WO2023172514A1

WO2023172514A1 - Engineered immune cell therapeutics targeted to her2 and methods of use thereof

Info

Publication number: WO2023172514A1
Application number: PCT/US2023/014636
Authority: WO
Inventors: Celeste Jeanne RICHARDSON; Eugene Daehee CHOI; Jared Allan SEWELL; Jennifer Ann Johnson; Luke Martin BARRON
Original assignee: Catamaran Bio, Inc.
Priority date: 2022-03-07
Filing date: 2023-03-06
Publication date: 2023-09-14

Abstract

The present disclosure provides HER2 specific chimeric antigen receptors (CARs), immune cells (e.g., natural killer (NK) cells) comprising a HER2 specific CAR, and methods of use thereof. The disclosure further provides methods for treating a HER2 -positive cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the immune cell (e.g., NK cell) comprising a HER2 specific CAR.

Description

ENGINEERED IMMUNE CELL THERAPEUTICS TARGETED TO HER2 AND METHODS OF USE THEREOF

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/317,316, filed on March 7, 2022; U.S. Provisional Application No. 63/322,027, filed on March 21, 2022; U.S. Provisional Application No. 63/350,943, filed on June 10, 2022; and U.S. Provisional Application No. 63/422,066, filed on November 3, 2022, the entire contents of each of which are expressly incorporated herein by reference.

FIELD OF THE INVENTION

The present disclosure relates to HER2-specific chimeric antigen receptors (CARs), immune cells (e.g., natural killer (NK) cells) comprising a HER2-specific CAR, and methods of use thereof.

BACKGROUND

The adoptive transfer of autologous human T lymphocytes engineered to express chimeric antigen receptors (CARs) has resulted in remarkable clinical responses for the treatment of hematological malignancies. However, challenges remain, including systemic toxicity, target antigen-negative relapse, and logistical autologous therapy manufacturing hurdles. Furthermore, despite the success of HER2-targeted therapies for HER2-positive breast and gastric cancer, additional therapies are needed to address treatment-resistant metastatic disease and the immunosuppressive tumor microenvironment - a major barrier to effectively address solid tumors. Natural killer (NK) cells mediate effective cytotoxicity against tumor cells and unlike T cells, lack the potential to cause graft versus host disease (GVHD) in the allogeneic setting. Thus, NK cells expressing a CAR (CAR-NK cells) are a promising candidate for off-the-shelf cellular therapies. There is a need in the art for alternative HER2-specific CAR-based therapies that overcome the limitations of current therapies, and provide a more effective and safer immunotherapies. SUMMARY

The present disclosure provides HER2-specific CARs, immune cells (e.g., NK cells) comprising a HER2-specific CAR, and methods of use thereof for immunotherapy, e.g., in the treatment of cancer.

In one aspect, the disclosure provides a nucleic acid molecule comprising a coding region flanked by a transposase binding site, wherein the coding region comprises: (a) a nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18, or wherein the CAR comprises an amino acid sequence that is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 1, (b) a nucleic acid sequence encoding a second polypeptide, wherein the second polypeptide comprises a transforming growth factor beta receptor 2 (TGFBR2) dominant negative receptor, and (c) a nucleic acid sequence encoding a third polypeptide, wherein the third polypeptide comprises a cytokine, or a functional fragment thereof.

In some embodiments, the nucleic acid sequence encoding the first polypeptide is 5’ upstream of both the nucleic acid sequence encoding the second polypeptide and the nucleic acid sequence encoding the third polypeptide, and wherein the nucleic acid sequence encoding the second polypeptide is 5’ upstream of the nucleic acid sequence encoding the third polypeptide.

In some embodiments, the nucleic acid sequence encoding the second polypeptide is 5’ upstream of both the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the third polypeptide, and wherein the nucleic acid sequence encoding the first polypeptide is 5’ upstream of the nucleic acid sequence encoding the third polypeptide.

In some embodiments, the CAR comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

In some embodiments, the CAR comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-6 or SEQ ID NOs: 13-18.

In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 1-6 or SEQ ID NOs: 13-18. In some embodiments, the CAR consists of an amino acid sequence of any one of SEQ ID NOs: 1-6 or SEQ ID NOs: 13-18.

In some embodiments, the transposase binding site comprises a 5’ transposase binding site and/or a 3’ transposase binding site that are specifically recognized by a transposase.

In some embodiments, the transposase is selected from the group consisting of a TcBuster transposase, a piggyBac transposase, a Sleeping Beauty transposase, a Tn3 transposase, a Tn5 transposase, a Tn7 transposase, a TnlO transposase, a Frog Prince transposase, an IS5 transposase, a TnlO transposase, a Tn903 transposase, a SPIN transposase, a hAT transposase, a Hermes transposase, a Hobo transposase, an AeBuster transposase, a BtBuster transposase, a CfBuster transposase, a Tol2 transposase, a Tc3 transposase, a Mosl transposase, a MuA transposase, a Himar I transposase, and a Helitron transposase.

In some embodiments, the 5’ transposase binding site comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of any one of SEQ ID NOs: 148, 149, 160, 161, 165, 168 or 181-183.

In some embodiments, the 5’ transposase binding site comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 148, 149, 160, 161, 165, 168 or 181-183.

In some embodiments, the 5’ transposase binding site comprises the nucleic acid sequence of any one of SEQ ID NOs: 148, 149, 160, 161, 165, 168 or 181-183.

In some embodiments, the 3’ transposase binding site comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of any one of SEQ ID NOs: 150, 151, 162, 163, 166, 169 or 184-186.

In some embodiments, the 3’ transposase binding site comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 150, 151, 162, 163, 166, 169 or 184-186.

In some embodiments, the 3’ transposase binding site comprises the nucleic acid sequence of any one of SEQ ID NOs: 150, 151, 162, 163, 166, 169 or 184-186.

In some embodiments, the coding region is operably linked to a promoter.

In some embodiments, the promoter is selected from the group consisting of an MND promoter, an EF-la promoter, an EFS promoter, a MSCV promoter, a CMV promoter, a PGK promoter, a CAG promoter, a SFFV promoter, a CBH promoter, a SV40 promoter, a UBC promoter, or a RPBSA promoter. In some embodiments, the coding region further comprises a nucleic acid sequence encoding a fourth polypeptide.

In some embodiments, the fourth polypeptide comprises a cytokine receptor or a fragment thereof that specifically binds to a cytokine.

In some embodiments, the fourth polypeptide comprises IL- 15 receptor alpha or a fragment thereof that specifically binds to IL- 15.

In some embodiments, the fourth polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 102-103 or 194-198.

In some embodiments, the nucleic acid sequence encoding the fourth polypeptide is 3’ downstream of both the nucleic acid molecule encoding the first polypeptide and the nucleic acid molecule encoding the second polypeptide.

In some embodiments, the nucleic acid sequence encoding the fourth polypeptide is 5’ upstream of the nucleic acid molecule encoding the third polypeptide.

In some embodiments, the coding region further comprises at least one nucleic acid sequence encoding a self-cleaving peptide.

In some embodiments, the self-cleaving peptide is selected from the group consisting of E2A, T2A, P2A and F2A.

In some embodiments, the coding region comprises at least one nucleic acid sequence encoding an internal ribosomal entry site (IRES).

In some embodiments, the cytokine, or a functional fragment thereof comprises IL- 15, or a functional fragment thereof.

In some embodiments, the cytokine, or the functional fragment thereof, comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 100-101.

In some embodiments, the cytokine, or the functional fragment thereof, comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 100-101.

In some embodiments, the cytokine, or the functional fragment thereof, comprises the amino acid sequence of any one of SEQ ID NOs: 100-101.

In some embodiments, the cytokine, or the functional fragment thereof, consists of the amino acid sequence of any one of SEQ ID NOs: 100-101. In some embodiments, the third polypeptide comprises a fusion protein comprising IL- 15 or a functional fragment thereof, and IL-15RA or a fragment thereof that specifically binds to IL- 15.

In some embodiments, the TGFBR2 dominant negative receptor comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 108-113.

In some embodiments, the TGFBR2 dominant negative receptor comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108-113.

In some embodiments, the TGFBR2 dominant negative receptor comprises the amino acid sequence of any one of SEQ ID NOs: 108-113.

In some embodiments, the TGFBR2 dominant negative receptor consists of the amino acid sequence of any one of SEQ ID NOs: 108-113.

In another aspect, the disclosure provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

In some embodiments, the CAR comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

In some embodiments, the CAR consists of an amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

In another aspect, the disclosure provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the CAR comprises an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 1.

In some embodiments, the CAR consists of an amino acid sequence of SEQ ID NO:

1. In another aspect, the disclosure provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

In some embodiments, the CAR comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

In some embodiments, the CAR consists of an amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

In another aspect, the disclosure provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the CAR comprises an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 7.

In some embodiments, the CAR consists of an amino acid sequence of SEQ ID NO: 7.

In another aspect, the disclosure provides a nucleic acid molecule comprising: (a) a nucleic acid sequence encoding a first polypeptide comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18 or wherein the CAR comprises an amino acid sequence that is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 1; (b) a nucleic acid sequence encoding a second polypeptide comprising a TGFBR2 dominant negative receptor; and (c) a nucleic acid sequence encoding a third polypeptide comprising a cytokine, or a functional fragment thereof.

