or expressed in replaceable organs (e.g. thyroid) as published by the GTEx consortium, 4) are consistently expressed in colorectal cancer primary tissues catalogued in TCGA, or in CRC-derived cell lines catalogued in the Cancer Cell Line Encyclopedia (CCLE), and 5) are associated with fetal WNT program or essential cellular functions (FIG. 3A). This approach distinguished a total of 49 peptides from 45 proteins exclusively found in primary tumor and / or metastatic lesions (FIG. 3B-3C). Of these, target peptide screening analysis successfully discovered 2 peptides from NKD1 (SLLHTIYEV (SEQ ID NO: 1), FLDTPIAKV (SEQ ID NO: 2)) as a strong therapeutic candidate given its prevalence and relevance in CRC. It is absent in normal but significantly upregulated in tumor PDOs, is highly expressed and prevalent in colorectal cancer samples and CRC cell-lines catalogued in TCGA and CCLE (FIGs. 3D-3F). We also validated its expression and translation in organoid lines by RNAseq and western blot (FIGs. 4A-4C). We further confirmed its expression and presentation directly from surgical specimens by FISH as well as by our immunopeptidomics pipeline (FIG. 4D).Example 3: Identification of NKDl-restricted TCRs

[0196] We isolated NKD1 specific TCRs from HLA-A2+ healthy donor PBMCs by isolating and co-incubating CD8+ T cells with autologous matured monocyte derived dendritic cells (moDCs) plus NKD1 -derived peptides. Samples were stained with pMHC dextramers directly conjugated to fluorophores and unique DNA barcodes allowing 10X single cell TCR and mRNA sequencing. We retrieved a total of 7 sequences with high dextramer counts per cell from a total of 3 out of 4 healthy donors (FIGs. 5A-5B). Variable regions from candidate TCRs were subsequently cloned after murine TCR constant region for downstream assays.

[0197] Polyclonal CD8+ T cells were transduced retrovirally with individual candidate TCRs to determine their specificity, functional avidity and cytolytic activity. Dextramer staining confirmed pMHC specific staining in clonotypes #1, #3, #5, and #6 (FIGs. 6A-6D).Each TCR’s relative antigen sensitivity was measured by TNFa production upon co-culture with HLA-A*02:01+ T2 cells pulsed with titrated amounts of respective peptides.Clonotypes #5 and #6 demonstrated the highest and significant activation consistently across all donors, while clone #7, #3, #4, #1 were more donor dependent. We then sought to -66- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406determine cytolytic activity by co-culturing transduced CD8+ T cells with an HLA-A*02:01+ restricted metastatic colorectal cancer cell line, SW620. Immunopeptidomics analysis of SW620 cells confirmed presentation of target peptides. Clonotype #5 and #2 demonstrated significant killing of SW620 by 72hrs in all three donors, while clonotype #7, #4, #6, #1 were donor dependent. Parallelly, non-specific killing was assessed by comparing their cytolytic activity against HLA-A*02:01+ melanoma cell line A375 and none of the TCRs mediated non-scpecific cytolysis of A375 cells. Of the seven identified, TCR#5 has the highest level of pMHC staining, induces the strongest cytokine production and harbors the most efficient cytolytic activity with minimal non-specific killing. This is followed by TCR #6, which has a weaker dextramer staining but retains strong cytokine secretion and cytolytic activity in donor 2. Although TCR#2 had exceptional killing activity it failed to induce cytokine secretion when co-incubated with pulsed T2 cells and had negligible dextramer staining, suggesting specific SW620 cell killing through a non-specific target detection other than NKD1 derived peptides. Clonotype #7 showed some moderate specific killing activity also had moderate cytokine and dextramer binding capacity, suggesting a lower active TCR. Together, these experiments demonstrate the existence of an immunogenic oncofetal peptide that is targetable using TCRs with diverse functional attributes.