In some embodiments, the nucleic acid sequence encoding the first polypeptide is 5’ upstream of both the nucleic acid sequence encoding the second polypeptide and the nucleic acid sequence encoding the third polypeptide, and wherein the nucleic acid sequence encoding the second polypeptide is 5’ upstream of the nucleic acid sequence encoding the third polypeptide. In some embodiments, the nucleic acid sequence encoding the second polypeptide is 5’ upstream of both the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the third polypeptide, and wherein the nucleic acid sequence encoding the first polypeptide is 5’ upstream of the nucleic acid sequence encoding the third polypeptide.

In some embodiments, the CAR comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 1-6 or SEQ ID NOs: 13-18.

In some embodiments, the CAR consists of an amino acid sequence of any one of SEQ ID NOs: 1-6 or SEQ ID NOs: 13-18.

In some embodiments, the TGFBR2 dominant negative receptor consists of an amino acid sequence of any one of SEQ ID NOs: 108-113.

In some embodiments, the cytokine is selected from the group consisting of IL- 15, IL- 2, IL- 12, IL- 18, IL-21, LIGHT, CD40L, FLT3L, 4-1BBL and FASL, or a functional fragment thereof.

In some embodiments, the cytokine is IL-15, IL-2, IL-12, IL-18 or IL-21, and the cytokine is expressed as a fusion protein with a transmembrane domain.

In some embodiments, the cytokine is IL- 15, or a functional fragment thereof. In some embodiments, the IL-15, or the functional fragment thereof, comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 100-101.

In some embodiments, the IL-15, or the functional fragment thereof, comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 100-101.

In some embodiments, the IL-15, or the functional fragment thereof, comprises the amino acid sequence of any one of SEQ ID NOs: 100-101.

In some embodiments, the IL-15, or the functional fragment thereof, consists of the amino acid sequence of any one of SEQ ID NOs: 100-101.

In some embodiments, the third polypeptide comprises a fusion protein comprising IL- 15 or a functional fragment thereof, and IL-15RA or a fragment thereof that specifically binds to IL- 15.

In some embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding a fourth polypeptide.

In some embodiments, the nucleic acid molecule further comprises at least one nucleic acid sequence encoding a self-cleaving peptide.

In some embodiments, the self-cleaving peptide is selected from E2A, T2A, P2A and F2A.

In some embodiments, the nucleic acid molecule further comprises at least one nucleic acid sequence encoding an internal ribosomal entry site (IRES). In another aspect, the disclosure provides a nucleic acid molecule encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 133-144.

In another aspect, the disclosure provides a nucleic acid molecule encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 133-144.

In another aspect, the disclosure provides a nucleic acid molecule encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 133-144.

In some embodiments, one or more of the nucleic acid molecules described herein comprise an origin of replication.

In some embodiments, one or more of the nucleic acid molecules described herein is a DNA molecule.

In some embodiments, one or more of the nucleic acid molecules described herein is an RNA molecule.

In some embodiments, one or more of the nucleic acid molecules described herein is a circular molecule.

In another aspect, the disclosure provides an expression vector comprising one or more of the nucleic acid molecules described herein.

In some embodiments, one or more of the nucleic acid molecules described herein is a linear nucleic acid molecule.

In another aspect, the disclosure provides an engineered immune cell comprising one or more of the nucleic acid molecules described herein.

In some embodiments, the immune cell is a natural killer (NK) cell, a T cell, or a NKT cell.

In another aspect, the disclosure provides an engineered natural killer (NK) cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

In some embodiments, the CAR comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18. In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

In another aspect, the disclosure provides an engineered natural killer (NK) cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence that is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the CAR comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 1.

In some embodiments, the CAR consists of an amino acid sequence of SEQ ID NO: 1.

In another aspect, the disclosure provides an engineered natural killer (NK) cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

In some embodiments, the CAR comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

In some embodiments, the polypeptide consists of an amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

In another aspect, the disclosure provides an engineered natural killer (NK) cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence that is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 7.

In some embodiments, the CAR consists of an amino acid sequence of SEQ ID NO:

7. In another aspect, the disclosure provides a population of cells comprising one or more of the engineered immune cells and/or one or more of the engineered NK cells described herein.

In some embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the cells in the population express a CAR on the cell surface.

In some embodiments, the population of cells comprises the engineered NK cell, and wherein the engineered NK cell exhibits an NK cell effector function.

In some embodiments, the NK cell effector function is selected from the group consisting of HER2-dependent cytotoxicity, directed secretion of cytolytic granules, expression of CD107a, expression of CD69, production of tumor necrosis factor (TNF)- alpha, and production of interferon (IFN)-gamma.

In some embodiments, the population exhibits one or more NK cell effector functions at a level that is at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5- fold, at least 3 -fold, at least 5-fold, or at least 10-fold higher relative to a population of cells that do not comprise NK cells expressing a CAR.

In some embodiments, the population exhibits HER2-dependent cytotoxicity at a level that is at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3 -fold, at least 5-fold, or at least 10-fold higher relative to a population of cells that do not comprise NK cells expressing a CAR.

In another aspect, the disclosure provides a pharmaceutical composition comprising the engineered immune cell, the engineered NK cell, and/or the population of cells described herein, and a pharmaceutically acceptable carrier.

In another aspect, the disclosure provides a method for treating a HER2 -positive cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the engineered immune cell, the engineered NK cell, the population of cells, and/or the pharmaceutical composition described herein, thereby treating the HER2-positive cancer in the subject.

In some embodiments, the cancer comprises a solid tumor.

In some embodiments, the cancer is selected from the group consisting of a breast cancer, a gastric cancer, an esophageal cancer, an esophagogastric junction (GEJ) cancer, an ovarian cancer, a medulloblastoma, an osteosarcoma, a non-small cell lung carcinoma, a colorectal rectal cancer, a bladder cancer, and a prostate cancer.

In some embodiments, the engineered immune cell or the engineered NK cell is an allogeneic cell.

In some embodiments, the engineered immune cell or the engineered NK cell is an autologous cell.

In some embodiments, the method further comprises administering an additional therapeutic agent to the subject.

In some embodiments, the additional therapeutic agent is selected from the group consisting of an immune activator, a tyrosine kinase inhibitor, a metabolic inhibitor, an immune checkpoint inhibitor, a cytokine, a hypomethylating agent, and a therapeutic agent that targets HER2.

In some embodiments, (a) the additional therapeutic is the immune activator and wherein the immune activator is selected from the group consisting of 4-1BBL and OX-40; (b) the additional therapeutic is the metabolic inhibitor, and wherein the metabolic inhibitor is selected from the group consisting of an A2AR inhibitor and an IDO inhibitor; (c) the additional therapeutic is the checkpoint inhibitor and wherein the checkpoint inhibitor is an inhibitor of a protein selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA4, B7-H3, BTLA, KIR, LAG3, TIM-3, VISTA, AHR, c-cbl, and HPK1; (d) the additional therapeutic is the cytokine and wherein the cytokine is selected from the group consisting of IL-2, IL-15, IL-12, IL-18, IL-21, and a functional fragment thereof; and/or (e) the additional therapeutic is the therapeutic agent that targets HER2 and wherein the therapeutic agent that targets HER2 is selected from the group consisting of trastuzumab, pertuzumab, margetuximab, trastuzumab-DM-1, lapatinib, neratinib and tucatinib.

In another aspect, the disclosure provides a method for generating an engineered natural killer (NK) cells, the method comprising: (a) providing an NK cell or a precursor thereof; (b) contacting the NK cell or the precursor thereof with one or more of the nucleic acid molecules described herein, under conditions sufficient to transfer the nucleic acid molecule across a cell membrane of the NK cell or the precursor thereof; and (c) culturing the NK cell or the precursor thereof under conditions suitable for expression of the one or more of the nucleic acid molecules described herein, thereby generating an engineered NK cell.

In some embodiments, the method further comprises contacting the NK cell with a transposase or a nucleic acid molecule encoding a transposase. In some embodiments, the transposase is selected from the group consisting of of a TcBuster transposase, a piggyBac transposase, a Sleeping Beauty transposase, a Tn3 transposase, a Tn5 transposase, a Tn7 transposase, a TnlO transposase, a Frog Prince transposase, an IS5 transposase, a TnlO transposase, a Tn903 transposase, a SPIN transposase, a hAT transposase, a Hermes transposase, a Hobo transposase, an AeBuster transposase, a BtBuster transposase, a CfBuster transposase, a Tol2 transposase, a Tc3 transposase, a Mosl transposase, a MuA transposase, a Himar I transposase, and a Helitron transposase.

In another aspect, the disclosure provides a polypeptide comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

In another aspect, the disclosure provides a polypeptide comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 1.

In another aspect, the disclosure provides a polypeptide comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19- 24.

In some embodiments, the CAR comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24. In some embodiments, the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

In another aspect, the disclosure provides a polypeptide comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the CAR comprises an amino acid sequence of SEQ ID NO: 7.

In another aspect, the disclosure provides a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 133-144.

In some embodiments, the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 133-144.

In some embodiments, the polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 133-144.

In some embodiments, the polypeptide consists of an amino acid sequence of any one of SEQ ID NOs: 133-144.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic of a coding region of a transposon comprising a nucleic acid sequence encoding a HER2-specific chimeric antigen receptor (“HER2-CAR”), a TGFBR2 dominant negative receptor, and IL- 15, separated by self-cleaving peptide sequences.

FIG. IB is a bar graph showing the levels of expression of HER2-specific CAR (“CAR”) and TGFBR2 DNR (“DNR”) by the engineered peripheral blood NK cells.

FIG. 1C is a bar graph showing the levels of secreted IL- 15 in supernatants of engineered peripheral blood NK cells (“Engineered NK”) and unmodified, mock electroporated, control NK cells (“EP only”).

FIG. 2A is a graph showing the CAR-dependent cytotoxic activity of engineered NK cells (“engineered NK”) and unmodified, mock electroporated, control NK cells (“EP only”) against HER2⁺ SKOV3 cells in vitro (depicted as percentage target cell killing). FIG. 2B is a bar graph showing levels of interferon-y (IFNy) production (pg/mL) of engineered NK cells (“engineered NK”) and unmodified, mock electroporated, control NK cells (“EP only”) following co-culture with HER2⁺ SK0V3 cells.

FIG. 3A is a graph showing the level of phosphorylated SMAD2 in engineered NK cells expressing TGFBR2 DNR including a tag (“DNR”) or untransduced control NK cells (“Control”) following exposure to the indicated concentration of recombinant human TGF- Pl.

FIG. 3B is a graph showing the level of phosphorylated SMAD2 in non-engineered NK cells neighboring engineered NK cells as determined after gating on non-engineered NK cells (“DNR”) or untransduced control NK cells (“Control”) following exposure to the indicated concentration of recombinant human TGF-pi.

FIG. 3C is a bar graph showing the level of DNAM-1 in engineered NK cells expressing TGFBR2 DNR including a tag (“DNR”) or engineered NK cells expressing a fusion protein including truncated CD19 (CD191-319) and a tag (“Control”) following exposure to 10 ng/mE recombinant TGF-pi (“+TGFP”) or no exposure to recombinant TGF-pi (“No TGFP”). MFI = mean fluorescence intensity.

FIG. 3D is a bar graph showing the cytotoxic activity against K562-luc target cells of engineered NK cells expressing TGFBR2 DNR including a tag (“DNR”) or engineered NK cells expressing a fusion protein including truncated CD19 (CD191-319) and a tag (“Control”) following exposure to 10 ng/mL recombinant TGF-pi (“+TGFP”) or no exposure to recombinant TGF-pi (“No TGFP”).

FIG. 4A is a graph showing the in vitro survival, as represented by the number of total viable cells, of engineered NK cells (“engineered NK”) and unmodified, mock electroporated, control NK cells (“EP only”) following the indicated days of culture in the absence of exogenous cytokines.

FIG. 4B is a graph showing the number of engineered NK cells (“engineered NK”) and unmodified, mock electroporated, control NK cells (“EP only”) per pL of blood in NSG mice at the indicated day post-administration.

FIG. 5A is a schematic of the in vivo efficacy study design.

FIG. 5B is a graph showing tumor burden in mice that were administered the engineered NK cells (“Engineered NK”), unmodified, mock electroporated control NK cells (“EP only”), or saline (“Saline control”) at the indicated day post-first dose. Tumor cell luciferase signal is reported as flux (photons/second). * = p<0.05 using non-parametric t-test at individual time points between the mice receiving the engineered NK cells and both control groups of mice.

FIG. 5C shows representative bioluminescence images of tumors in mice.

FIG. 5D shows a Kaplan-Myer survival plot demonstrating prolonged survival of mice administered the engineered NK cells (“Engineered NK”) as compared to mice administered unmodified, mock electroporated control NK cells (“EP only”), or saline (“Saline control”). *** = p<0.001 using Mantel Cox log-rank non-parametric test.

FIG. 6A is a graph showing HER2⁺ SK0V3 target cell survival in vitro following co- culture of the target cells with either (a) unmodified, mock electroporated, control NK cells (“Control cells”) or (b) engineered NK cells (“Engineered NK”) at the indicated effector cell to target cell (E:T) ratios over time. The survival of target HER2⁺ SK0V3 cells that were not co-cultured with any NK effector cells is also shown (“Tumor Alone”).

FIG. 6B is a bar graph showing the production of interferon gamma (IFNy) by unmodified, mock electroporated, control NK cells (“Control cells”) or engineered NK cells (“Engineered NK”) following a 24-hour co-culture of the NK cells with target cell lines of gastric cancer (NCI-N87), breast cancer (HCC1954 and SKBR3) or ovarian cancer (SK0V3) origin at an E:T ratio of 2.5:1.

FIG. 7A is a schematic depicting the in vivo persistence study design with longitudinal blood collection to assess NK cell levels in circulation.

FIG. 7B is a graph showing the number of human NK cells per pL of peripheral blood in NSG mice following intravenous (IV) administration of either 2xl0⁶ (squares), 4 x 10⁶ (triangles), or 8 x 10⁶ (circles) CAR⁺ engineered NK cells (“Engineered NK”), or unmodified, mock electroporated, control NK cells (“Control cells”; diamonds) at the indicated days post IV injection.

FIG. 7C is a graph showing the detected concentration of IL- 15 in plasma of NSG mice following intravenous (IV) administration of either 2xl0⁶ (squares), 4 x 10⁶ (triangles), or 8 x 10⁶ (circles) CAR⁺ engineered NK cells (“Engineered NK”), or unmodified, mock electroporated, control NK cells (“Control cells”; diamonds) at the indicated days post IV injection.

FIG. 8A is a schematic depicting the in vivo study design with longitudinal blood collections for at least two months prior to an intraperitoneal (IP) tumor cell injection.

FIG. 8B is a graph showing tumor burden in mice that were administered the engineered NK cells (“Engineered NK”; squares), unmodified, mock electroporated control NK cells (“Control cells”; circles) at the indicated day post-tumor initiation. Tumor cell luciferase signal is reported as flux (photons/second).

FIG. 9A is a schematic depicting the in vivo study design of a tumor efficacy study with a therapeutic dosing schedule.

FIG. 9B is a graph showing tumor burden in mice that were administered engineered NK cells (“Engineered NK”; circles), unmodified, mock electroporated control NK cells (“Control cells”; squares), or saline (“Vehicle control”; triangles) at the indicated days post- tumor initiation. Tumor cell luciferase signal is reported as flux (photons/second). * = p<0.05 using non-parametric Kruskal-Wallis at the indicated timepoint (Day 33) between the mice that were administered the engineered NK cells and both control groups of mice. Upward pointing triangles on the X-axis indicate the dosing days along the study.

FIG. 9C shows representative bioluminescence image series of tumors in mice.

FIG. 10A is a schematic depicting the in vivo study design with N87 tumor cell line xenograft injections occurring at day -10 followed by intravenous injection treatment beginning at Day 0. Study was performed until tumors reached a humane point (tumor volume of approximately 2,000 mm³).

FIG. 10B is a graph showing mean subcutaneous tumor volume (+/- standard error of measurement) in mice that were administered either engineered NK cells (“Engineered NKs”; empty circles), unmodified, mock electroporated control NK cells (“Control Cells”; squares), or saline (“Vehicle Control”; filled-in circles).

FIG. 10C is a Kaplan-Meier survival plot demonstrating prolonged survival of mice administered engineered NK cells (“Engineered NKs”; dashed line) as compared to mice administered the unmodified, mock electroporated control NK cells (“Control Cells”; dotted line), or saline (“Vehicle Control”; solid line).

FIG. 10D is a graph showing the correlation between NK cell levels in the circulation and the subcutaneously growing tumor volume in mice administered with either the engineered NK cells (“Engineered NK”), the unmodified, mock electroporated control NK cells (“Control Cells”), or saline (“Vehicle Control”) at day 60 post-intravenous administration of treatment.

FIG. 10E is a bar graph showing the total NK cell count (cells) found in the indicated organs of the mice treated with engineered NK cells at day 90 post-intravenous administration of treatment. FIG. HA is a bar graph showing relative HER2 surface protein expression levels of SK0V3, NCI-N87, and HT-29 cell lines, which were determined by staining with a HER2- specific antibody and measured by flow cytometry.

FIG. 11B is a bar graph showing that DNR/CAR/IL-15 expression enabled greater IFN-yby engineered than control NK cells across a > 100-fold range of HER2 expression levels by the tumor target cells.

FIG. 11C is a graph showing that DNR/CAR/IL-15 expression enabled greater cytotoxic activity by engineered NK cells compared to control NK cells stimulated by HER2- high SK0V3 cells.

FIG. HD is a graph showing that DNR/CAR/IL-15 expression enabled greater cytotoxic activity by engineered NK cells compared to control NK cells stimulated by HER2- intermediate NCI-N87 cells.

FIG. HE is a graph showing that DNR/CAR/IL-15 expression enabled greater cytotoxic activity by engineered NK cells compared to control NK cells stimulated by HER2- low HT-29 cells.

FIG. 12A is a graph showing that engineered NK cells harvested on day 10 post electroporation were able to regress established subcutaneous tumors from a peak tumor volume of approximately 300-400mm³.

FIG. 12B is a graph showing that a single dose of 2xl0⁶ engineered NK cells with a nucleic acid sequence encoding DNR first, CAR second, and IL- 15 third (Construct 2) achieved similar strong anti-tumor activity and tumor regression as a 4-fold higher dose of 8xl0⁶ engineered NK cells with a nucleic acid sequence encoding CAR first, DNR second, and IL- 15 third (Construct 1).