[0198] Finally, we established the basis of peptide recognition of each TCR by performing an alanine scan. CD8+ T cells transduced with Individual TCRs were coincubated with T2 cells separately pulsed with Ipg / ml of peptide sequences containing an alanine or glycine substitution at each position of the original corresponding NKD1 amino acid sequence, and determined their effect on TNFa secretion. TCR #5 is dependent on P2, 3, 5, 6 amino acids to induce TCR signaling and activation. TCR#6 also relies on amino acids in the middle such as P2, 3, 4, 5, 6, 7, 8. On the other hand, TCR#7 relies on the first 5 amino acids for recognition but not the last 3. Together, these data establish TCR#5 as a leading candidate for further development, with TCR#6 as a secondary option, providing a foundation for targeted immunotherapeutic strategies against NKD1 -presenting metastatic colorectal cancer cells.EQUIVALENTS

[0199] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of -67- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0200] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0201] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0202] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.-68- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406REFERENCES1. Siegel, R. L., Miller, K. D., Wagle, N. S. & Jemal, A. Cancer statistics, 2023. CA. Cancer J. Clin. 73, 17-48 (2023).2. Neugut, A. I. et al. FOLFOX and FOLFIRI Use in Stage IV Colon Cancer: Analysis of SEER-Medicare Data. Clin. Colorectal Cancer 18, 133-140 (2019).3. Abramson, J. S. et al. Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study. The Lancet 396, 839-852 (2020).4. Maude, S. L. et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N. Engl. J. Med. 378, 439-448 (2018).5. Neelapu, S. S. et al. Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N. Engl. J. Med. 377 , 2531-2544 (2017).6. Hassan, R. et al. Mesothelin-targeting T cell receptor fusion construct cell therapy in refractory solid tumors: phase 1 / 2 trial interim results. Nat. Med. 29, 2099-2109 (2023). 7. Zhang, C. et al. Phase I Escalating-Dose Trial of CAR-T Therapy Targeting CEA+ Metastatic Colorectal Cancers. Mol. Ther. 25, 1248-1258 (2017).8. Xu, J. et al. HER2-specific chimeric antigen receptor-T cells for targeted therapy of metastatic colorectal cancer. Cell Death Dis. 12, 1-11 (2021).9. Hunder, N. N. et al. Treatment of Metastatic Melanoma with Autologous CD4+ T Cells against NY-ESO-1. N. Engl. J. Med. 358, 2698-2703 (2008).10. Rapoport, A. P. et al. NY-ESO-l-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma. Nat. Med. 21, 914-921 (2015). 11. Kim, G. B. et al. Quantitative immunopeptidomics reveals a tumor stroma-specific target for T cell therapy. Sci. Transl. Med. 14, eabo6135 (2022).12. Tran, E. et al. T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer. N. Engl. J. Med. 375, 2255-2262 (2016).13. Sim, M. J. W. et al. High-affinity oligocl onal TCRs define effective adoptive T cell therapy targeting mutant KRAS-G12D. Proc. Natl. Acad. Sci. 117, 12826-12835 (2020).-69- 4906-6039-7446.1Atty. Dkt. No.: 115872-340614. Chandran, S. S. et al. Immunogenicity and therapeutic targeting of a public neoantigen derived from mutated PIK3CA. Nat. Med. 28, 946-957 (2022).15. Loftier, M. W. et al. Mapping the HLA Ligandome of Colorectal Cancer Reveals an Imprint of Malignant Cell Transformation. Cancer Res. 78, 4627-4641 (2018).16. Cleyle, J. et al. Immunopeptidomic Analyses of Colorectal Cancers With and Without Microsatellite Instability. Mol. Cell. Proteomics 21 , 100228 (2022).17. Ganesh, K. et al. A rectal cancer organoid platform to study individual responses to chemoradiation. Nat. Med. 25, 1607-1614 (2019).18. Ganesh, K. et al. L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer. Nat. Cancer 1, 28-45 (2020).19. Moorman, A. R. et al. Progressive plasticity during colorectal cancer metastasis. 2023.08.18.553925 Preprint at https: / / doi.Org / 10.l 101 / 2023.08.18.553925 (2023).20. Kahn, M. Can we safely target the WNT pathway? Nat. Rev. Drug Discov. 13, 513— 532 (2014).21. Dao, T. et al. Therapeutic bispecific T-cell engager antibody targeting the intracellular oncoprotein WT1. Nat. Biotechnol. 33, 1079-1086 (2015).22. Dao, T. et al. Targeting the Intracellular WT1 Oncogene Product with a Therapeutic Human Antibody. Sci. Transl. Med. 5, 176ra33-176ra33 (2013).23. Dao, T. et al. A TCR mimic monoclonal antibody reactive with the “public” phospho-neoantigen pIRS2 / HLA-A* 02:01 complex. JCI Insight 7 , (2022).24. Klatt, M. G. etal. An input-controlled model system for identification of MHC bound peptides enabling laboratory comparisons of immunopeptidome experiments. J. Proteomics 228, 103921 (2020).25. Angonin, D. & Raay, T. J. V. Nkdl Functions as a Passive Antagonist of Wnt Signaling. PROS ONE 8, e74666 (2013).26. Li, J. et al. The NKDl / Racl feedback loop regulates the invasion and migration ability of hepatocarcinoma cells. Sci. Rep. 6, 26971 (2016).