DETAILED DESCRIPTION

The present disclosure provides HER2-specific chimeric antigen receptors (CARs), immune cells (e.g., NK cells) comprising HER2 specific CARs, methods of use thereof for immunotherapy, and methods of making engineered immune cells.

In one aspect, the disclosure provides nucleic acid molecules encoding a CAR, wherein the CAR comprises an amino acid sequence which is at least 98.5% identical to the entire amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 7; or is at least 92% identical to the entire amino acid sequence of any one of SEQ ID NOs: 2-6, 13-18, 8-12, or 19-24. In another aspect, the disclosure provides nucleic acid molecules comprising a coding region flanked by a transposase binding site, wherein the coding region comprises: (a) a nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a CAR, wherein the CAR comprises an amino acid sequence which is at least 98.5% identical to the entire amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 7; or is at least 92%' identical to the entire amino acid sequence of any one of SEQ ID NOs: 2-6, 13-18, 8-12 or 19-24, (b) a nucleic acid sequence encoding a second polypeptide, wherein the second polypeptide comprises a TGFBR2 dominant negative receptor, and (c) a nucleic acid sequence encoding a third polypeptide, wherein the third polypeptide comprises a cytokine, or a functional fragment thereof.

In another aspect, the disclosure provides immune cells (e.g., NK cells, T cells or NKT cells) comprising a CAR, wherein the CAR comprises an amino acid sequence which is at least 98.5% identical to the entire amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 7; or is at least 92%' identical to the entire amino acid sequence of any one of SEQ ID NOs: 2-6, 13-18, 8-12, or 19-24.

In another aspect, the present disclosure provides methods for treating a HER2- positive cancer in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of an immune cell (e.g., an NK cell, T cells or NKT cells) comprising a HER2-specific CAR described herein.

The immune cells (e.g., NK cells) provided herein may include (e.g., express) one or more exogenous polypeptides, e.g., a cytokine, in addition to a CAR. For example, to allow for the enhanced in vivo persistence of immune cells (e.g., NK cells) in vitro and/or in vivo, the cells may be engineered to include (e.g., express) a polypeptide comprising IL-15, IL-15 receptor alpha (IL-15Ra) or a fragment thereof that specifically binds to IL- 15, the sushi domain of IL-15Ra, a fusion protein comprising IL- 15 and IL-15Ra (or a functional fragment of IL-15Ra that specifically binds to IL- 15), a fusion protein comprising IL- 15 and a sushi domain of IL-15Ra. Additionally, the immune cells may also be engineered to include (e.g., express) an exogenous polypeptide comprising IL-2, IL- 12, IL- 18, IL-21, LIGHT, CD40L, FLT3L, 4-1BBL, or FASL, or a functional fragment of any of the foregoing. In some embodiments, the exogenous polypeptide comprises a domain (e.g., a transmembrane domain) that anchors to polypeptide the immune cell plasma membrane. In some embodiments, the immune cell comprises an exogenous polypeptide comprising IL-15, IL-2, IL- 12, IL- 18 and/or IL-21, wherein the exogenous polypeptide is membrane-bound (e.g., plasma membrane bound). In some embodiments, the immune cells may be engineered to express an exogenous polypeptide comprising IL- 15, wherein the exogenous polypeptide is secreted by the cell. In some embodiments, the immune cell is engineered to express an exogenous polypeptide comprising IL-15RA or a fusion protein comprising IL- 15 and IL- 15RA (or a functional fragment of IL-15Ra that specifically binds to IL- 15).

Further, to allow for enhanced in vivo ability of the immune cell to overcome an immunosuppressive tumor microenvironment, the immune cells provided herein may be engineered to comprise one or more additional exogenous polypeptides that reduce immunosuppression of the tumor microenvironment, such as a TGF-P dominant negative receptor. Such additional polypeptides reduce the effects of the immunosuppressive tumor microenvironment on the immune cell by binding to TGF-P, and reducing and/or preventing any downstream suppressive signaling events. In some embodiments, the TGF-P dominant negative receptor is a TGFBR1 dominant negative receptor or a TGFBR2 dominant negative receptor.

In some embodiments, the modified immune cells (e.g., NK cells) includes (e.g., expresses) a HER-2 specific CAR, and further expresses a cytokine, e.g., IL15, and/or a TGFBR2 dominant negative receptor.

In another aspect, the disclosure relates to nucleic acid molecules encoding the CARs and one or more additional polypeptides described herein. The disclosure also relates to vectors {e.g., plasmids, retroviral vectors, mini-circle vectors and nanoplasmids) comprising the nucleic acid molecules provided herein.

Genetic reprogramming of immune cells {e.g., NK cells or T cells) for adoptive cancer immunotherapy has clinically relevant applications and benefits such as 1) innate anti- tumor surveillance without prior need for sensitization, 2) allogeneic efficacy without graft versus host reactivity (in the case of al logeneic NK cells), and 3) direct cell-mediated cytotoxicity and cytolysis of target antigen-expressing cancer cells, including solid tumors {e.g., HER2-positive cancer cells). Accordingly, the present disclosure also provides methods for treating immune-related disorders, such as cancer, comprising adoptive cell immunotherapy with any of the engineered immune cells {e.g., NK cells, T cells, or NKT cells) described herein. I. DEFINITIONS

Unless otherwise specified, each of the following terms have the meaning set forth in this section.

The indefinite articles “a” and “an” refer to one or to more than one one (i.e., at least one) of the grammatical object of the article.

The conjunctions “or” and “and/or” are used interchangeably as non-exclusive disjunctions.

The term “about” when referring to a measurable value (e.g., amount or duration) is meant to encompass variations of in some instances + 20 %, or in some instances ± 10 %, or in some instances ± 5 %, or in some instances + 1 %, or in some instances + 0.5 %, or in some instances + 0.1% from the specified value.

As used herein, the term "antigen recognition domain" refers to a molecule or portion of a molecule (e.g., a CAR) that specifically binds to an antigen (e.g., HER2).

As used herein, the term "antibody" refers to a polypeptide sequence derived from an immunoglobulin molecule, which specifically binds to an an antigen. Antibodies can be monoclonal or polyclonal, multiple or single chain, and/or intact immunoglobulins, and may be derived from recombinant or natural sources. A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N- terminal variable (VH) region and three C-terminal constant (CHL CH2 and CH3) regions, and each light chain contains one N Terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have a similar general structure, with each region comprising four framework regions, whose sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody, which is responsible for antigen binding.

As used herein, the term "antibody fragment" refers to at least one portion of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hinderance, stabilizing, destabilizing and/or spatial hinderance) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, F(ab')2, Fv fragments, single chain Fv (scFv), disulfide-linked Fvs, a FD fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies (e.g., sdAb (either VH or VL), camelid VHH domains, isolated CDRs, or other epitope binding fragments of an antibody. Antibody fragments can be incorporated into maxibodies, diabodies, triabodies, tetrabodies, minibodies, intrabodies, v- NAR, and bis-scFvs or grageted inot polypeptide scaffolds.

The term “exogenous,” when used in relation to a polypeptide or nucleic acid in a cell or organism refers to a polypeptide or nucleic acid that has been introduced into the cell or organism by artificial or natural means.

As used herein, the term "chimeric antigen receptor” or “CAR” refers to a fusion polyptide which when present in an immune cell (e.g., a T cell or an NK cell) provides the cell with specificity for a target cell (e.g., a cancer cell) and with intracellular signal generation. In some embodiments, a CAR comprises an extracellular portion (e.g., an extracellular antigen recognition domain), a tansmembrane domain, and a cytoplasmic domain (e.g., comprising an intracellular' signaling domain). In some embodiments, the cytoplasmic domain comprises an intracellular signaling domain derived from a stimulatory polypeptide or co- stimulatory polypeptide. In some embodiments, the intracellular signaling domain is derived from the zeta chain associated with the T cell receptor complex (i.e., CD3Q, common FcR gamma (FCER1G), CD28, 4-1BB, DNAX-activating protein 10 (DAP10), DNAX-activating protein 12 (DAP12), 2B4, and/or 0X40. In some embodiments, the CAR comprises a signal peptide at the amino terminus (N-terminus). In some embodiments, the CAR comprises a signal peptide at the N-terminus of the extracellular portion of the C AR. Signal peptides are generally cleaved off the CAR during cellular processing and localization of the CAR to a cellular membrane (e.g., plasma membrane) of the immune cell. In some embodiments, the CAR is selected from the CARs of any one of SEQ ID NOs:l-24.

The term “signaling domain” as used herein refers to the functional portion of a protein which acts by transmitting information within a cell to regulate a cellular activity (e.g., an effector function) via a signaling pathway (e.g., by generating second messengers) or functioning as an effector (e.g., by responding to a second messenger). A signaling domain can be a co- stimulatory domain and/or an activation domain.

As used herein, the term "coding region" refers to a portion of a nucleic acid molecule that is transcribed, and optionally also translated into a gene product, e.g., a polypeptide, in vitro or in vivo when under the control of an appropriate regulatory sequences. In some embodiments, the nucleic acid molecule including a coding region may be cDNA, genomic DNA, or an RNA, and may be single-stranded or double-stranded. In some embodiments, the boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence is usually located 3' of the translation stop codon. In some embodiments, the coding region encodes a polycistronie m RN A.

The terms “peptide,” “polypeptide” and “protein” are used interchangeably herein and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.

As used herein, the term "functional fragment," refers to a portion of a reference polypeptide that is substantially identical to, but shorter in length, than the reference polypeptide and retains at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of one activity (e.g., cognate binding partner (e.g., receptor) binding activity) of the reference polypeptide. A fragment can include an N- terminal truncation, a C-terminal truncation, or both N-terminal and C-terminal truncations relative to a reference polypeptide.

The terms “inverted terminal repeat” and “ITR” refer to a nucleic acid sequence located at one end of a transposable unit that can be cleaved by a transposase when used in combination with a complementary sequence that is located on the opposing end of the transposable unit. In some embodiments, at least one pair of ITRs are the minimum sequence required for transposition activity by a transposase. In some embodiments, the ITR comprises the sequence flanking the transposable unit before recognition and/or cleavage by a transposase. In some embodiments, the ITRs comprise the sequence flanking the transposable after recognition and/or cleavage by a transposase. In some embodiments, a transposase binding site comprises one or more (e.g., 1, 2 or 3) ITRs.

As used herein, the term “transposase binding site” refers to a nucleic acid sequence that can be selectively bound by and/or cleaved by a transposase. In some embodiments, the sequence is a DNA sequence. In some embodiments, the transposase binding site comprises one or more inverted terminal repeats (ITRs). In some embodiments, the transposase binding site is a 5’ transposase binding site. In some embodiments, the 5’ transposase binding site comprises one or more 5’ ITRs. In some embodiments, the transposase binding site (e.g., 5’ transposase binding site) comprises the nucleic acid sequence of any one of SEQ ID NOs: 148, 149, 160, 161, 165, 168 and 181-183. In some embodiments, the transposase binding site is a 3’ transposase binding site. In some embodiments, the 3’ transposase binding site comprises one or more 3’ ITRs. In some embodiments, the transposase binding site (e.g., a 3’ transposase binding site) comprises the nucleic acid sequence of any one of SEQ ID NOs: 150, 151, 162, 163, 166, 169 or 184-186.

The term “operably linked” as used herein refers to a physical or functional juxtaposition of the components so described as to permit them to function in their intended manner. In some embodiments, the term refers to a functional linkage between a promoter and a second polynucleotide sequence to direct the transcription of a nucleic acid corresponding to the second polynucleotide sequence.

As used herein, the term "pharmaceutically acceptable carrier" includes any and all carriers, for example, aqueous solvents (e.g., saline solutions, phosphate buffered saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, and such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition may be adjusted according to the intended use.

The term “subject” refers to any mammal. In some embodiments, the “subject or “subject in need” of a treatment can be a primate (e.g., a human, a simian (e.g., a monkey (e.g., marmoset or baboon), or an ape (e.g., a gorilla, chimpanzee, orangutan, or gibbon)), a rodent (e.g., a mouse, a guinea pig, a hamster, or a rat), a rabbit, a dog, a cat, a horse, a sheep, a cow, a pig, or a goat. In some embodiments, the subject is a non-human mammal, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., a mouse, a pig, a rat, or a non-human primate). In some examples, a subject has been previously diagnosed or identified as needing treatment by a medical professional.

As used herein, the terms “treat”, “treating”, and/or “treatment” means a reduction in the number, severity, frequency, and/or duration of one or more symptoms of a medical disease or condition in a subject.

IL NUCLEIC ACID COMPOSITIONS

In one aspect, the disclosure provides nucleic acid molecules encoding a CAR, wherein the CAR comprises an amino acid sequence which is at least 98.5% identical to the entire amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 7; or is at least 92% identical to the entire amino acid sequence of any one of SEQ ID NOs: 2-6, SEQ ID NOs: 13-18, SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24. 1. Chimeric Antigen Receptors

In another aspect, the disclosure provides nucleic acid molecules encoding a CAR, wherein the CAR comprises an extracellular' portion comprising an antigen recognition domain that specifically binds human HER2; a transmembrane domain; and an intracellular domain. In some embodiments, the CAR further comprises a hinge domain. In some embodiments, the CAR further comprises a signal peptide. In some embodiments, the intracellular domain comprises one or more (e.g., 1, 2 or 3 signaling domains ). In some embodiments, the intracellular domain comprises one or more signaling domains (e.g., one or more costimulatory orone or more activation domains.

A, Antigen Recognition Domains

In some embodiments, the antigen recognition domain of a CAR described herein specifically binds to HER2. hi some embodiments, the antigen recogniation domain of a CAR described herein comprises or consists of a single chain variable fragment (scFv) (e.g., derived from a anti-HER2 monoclonal antibody). In some embodiments, the antigen recognition domain of a CAR provided herein comprises a heavy chain variable (VH) domain and light chain variable (VL) domain that specifically bind to HER2. Optionally, the VL and VH are joined by a flexible linker, such as a glycine- serine linker or a Whitlow linker. Any suitable linker may be disposed between the VH domain amino acid residues and the VL domain amino acid residues of the CAR (e.g., (Gly3-Ser)_n, wherein n is a positive integer equal to or greater than 1 (e.g., n may be equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) (SEQ ID NO: 191) or (Gly4-Ser)_n, wherein n is a positive integer equal to or greater than 1 (e.g., n may be equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) (SEQ ID NO: 192). In some embodiments, the linker comprises the amino acid sequence (Gly4Ser)4 (SEQ ID NO: 132) or (Gly4Ser)3 (SEQ ID NO: 193). In some embodiments, the linker comprises multiple repeats (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) of (GlySer), (GlyzSer), or (GlysSer).

In some embodiments, the antigen recognition domain of a CAR provided herein is encoded by a nucleic acid sequence that is codon-optimized for human codon usage.

The CARs described herein are engineered to target HER2 and confer immune cells expressing the CARs the ability to HER2-positive target cells (e.g., HER2-positive cancer cells). Human epidermal growth factor receptor 2 or HER2 is a member of the HER family of epidermal growth factor receptors and is encoded by the ErbB-2 gene. The HER family of proteins regulate cell growth and survival, as well as adhesion, migration, differentiation, and other cellular responses. HER2 (also known as receptor tyrosine-protein kinase erbB-2, Her2/neu or ErbB-2) is a 185 kDa cytoplasmic transmembrane tyrosine kinase receptor (see, e.g., Akiyama el al. Science 232(4758): 1644-6 (1986), incorporated in its entirety herein by reference). Overexpression and/or amplification of HER2 is observed in the development of a variety of solid cancers including breast, gastric, stomach, colorectal, ovarian, pancreatic, endometrial, and non-small cell lung cancers. An exemplary amino acid sequence of human

HER2 is provided below:

In some embodiments, the antigen recognition domain of a CAR provided herein specifically binds to a polypeptide comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 25.

In some embodiments, the antigen recognition domain of a CAR provided herein comprises a heavy chain variable (VH) domain comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the the amino acid sequence of SEQ ID NO: 187 (shown below with the CDR1 CDR2 and CDR3 sequences underlined):

( Q )

In some embodiments, the antigen recognition domain of a CAR provided herein comprises a heavy chain variable (VH) domain comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the the amino acid sequence of SEQ ID NO: 26 (shown below with the CDR1, CDR2 and CDR3 sequences underlined):

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS (SEQ ID NO: 26).

In some embodiments, the antigen recognition domain of a CAR provided herein comprises a VH domain comprising one or more (e.g., one, two or all three) of a complementarity determining region 1 (CDR1) comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence: DTYIH (SEQ ID NO: 27), a complementarity determining region 2 (CDR2) comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence: RIYPTNGYTRYADSVKG (SEQ ID NO: 28), and a complementarity determining region 3 (CDR3) comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of the amino acid sequences: WGGDGFYAMDV (SEQ ID NO: 29) or WGGDGFYAMDY (SEQ ID NO: 188). In some embodiments, the antigen recognition domain of a CAR provided herein comprises a VH domain comprising one or more (e.g., one, two or all three) of a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 27, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 28, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antigen recognition domain of the CAR comprises a VH domain comprising one or more (e.g., one, two or all three) of a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 27, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 28, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 188.

In some embodiments, the antigen recognition domain of a CAR provided herein comprises a light chain variable (VL) domain comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 215 (shown below with the CDR1, CDR2 and CDR3 sequences underlined): DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 215).

In some embodiments, the antigen recognition domain of a CAR provided herein comprises a VL domain comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 30 (shown below with the CDR1, CDR2 and CDR3 sequences underlined): DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSG TDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT (SEQ ID NO: 30).

In some embodiments, the antigen recognition domain of a CAR provided herein comprises a VL domain comprising one or more (e.g., one, two or all three) of a CDR1 comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence: RASQDVNTAVA (SEQ ID NO: 31), a CDR2 comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence: SASFLYS (SEQ ID NO: 32), and a CDR3 comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence: QQHYTTPPT (SEQ ID NO: 33). In some embodiments, the antigen recognition domain of a CAR provided herein comprises a VL domain comprising one or more (e.g., one, two or all three) of a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 31, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 32, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 33.

In some embodiments, the antigen recognition domain of a CAR provided herein comprises both a VH domain and a VL domain, wherein the VH domain comprises a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 27, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 28, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 29, and wherein the VL domain comprises a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 31, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 32, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 33.

In some embodiments, the antigen recognition domain of a CAR provided herein comprises a VH domain and a VL domain, and a linker is disposed between VH domain and the VL domain. In some embodiments, the linker disposed between VH domain and the VL domain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of the amino acid sequences: GSTSGGGSGGGSGGGGSS (SEQ ID NO: 189) and GSTSGSGKPGSGEGS (SEQ ID NO: 34).