-70- 4906-6039-7446.1Atty. Dkt. No.: 115872-340627. Twaroski, K. et al. FGF2 mediates hepatic progenitor cell formation during human pluripotent stem cell differentiation by inducing the WNT antagonist NKDL Genes Dev.29, 2463-2474 (2015).28. Chan, C.-C., Zhang, S., agatay, T. & Wharton, K. A. Cell-autonomous, myristyl-independent activity of the Drosophila Wnt / Wingless antagonist Naked cuticle (Nkd). Dev. Biol. 311, 538-553 (2007).29. Ishikawa, A. et al. Mouse Nkdl, a Wnt antagonist, exhibits oscillatory gene expression in the PSM under the control of Notch signaling. Meeh. Dev. 121, 1443-1453 (2004).30. Newey, A. et al. Immunopeptidomics of colorectal cancer organoids reveals a sparse HLA class I neoantigen landscape and no increase in neoantigens with interferon or MEK-inhibitor treatment. J. Immunother. Cancer 7 , 309 (2019).31. Jaeger, A. M. et al. Deciphering the immunopeptidome in vivo reveals new tumour antigens. Nature 607, 149-155 (2022).32. Rosati, E. et al. Overview of methodologies for T-cell receptor repertoire analysis. BMC Biotechnol. 17, 61 (2017).33. Gerstberger, S., Jiang, Q. & Ganesh, K. Metastasis. Cell 186, 1564-1579 (2023). 34. Murakami, K. et al. A genome-scale CRISPR screen reveals factors regulating Wnt-dependent renewal of mouse gastric epithelial cells. Proc. Natl. Acad. Sci. 118, e2016806118 (2021).35. Heino, S. et al. Lefl restricts ectopic crypt formation and tumor cell growth in intestinal adenomas. Sci. Adv. 7, eabj0512 (2021).36. Larraguibel, J. et al. Wnt ligand-dependent activation of the negative feedback regulator Nkdl. Mol. Biol. Celll , 2375-2384 (2015).37. Raay, T. J. V. et al. Nakedl Antagonizes Wnt Signaling by Preventing Nuclear Accumulation of P-Catenin. PLOS ONE 6, el8650 (2011).38. Chen, X., Xu, P., Zhu, J. & Liu, F. Downregulation of NKD1 in human osteosarcoma and its clinical significance. Mol. Med. Rep. 17, 1111-1117 (2018).-71- 4906-6039-7446.1Atty. Dkt. No.: 115872-340639. Zhang, S., Wang, Y., Dai, S.-D. & Wang, E.-H. Down-regulation of NKD1 increases the invasive potential of non-small-cell lung cancer and correlates with a poor prognosis. BMC Cancer 11, 186 (2011).40. Chen, B. et al. Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps. Cell 184, 6262-6280. e26 (2021).41. Joanito, I. et al. Single-cell and bulk transcriptome sequencing identifies two epithelial tumor cell states and refines the consensus molecular classification of colorectal cancer. Nat. Genet. 54, 963-975 (2022).42. Caldwell, G. M. et al. Wnt signalling in adenomas of familial adenomatous polyposis patients. Br. J. Cancer 103, 910-917 (2010).43. Wang, Y. et al. Identification of colon tumor marker NKD1 via integrated bioinformatics analysis and experimental validation. Cancer Med. 10, 7383-7394 (2021).44. Tan, H. L. et al. Conservation of oncofetal antigens on human embryonic stem cells enables discovery of monoclonal antibodies against cancer. Set. Rep. 8, 11608 (2018). 45. Akers, S. N., Odunsi, K. & Karpf, A. R. Regulation of cancer germline antigen gene expression: implications for cancer immunotherapy. Future Oncol. 6, 717-732 (2010). 46. Yan, Q., Fang, X., Li, C., Lan, P. & Guan, X. Oncofetal proteins and cancer stem cells. Essays Biochem. 66, 423-433 (2022).47. Sharma, A., Bleriot, C., Currenti, J. & Ginhoux, F. Oncofetal reprogramming in tumour development and progression. Nat. Rev. Cancer 22, 593-602 (2022).48. Yarmarkovich, M. et al. Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs. Nature 599, 477-484 (2021).49. Cameron, B. J. et al. Identification of a Titin-Derived HLA-Al-Presented Peptide as a Cross-Reactive Target for Engineered MAGE A3-Directed T Cells. Set. Transl. Med. 5, (2013).50. Willemsen, R., Ronteltap, C., Heuveling, M., Debets, R. & Bolhuis, R. Redirecting human CD4+ T lymphocytes to the MHC class I-restricted melanoma antigen MAGE-A1 by TCR aP gene transfer requires CD8a. Gene Ther. 12, 140-146 (2005).-72- 4906-6039-7446.1Atty. Dkt. No.: 115872-340651. Robbins, P. F. et al. Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific T Cell Functions. J. Immunol. 180, 6116-6131 (2008).52. Gejman, R. S. et al. Identification of the Targets of T-cell Receptor Therapeutic Agents and Cells by Use of a High-Throughput Genetic Platform. Cancer Immunol. Res. 8, 672-684 (2020).-73- 4906-6039-7446.1

Claims

Atty. Dkt. No.: 115872-3406WHAT IS CLAIMED IS1. A T cell receptor (TCR) construct comprising a TCR alpha chain variable region and a TCR beta chain variable region, wherein:a) (i) the TCR alpha chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 4, a CDR2 amino acid sequence of SEQ ID NO: 5, and a CDR3 amino acid sequence of SEQ ID NO: 6 or having at least 80 % identity to SEQ ID NO: 6, and (ii) the TCR beta chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 8, a CDR2 amino acid sequence of SEQ ID NO: 9, and a CDR3 amino acid sequence of SEQ ID NO: 10, or having at least 80 % identity to SEQ ID NO: 10; orb) (i) the TCR alpha chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 12, a CDR2 amino acid sequence of SEQ ID NO: 13, and a CDR3 amino acid sequence of SEQ ID NO: 14 or having at least 80 % identity to SEQ ID NO: 14, and (ii) the TCR beta chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 16, a CDR2 amino acid sequence of SEQ ID NO: 17, and a CDR3 amino acid sequence of SEQ ID NO: 18, or having at least 80 % identity to SEQ ID NO: 18; orc) (i) the TCR alpha chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 20, a CDR2 amino acid sequence of SEQ ID NO: 21, and a CDR3 amino acid sequence of SEQ ID NO: 22 or having at least 80 % identity to SEQ ID NO: 22, and (ii) the TCR beta chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 24, a CDR2 amino acid sequence of SEQ ID NO: 25, and a CDR3 amino acid sequence of SEQ ID NO: 26, or having at least 80 % identity to SEQ ID NO: 26; ord) (i) the TCR alpha chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 28, a CDR2 amino acid sequence of SEQ ID NO: 29, and a CDR3 amino acid sequence of SEQ ID NO: 30 or having at least 80 % identity to SEQ ID NO: 30, and (ii) the TCR beta chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 32, a CDR2 amino acid sequence of SEQ ID NO: 33, and a CDR3 amino acid sequence of SEQ ID NO: 34, or having at least 80 % identity to SEQ ID NO: 34; or-74- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406e) (i) the TCR alpha chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 36, a CDR2 amino acid sequence of SEQ ID NO: 37, and a CDR3 amino acid sequence of SEQ ID NO: 38 or having at least 80 % identity to SEQ ID NO: 38, and (ii) the TCR beta chain variable region comprises a CDR1 amino acid sequence of SEQ ID NO: 40, a CDR2 amino acid sequence of SEQ ID NO: 41, and a CDR3 amino acid sequence of SEQ ID NO: 42, or having at least 80 % identity to SEQ ID NO: 42.