In some embodiments, the antigen recognition domain of a CAR provided herein comprises: (a) a VH domain comprising an amino acid sequence that at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 187 or SEQ ID NO: 26, and (b) a VL domain comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 713 or SEQ ID NO: 30. In some embodiments, the antigen recognition domain of a CAR provided herein comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 187, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 713. In some embodiments, the antigen recognition domain of a CAR provided herein comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 26, and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 30.

In some embodiments, the antigen recognition domain of a CAR provided herein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35 (shown below with the CDRs and linker underlined): D I QMTQSP S SLSASVGDRVT I TCRASQDVNTAVAWYQQKP GKAPKLL I YSASFLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTGSTSGSGKPGSGEGS E VQLVE S GGGLVQP GGS LRL S CAAS GFN I KDTYIHWVRQAP GKGLE WVARI YP TNG YTRYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDVWGQGTLVTVSS (SEQ ID NO: 35).

In some embodiments, the antigen recognition domain of a CAR provided herein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 190 (shown below with the CDRs and linker underlined):

D I QMTQSP S SLSASVGDRVT I TCRASQDVNTAVAWYQQKP GKAPKLL I YSASFLYSGVPSRF SGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGGGSGGGSGGGGS SEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARI YPTNGYTRYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 190).

In some embodiments, the antigen recognition domain of a CAR provided herein comprises a VH domain comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the the amino acid sequence of SEQ ID NO: 36 (shown below with the CDR1, CDR2 and CDR3 sequences underlined): EVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFAD DFKGRFDFSLETSANTAYLQINNLKSEDMATYFCARWEVYHGYVPYWGQGTTVTVSS (SEQ ID NO: 36).

In some embodiments, the antigen recognition domain of a CAR provided herein comprises a VH domain comprising one or more (e.g., one, two or all three) of a CDR1 comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence: NYGMN (SEQ ID NO: 37); a CDR2 comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence: WINTSTGESTFADDFKG (SEQ ID NO: 38), and a CDR3 comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence: WEVYHGYVPY (SEQ ID NO: 39).

In some embodiments, the antigen recognition domain of a CAR provided herein comprises a VL domain comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40 (shown below with the CDR1, CDR2 and CDRL sequences underlined):

D I QL TQ S HKF L S T S VGD RVS I T CKASQDVYNAVAW YQQKP GQ S P KL L I Y SASSRYT GVP S RF TGSGSGPDFTFTI SSVQAEDLAVYFCQQHFRTPFTFGSGTKLE IKAL (SEQ ID NO: 40).

In some embodiments, the antigen recognition domain of a CAR provided herein comprises a VL domain comprising one or more (e.g., one, two or all three) of a CDR1 comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence: KASQDVYNAVA (SEQ ID NO: 41), a CDR2 comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence: SASSRYT (SEQ ID NO: 42), and a CDR3 comprising or consisting of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence: QQHFRTPFT (SEQ ID NO: 43).

In some embodiments, the antigen recognition domain of a CAR provided herein comprises both a VH domain and a VL domain, wherein the VH domain comprises a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 38, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 39, and wherein the VL domain comprises a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 41, a CDR2 comprising or consisting of the amino acid sequence of SEQ ID NO: 42, and a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 43.

In some embodiments, the antigen recognition domain of a CAR provided herein comprises a VH domain and a VL domain, and a linker is disposed between VH domain and the VL domain. In some embodiments, the linker disposed between VH domain and the VL domain comprises or consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence: GGGGSGGGGSGGGGS (SEQ ID NO: 45).

In some embodiments, the antigen recognition domain of a CAR provided herein comprises: (a) a VH domain comprising an amino acid sequence that at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 36, and (b) a VL domain comprising an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40.

In some embodiments, the antigen recognition domain of a CAR provided herein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 47 (shown below with the CDRs and linker underlined):

E VQLQQS GP E LKKP GE TVKI S CKAS G YP F TNYGMNWVKQAP GQGLKWMGWINTSTGESTFAD DFKGRFDFSLETSANTAYLQINNLKSEDMATYFCARWEVYHGYVPYWGQGTTVTVSSGGGGS GGGGSGGGGSD IQLTQSHKFLSTSVGDRVSIT CKASQDVYNAVAW YQQKP GQSPKLLI YSAS SRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKAL (SEQ ID NO: 47).

B. Signal Peptides

In some embodiments, a polypeptide provided herein comprises a signal peptide located at the N-terminus of the polypeptide. Signal peptides may be cleaved from the polypeptide during cellular processing in an immune cell. In some embodiments, a CAR described herein comprises a signal peptide located at the N-terminus of the CAR. In some embodiments, the antigen recognition domain of the CAR described herein comprises a signal peptide located at the N-terminus of the antigen recognition domain. Examples of signal peptides include, but are not limited to, a granulocyte-macrophage colony- stimulating factor receptor (GMCSFRA) signal peptide, a CD27 signal peptide, a CD8alpha signal peptide, a human IgG heavy chain signal peptide, a CD28 signal peptide, an IL-2 signal peptide, a TGFBR2 signal peptide, and/or an IL- 15 signal peptide. In some embodiments, a polypeptide (e.g., a CAR or an exogenous polypeptide) provided herein comprises a signal peptide comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 48-56. In some embodiments, a polypeptide (e.g., a CAR) provided herein comprises a GMCSFRA signal peptide comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%' identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, a polypeptide provided herein comprises a CD27 signal peptide sequence comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 49. In some embodiments, a polypeptide provided herein comprises a CD8alpha signal peptide sequence comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%' identical to the amino acid sequence of SEQ ID NO: 50. In some embodiments, a polypeptide provided herein comprises a human IgG heavy chain signal peptide sequence comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, a polypeptide provided herein comprises a human IgG heavy chain signal peptide sequence comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%', 99% or 100% identical to the amino acid sequence of SEQ ID NO: 52. In some embodiments, a polypeptide provided herein comprises a CD28 signal peptide sequence comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 53. In some embodiments, a polypeptide provided herein comprises an IL-2 signal peptide sequence comprising or consisting of an amino acid sequence that is at least 80%. 85%, 90%, 91%, 92%, 93%. 94%, 95%. 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 54. In some embodiments, a polypeptide provided herein comprises a TGFBR2 signal peptide sequence comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%', 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55. In some embodiments, a polypeptide provided herein comprises an IL- 15 signal peptide sequence comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 56. In some embodiments, any one of the CARs provided herein do not comprise a signal peptide.

C. Hinge Domains

In some embodiments, a CAR provided herein includes a hinge domain between the antigen recognition domain and the transmembrane domain of the CAR. In some embodiments, the hige domain provides flexibility for the antigen recognition domain to bind to a target antigen epitope (e.g., a HER2 epitope). In some embodiments, the hinge domain is up to about 300 amino acid residues in length. In some embodiments, the hinge domain is from about 10 to about 100 amino acid residues in length. In some embodiments, the hinge domain is from about 25 to about 50 amino acid residues in length. In some embodiments, the hinge domain comprises or consists of a CD8a (e.g., a human CD8a hinge domain), IgGl (e.g., an IgGl hinge domain or IgGl short hinge domain), IgG4 (e.g., an IgG4 hinge domain, an IgG4 short hinge domain, an IgG4 hinge-CH3, IgG4 mutant hinge domain, an IgG4 mutant- 1 hinge domain, or an IgG4 mutant-2 hinge domain) or CD28 hinge domain.

Hinge domains may be derived from CD8, CD8alpha, CD4, CD28, 4-1BB, FcyRIIIa, or an IgG (e.g., IgGl, IgG2 or lgG4), and from an antibody heavy-chain constant region. Alternatively, the hinge domain may be a synthetic sequence. In some embodiments, the hinge domain compri ses all or part of the hinge region of an immunoglobulin (e.g., human IgGl, IgG2, IgG3, or IgG4). In some embodiemtns, the hinge domain comprises an immunoglobulin CH2 and/or CH3 domain. In some embodiments, the hinge domain comprises a CH3 hinge region of a human immunoglobulin. In some embodiments, the hinge domain is from an IgG (e.g., IgGl, IgG2, IgG3 or IgG4) and the domain comprises one or more mutations (e.g., amino acid substitutions (e.g., in its CH2 domain) so as to prevent or reduce off-target binding of the hinge domain and/or a CAR comprising the hinge domain to an Fc receptor. In some embodiments, the hinge domain is derived from an IgGl, IgG2, IgG3, or IgG4 Fc region and includes one or more amino acid substitutions as compared to the wild-type protein from which the hinge domain was derived. In some embodiments, the hinge domain is derived from an IgGl, IgG2, IgG3, or IgG4 Fc region and includes one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, thirty, or more) amino acid substitutions at an amino acid residue at position 220, 226, 228, 229, 230, 233, 234, 235, 234, 237, 238, 239, 243, 247, 267, 268, 280, 290, 292, 297, 298, 299, 300, 305, 309, 318, 326, 330, 331, 332, 333, 334, 336, and/or 339 (amino acid residue positions indicated in the EU index proposed in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda). In some embodiments, the hinge domain is derived from an IgGl, IgG2, IgG3, or IgG4 Fc region and includes one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, thirty, or more) of the following amino acid substitutions C220S, C226S, S228P, C229S, P230S, E233P, V234A, L234V, L234F, E234A, E235A, E235E, G236A, G237A, P238S, S239D, F243E, P247I, S267E, H268Q, S280H, K290S, K290E, K290N, R292P, N297A, N297Q, S298A, S298G, S298D, S298V, T299A, Y300E, V305I, V309E, E318A, K326A, K326W, K326E, E328F, A33OE, A33OS, A331S, P331S, I332E, E333A, E333S, E333S, K334A, A339D, A339Q, and P396E. In some embodiments, the hinge domain is derived from an IgGl, IgG2, IgG3, or IgG4 Fc region and includes one or more of the following combinations of amino acid substitutions: S228P and E235E; S228P and N297Q; E235E and N297Q; S228P, E235E, and N297Q. Examples of hinge domains are provided below.