2. The TCR construct of claim 1, wherein the NKD1 peptide is bound to a HLA-A2 molecule.

3. The TCR construct of claim 2, wherein the HLA-A2 molecule is a HLA-A*02:01, HLA-A*02:02 or HLA-A*02:04 molecule.

4. The TCR construct of any one of claims 1-3, wherein:the TCR alpha chain variable region comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 7, and the TCR beta chain variable region comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 11; orwherein the TCR alpha chain variable region comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 15, and b) the TCR beta chain variable region comprises the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 19.

5. The TCR construct of any one of claims 1-3, wherein:the TCR alpha chain variable region comprises the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 23, and the TCR beta chain variable region comprises the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 27; orwherein the TCR alpha chain variable region comprises the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 31, and b) the TCR beta chain variable region comprises the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 35; or-75- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406wherein the TCR alpha chain variable region comprises the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 39, and b) the TCR beta chain variable region comprises the amino acid sequence of SEQ ID NO: 43 or an amino acid sequence having at least 80 % identity to SEQ ID NO: 43.

6. The TCR construct of any one of claims 1-5, wherein the TCR construct specifically binds to aNKDl peptide having the amino acid sequence SLLHTIYEV (SEQ ID NO: 1) or FLDTPIAKV (SEQ ID NO: 2).

7. The TCR construct of any one of claims 1-6, wherein one or more TCR alpha chain variable regions and one or more TCR beta chain variable regions are covalently linked to each other to form TCR heterodimers or multimers.

8. The TCR construct of any one of claims 1-7, further comprising an antibody or antigen binding fragment that specifically binds an additional target antigen, optionally wherein the additional target antigen is notNKDl.

9. The TCR construct of claim 8, wherein the antigen binding fragment is a scFv.

10. The TCR construct of claim 9, wherein the scFv is a humanized anti-CD3 scFv.

11. The TCR construct of any one of claims 1-10, wherein the additional target antigen is selected from the group consisting of CD3, CD28, CD5, CD16 or CD56.

12. The TCR construct of any one of claims 1-11, wherein the TCR construct is linked to at least one molecular marker or reporter via a linker.

13. The TCR construct of any one of claims 1-12, wherein the TCR construct is soluble or comprises a transmembrane domain.

14. A fusion protein comprising the TCR construct of any one of claims 1-13 operably linked to one or more polypeptides selected from among Fc receptors; Fc domains, including IgA, IgD, IgG, IgE, and IgM; cytokines, including IL-2 or IL-15; toxins; enzymes; CD247 (CD3-zeta), CD28, CD137, CD134 domain, or combinations thereof, optionally wherein the TCR construct is linked to the one or more polypeptides via a linker.

15. A composition comprising the TCR construct of any one of claims 1-13 and a pharmaceutically-acceptable carrier, wherein the TCR construct is optionally conjugated -76- 4906-6039-7446.1Atty. Dkt. No.: 115872-3406to an isotope, a dye, a drug, a chromagen, a contrast agent, a toxin, an enzymes, an enzyme inhibitor, a hormone, a hormone antagonist, a growth factor, a radionuclide, a metal, a liposome, a nanoparticle, RNA, DNA or any combination thereof.

16. A composition comprising the fusion protein of claim 14 and a pharmaceutically-acceptable carrier.

17. An engineered immune cell comprising the TCR construct of any one of claims 1-13 and / or a nucleic acid encoding the TCR construct.

18. The engineered immune cell of claim 17, wherein the engineered immune cell is a lymphocyte or a myeloid cell (e.g., macrophage).

19. The engineered immune cell of claim 18, wherein the lymphocyte is a T cell, a CD4+ T cell, a CD8+ T cell, a B cell, a tumor infiltrating lymphocyte, or a natural killer (NK) cell.

20. The engineered immune cell of any one of claims 17-19, wherein the engineered immune cell is derived from an autologous donor, an allogenic donor or an iPS engineered cell.

21. The engineered immune cell of any one of claims 17-20, further comprising at least one co-stimulatory receptor or a TCR that binds to an additional target antigen that is notNKDl.

22. A nucleic acid encoding the TCR construct of any one of claims 1-13 or the fusion protein of claim 14.

23. The nucleic acid of claim 22, wherein the nucleic acid encoding the TCR construct or the fusion protein is operably linked to a promoter.

24. The nucleic acid of claim 23, wherein the promoter is a constitutive promoter or a conditional promoter.

25. A vector comprising the nucleic acid of any one of claims 22-24.

26. The vector of claim 25, wherein the vector is a viral vector, a plasmid, an extracellular vesicle (EV) vector, or a nanoparticle.

27. The vector of claim 25, wherein the vector is a retroviral vector or a lentiviral vector.-77- 4906-6039-7446.1Atty. Dkt. No.: 115872-340628. A host cell comprising the nucleic acid of any one of claims 22-24 or the vector of any one of claims 25-27.

29. A kit comprising the nucleic acid of any one of claims 22-24 or the vector of any one of claims 25-27, and instructions for use.

30. A method for preparing immune cells for cancer therapy comprising isolating immune cells from a donor subject; and transducing the immune cells with (a) the nucleic acid of any one of claims 22-24 or (b) the vector of any one of claims 25-27.

31. A method of treatment comprising isolating immune cells from a donor subject; transducing the immune cells with (a) the nucleic acid of any one of claims 22-24 or (b) the vector of any one of claims 25-27; and administering the transduced immune cells to a recipient subject.

32. The method of claim 31, wherein the donor subject and the recipient subject are the same or different.

33. The method of any one of claims 30-32, wherein the immune cells isolated from the donor subject comprise one or more lymphocytes or myeloid cells.

34. The method of claim 33, wherein the one or more lymphocytes is a T cell, a B cell, or a natural killer (NK) cell.

35. The method of claim 33, wherein the one or more myeloid cells is a macrophage or a monocyte.

36. A method for treating a NKD1 -expressing cancer in a subject in need thereof comprising administering to the subject an effective amount of the TCR construct of any one of claims 1-13, the fusion protein of claim 14, the composition of claim 15 or 16, or the engineered immune cell of any one of claims 17-21, optionally wherein the cancer is a relapsed or refractory cancer.

37. A method for treating a NKD1 -expressing cancer in a subject in need thereof comprising administering to the subject an effective amount of the vector of any one of claims 25-27, wherein the vector is configured to deliver the nucleic acid of any one of claims 22-24 to T cells in situ, optionally wherein the cancer is a relapsed or refractory cancer.-78- 4906-6039-7446.1Atty. Dkt. No.: 115872-340638. A method for treating of inhibiting tumor growth or metastasis in a subject with a NKD1 -expressing cancer comprising contacting a tumor cell with an effective amount of the TCR construct of any one of claims 1-13, the fusion protein of claim 14, the composition of claim 15 or 16, or the engineered immune cell of any one of claims 17-21.

39. A method for treating of inhibiting tumor growth or metastasis in a subject with a NKD1 -expressing cancer comprising contacting a tumor cell with an effective amount of the vector of any one of claims 25-27, wherein the vector is configured to deliver the nucleic acid of any one of claims 22-24 to T cells in situ, optionally wherein the cancer is a relapsed or refractory cancer.

40. The method of any one of claims 36-39, wherein the TCR construct, the fusion protein, the composition, the engineered immune cell or the vector is administered intravenously, intraperitoneally, subcutaneously, intramuscularly, or intratum orally.

41. The method of any one of claims 36-40, further comprising administering an additional cancer therapy.

42. The method of claim 41, wherein the additional cancer therapy is selected from among surgery, chemotherapy, radiation therapy, immunotherapy, monoclonal antibodies, anti-cancer nucleic acids or proteins, anti-cancer viruses or microorganisms, and any combinations thereof.

43. The method of any one of claims 36-42, further comprising administering a cytokine to the subject.

44. The method of claim 43, wherein the cytokine is selected from the group consisting of interferon a, interferon P, interferon y, complement C5a, IL-2, TNF alpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7, CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL9, CXCR1, CXCR2, CXCR4, CXCR5, CXCR6, CXCR7 and XCL2.-79- 4906-6039-7446.1Atty. Dkt. No.: 115872-340645. The method of any one of claims 36-44, further comprising sequentially, separately, or simultaneously administering to the subject at least one therapeutic agent.

46. The method of any one of claims 36-44, wherein the cancer or tumor is colorectal cancer.

47. The method of claim 46, wherein the colorectal cancer is primary or metastatic.

48. A method for imaging a NKD1 -expressing tumor in a subject comprising administering to the subject an effective amount of the TCR construct of any one of claims 1-13, the fusion protein of claim 14, or the composition of claim 15 or 16, wherein the TCR construct, the fusion protein, or the composition is conjugated to a detectable label.-80- 4906-6039-7446.1

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