In some embodiments, a CAR provided herein comprises a hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of SEQ ID NOs: 57-68. In some embodiments, a CAR provided herein comprises a human CD8alpha hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 57. In some embodiments, a CAR provided herein comprises a human CD8alpha hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%' identical to the amino acid sequence of SEQ ID NO: 58. hi some embodiments, a CAR provided herein comprises a human IgGl hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 59. In some embodiments, a C AR provided herein comprises an IgG4 mutant- 1 hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 60.

In some embodiments, a CAR provided herein comprises a human IgGl short hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 61. In some embodiments, a CAR provided herein comprises a human IgG4 hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%', 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 62. In some embodiments, a CAR provided herein comprises a human IgG4 CH3 domain hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 63. In some embodiments, a CAR provided herein comprises a human IgG4 short hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 64. In some embodiments, a CAR provided herein comprises an IgG4 mutant hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 65. In some embodiments, a CAR provided herein comprises an IgG4 mutant-2 hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%;, 94%, 95%, 96%, 97%, 98%;, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 66. In some embodiments, a CAR provided herein comprises a human FcyRIIIa hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91%>, 92%, 93%, 94%;, 95%, 96%>, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 67. In some embodiments, a CAR provided herein comprises a human CD28 hinge domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%', 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 68.

Z). Transmembrane Domains

In some embodiments, a CAR described herein includes a transmembrane domain (e.g., linked to the extracellular portion of the CAR). A transmembrane domain can include one or more additional amino acid residues adjacent to the transmembrane region (e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid residues associated with the extracellular region of the protein from which the transmembrane domain was derived and/or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid residues associated with the intracellular region of the protein from which the transmembrane domain was derived). In some embodiments, the transmembrane domain may be derived from the same protein that a signaling domain of the CAR is derived. In some embodiments, the transmembrane domain is not derived from the same protein that a signaling domain of the CAR is derived. The transmembrane domain can be derived either from a natural or from a synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane domain can be a T-cell receptor alpha chain transmembrane domain, a T-cell receptor beta chain transmembrane domain, a CD3zeta transmembrane domain, a CD8alpha transmembrane domain, a CD28 transmembrane domain, a NKG2D transmembrane domain, a CD 16 transmembrane domain, CD3 epsilon transmembrane domain, a CD3 gamma transmembrane domain, a CD3 delta transmembrane domain, a CD45 transmembrane domain, a CD4 transmembrane domain, a CD5 transmembrane domain, a CD9 transmembrane domain, a CD22 transmembrane domain, a CD28 transmembrane domain, a CD33 transmembrane domain, a CD37 transmembrane domain, a CD64 transmembrane domain, a CD80 transmembrane domain, a CD86 transmembrane domain, a CD134 transmembrane domain, a 4-1BB transmembrane domain, a CD154 transmembrane domain, a CD278 transmembrane domain, a CD357 transmembrane domain, a NKp44 transmembrane domain, a NKp46 transmembrane domain, a NKp30 transmembrane domain, a DNAM-1 transmembrane domain, a NKG2D transmembrane domain, a DAP10 transmembrane domain, a DAP12 transmembrane domain, or a erythropoietin receptor transmembrane domain.

In some embodiments, a CAR provided herein comprises a transmembrane domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 69-79. In some embodiments, a CAR provided herein comprises a human CD8alpha transmembrane domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 69. In some embodiments, a CAR provided herein comprises a human CD8alpha transmembrane domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 70. In some embodiments, a CAR provided herein comprises a human CD28 transmembrane domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 71. In some embodiments, a CAR provided herein comprises a human NKG2D transmembrane domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 72. hi some embodiments, a CAR provided herein comprises a human NKG2D transmembrane domain comprising or consisting of an amino acid sequence that is at least 90%, 91%', 92%, 93%, 94%, 95%', 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 73. In some embodiments, a CAR provided herein comprises a human CD16 transmembrane domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 74. In some embodiments, a CAR provided herein comprises a human NKp44 transmembrane domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 75. In some embodiments, a CAR provided herein comprises a human NKp46 transmembrane domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 76. In some embodiments, a CAR provided herein comprises a human DAP12 transmembrane domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 77. In some embodiments, a CAR provided herein comprises a human DAP10 transmembrane domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%' identical to the amino acid sequence of SEQ ID NO: 78. In some embodiments, a CAR provided herein comprises a human DNAM-1 transmembrane domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 79.

Optionally, a CAR provided herein may include a short polypeptide linker, in some embodiments, between 2 and 10 amino acid residues in length that may form the linkage between the transmembrane domain and a signaling domain of the CAR. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the linker comprises either the amino acid sequence (Gly3-Ser)_n, wherein n is a positive integer equal to or greater than 1 (e.g., n may be equal to 1, 2 , 3, 4, 5, 6, 7, 8, 9, or 10) (SEQ ID NO: 191) or (Gly4-Ser)_n, wherein n is a positive integer equal to or greater than 1 (e.g., n may be equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) (SEQ ID NO: 192). In some embodiments, the linker comprises the amino acid sequence (Gly₄Ser)₄ (SEQ ID NO: 132) or (Gly₄Ser)₃ (SEQ ID NO: 193). In some embodiments, the linker comprises multiple repeats (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) of (GlySer), (GlysSer), or (GlysSer).

E. Signaling Domains

The cytoplasmic domain or region of a CAR provided herein includes at least one signaling domain. In some embodiments, the signaling domain of the CAR mediates activation of at least one of the effector functions of the immune cell (e.g., an NK cell, a T cell or an NKT cell) in which the CAR is present. Effector functions include cytolytic activity or helper activity, including the secretion of cytokines. In some embodiments, the signaling domain comprises one or more (e.g., one, two, three, four, or more) immunoreceptor tyrosine-based activation motifs (IT AMs). In some embodiments, a CAR provided herein includes one or more (e.g., one, two, three, or more) signaling domains. When the CAR includes one or more signaling domains, the signaling domains may be linked to each other in a random or specified order. Optionally, a linker may be disposed between each signaling domain. In some embodiments, the linker is from between about 1 to about 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid residues in length. In some embodiments, the linker is a glycine-serine doublet. In some embodiments, the linker is a single amino acid residue (e.g., an alanine or glycine residue).

The CAR may include one or more signaling domains selected from a CD28 signaling domain, a 4- IBB signaling domain, a DAP10 signaling domain, a DAP12 signaling domain, a 2B4 signaling domain, an 0X40 signaling domain, a CD27 signaling domain, an OX40L signaling domain, a CD3zeta signaling domain, a FCER1G signaling domain, and a FCGR2A signaling domain, or a functional fragment of any of the foregoing.

In some embodiments, a CAR provided herein comprises a signaling domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 80-95. In some embodiments, a CAR provided herein comprises a human CD28 signaling domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%' identical to the amino acid sequence of SEQ ID NO: 80. In some embodiments, a CAR provided herein comprises a human CD28 signaling domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 81. In some embodiments, a CAR provided herein comprises a human 4- IBB signaling domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 82. In some embodiments, a CAR provided herein comprises a human 4- IBB signaling domain comprising or consisting of an amino acid sequence that is at least 90%. 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 83. In some embodiments, a CAR provided herein comprises a human DAP 10 costimulatory domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 84. In some embodiments, a CAR provided herein comprises a human DAP10 costimulatory domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 85. In some embodiments, a CAR provided herein comprises a human DAP12 costimulatory domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%' or 100% identical to the amino acid sequence of SEQ ID NO: 86. In some embodiments, a CAR provided herein comprises a human 2B4 costimulatory domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 87. In some embodiments, a CAR provided herein comprises a human 0X40 costimulatory domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 88. In some embodiments, a CAR provided herein comprises a human OX40L signaling domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%', 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 89. In some embodiments, a CAR provided herein comprises a human DNAM1 signaling domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 90.

In some embodiments, a CAR provided herein comprises a human CD3zeta signaling domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the CD3zeta signaling domain comprises a mutation in an ITAM domain (e.g., as described in Feucht et al., Nat Med. 2019; 25(1): 82—88; incorporated in its entirety herein by reference). In some embodiments, each of the two tyrosine residues in one or more of ITAM1, ITAM2, or ITAM3 domains of the CD3zeta signaling domain are point-mutated (e.g., substituted) to a phenylalanine residue. In some embodiments, the CD3zeta activation domain comprises a deletion of one or more of the ITAM1, ITAM2, or ITAM3 domains.

In some embodiments, a CAR provided herein comprises a human CD3zeta signaling domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%', 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NO: 92 and SEQ ID NO: 93. In some embodiments, a CAR provided herein comprises a human FCER1G signaling domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%;, 94%, 95%, 96%, 97%, 98%;, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 94. In some embodiments, a CAR provided herein comprises a human FCGR2A signaling domain comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%' or 100% identical to the amino acid sequence of SEQ ID NO: 95.

In embodiments, a CAR provided herein contains additional amino acid residues at the amino terminus (N-terminus) or carboxy terminus (C-terminus), or at both termini, which are not found in the amino acid sequence of the parent CAR. In some embodiments, the additional amino acid residues do not interfere with a biological function of the CAR, e.g., ability to recognize a target cell antigen (e.g, HER2), antigen-dependent cytotoxicity, antigen-dependent activation, and others.

Table 1 provides exemplary amino acid sequences of the CAR domains described herein.

Table 1. Exemplary Sequences of CAR Domains

G. HER2-Specific CARs

The present disclosure provides CARs, engineered immune cells (e.g., NK cells, T cells and NKT cells) including the CARs, and nucleic acid molecules encoding the CARs. In some embodiments, the disclosure provides a CAR comprising (a) an antigen recognition domain provided herein that specifically binds to HER2; (b) a hinge domain provided herein (e.g., an IgGl hinge domain or an an IgG4 mutant hinge domain); (c) a transmembrane domain provided herein (e.g., a CD28 transmembrane domain or a a CD8alpha transmembrane domain); (d) at least one (e.g., one, two or three) signaling domain provided herein (e.g., a 4- IBB signaling domain, a CD3zeta signaling domain, a DAP10 signaling domain, a DAP 12 signaling domain, an 0X40 signaling domain, an OX40L signaling domain, and a DAP12 signaling domain, or combinations thereof (e.g., a 4- IBB signaling domain and a CD3zeta singaling domain; a DAP10 signaling domain and a CD3zeta signaling domain; an 0X40 signaling domain and a CD3zeta signaling domain; a CD28 signaling domain and a DAP12 signaling domain; a CD28 signaling domain and a CD3zeta signaling domain a CD28 signaling domain and an OX40L signaling domain and a CD3zeta singaling domain). In some embodiments, the CAR further comprises a signal peptide (e.g., a GMCSFRA signal peptide or a CD8alpha signal peptide) at the N-terminus of the polypeptide. Also provided are nucleic acid molecules encoding the CARs and immune cells including (e.g., expressing the CARs).

Table 2, HER2-Specific CARs

In some embodiments, the disclosure provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5% identical, or 100% identical to any one of the amino acid sequences provided in Table 2. In some embodiments, the disclosure provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5% identical, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24.

In some embodiments, the disclosure provides a polypeptide comprising a CAR, wherein the CAR comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5% identical, or 100% identical to any one of the amino acid sequences provided in Table 2. In some embodiments, the disclosure provides a polypeptide comprising a CAR, wherein the CAR comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5% identical, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24.

In some embodiments, the CAR comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%;, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identical to the amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24. In some embodiments, the CAR comprises of an amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24. In some embodiments, the CAR consists of an amino acid sequence of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24.

2. Additional Polypeptides

In some embodiments, the present invention provides nucleic acid molecules which, in addition to the CAR, further encode one or more additional polypeptides. For example, in some embodiments, the present disclosure provides nucleic acid molecules (e.g., expression vectors, retroviral vectors, or plasmids) and protein constructs comprising a CAR, wherein the CAR comprises a) an antigen recognition domain provided herein, b) a hinge domain provided herein, c) a transmembrane domain provided herein, and d) at least one (e.g., one, two or three) signaling domain provided herein, and further comprises one or more additional polypeptides. In some embodiments, the CAR and the one or more additional polypeptides are each encoded by a separate nucleic acid molecule (e.g., a separate vector or plasmid). In some embodiments the CAR and the one or more additional polypeptides are encoded by the same nucleic acid molecule (e.g., the same vector or plasmid). In some embodiments, the one or more additional polypeptides are selected from a cytokine or a functional fragment thereof, a cytokine receptor or a fragment thereof that specifically binds to a cytokine, and a TGF-P dominant negative receptor.

In some embodiments of any of the engineered immune cells described herein, the engineered immune cell is an engineered NK cell, T cell, or natural killer T (NKT) cell. In some embodiments of any of the engineered immune cells described herein, the engineered immune cell includes one or more exogenous polypeptides (e.g., an additional polypeptide described herein).

A. Cytokines and Cytokine Receptors

In some embodiments, the additional polypeptide comprises a cytokine selected from the group consisting of IL-15, IL-2, IL-12, IL-18, IL-21, LIGHT, CD40L, FLT3L, 4-1BBL and FASL, or a functional fragment thereof. In some embodiments, the additional polypeptide is a fusion protein comprising any one of IL-15, IL-2, IL-12, IL-18 and IL-21, and the fusion protein is membrane bound on an immune cell (e.g., NK cell) of the disclosure. For example, in some embodiments, the additional polypeptide is a fusion protein comprising a transmembrane domain (e.g., a transmembrane domain provided herein) and any one of IL- 15, IL-2, IL-12, IL-18 and IL-21. In some embodiments, the additional polypeptide comprises a cytokine, and the polypeptide is a soluble polypeptide. In some embodiments, the additional polypeptide comprises a cytokine, wherein the additional polypeptide is secreted by an immune cell in which the polypeptide is expressed.

In some embodiments, the additional polypeptide comprises IL-15 or a functional fragment thereof. In some embodiments, the IL- 15 or a functional fragment thereof comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%', 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NO: 100 and SEQ ID NO: 101. In some embodiments, the additional polypeptide is a transmembrane polypeptide comprising IL- 15 or a functional fragment thereof.

In some embodiments, the additional polypeptide comprises IL- 15 receptor alpha (IL- 15Ra) or a functional fragment thereof (e.g., an IL- 15 Ra fragment that specifically binds to IL-15 or an IL-15 Ra sushi domain). In some embodiments, the IL-15Ra or the functional fragment thereof comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 102, 103, 194, or 195.

In some embodiments, the additional polypeptide comprises IL- 15 or a functional fragment thereof and an IL-15 Ra or a functional fragment thereof (e.g., an IL-15 Ra fragment that specifically binds to IL- 15 or an IL- 15 Ra sushi domain). In some embodiments, the additional polypeptide is a transmembrane protein comprising IL-15 or a functional fragment thereof and a sushi domain of IL- 15 receptor alpha. In some embodiments, the additional polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 196.

In some embodiments, the additional polypeptide is a transmembrane polypeptide comprising IL- 15 or a functional fragment thereof, the sushi domain of IL- 15 receptor alpha or a functional fragment thereof, and a transmembrane domain (e.g., a transmembrane domain provided herein). In some embodiments, the additional polypeptide comprises a linker disposed between the IL- 15 or the functional fragment thereof and the sushi domain of IL- 15 receptor alpha or the functional fragment thereof. In some embodiments, the additional polypeptide includes a linker disposed between the IL- 15 or the functional fragment thereof and the sushi domain of the IL- 15 receptor alpha or the functional fragment thereof, as well as a linker disposed between the sushi domain of the IL-15 receptor alpha or the functional fragment thereof and the transmembrane domain. In some embodiments, the linker comprises or consists of 1-20 amino acid residues in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length). In some embodiments, the linker comprises or consists of between about 5 and about 25 amino acids in length (e.g., between about 5 and about 20 amino acids in length, between about 10 and about 25 amino acids in length, or between about 10 and about 20 amino acids in length). In some embodiments, the linker comprises the amino acid sequence of any one of the linkers provided elsewhere herein. In some embodiments, the additional polypeptide comprises IL- 15 or a functional fragment thereof and an IL-15Ra or a functional fragment thereof (e.g., the amino acid sequence of any one of SEQ ID NOs: 102, 194 or 195). In some embodiments, the additional polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%', 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 197 or 198. In some embodiments, the additional polypeptide comprises a signal peptide at the N- terminus of the polypeptide. In some embodiments, the additional polypeptide comprises a signal peptide provided herein. In some embodimens, the additional polypeptide comprises a signal peptide selected from an IL- 15 signal peptide sequence, an IL-2 signal peptide sequence, and a CD8a signal peptide sequence. In some embodiments, the additional polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%', 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 48-56.

In some embodiments, the additional polypeptide comprises or consists of IL- 12 or a functional fragment thereof. In some embodiments, the additional polypeptide is a transmembrane protein comprising IL- 12 or a functional fragment thereof. In some embodiments, the additional polypeptide comprises or consists of IL- 12p40 or a functional fragment thereof. In some embodiments, the additional polypeptide comprises or consists of IL-12p35 or a functional fragment thereof. In some cases, the additional polypeptide is a fusion protein comprising IL-12p40 or a functional fragment thereof and IL-12p35 or a functional fragment thereof, fused via a linker (e.g., any one of the linkers provided herein). In some embodiments, the additional polypeptide comprises IL-12p40 or a functional fragment thereof, wherein the IL-12p40 or the functional fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%', 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the additional polypeptide comprises IL- 12p35 or a functional fragment thereof, wherein the IL-12p35 or the functional fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 99.

In some embodiments, the additional polypeptide comprises IL-18 or a functional fragment thereof. In some embodiments, the additional polypeptide is a transmembrane protein comprising IL- 18 or a functional fragment thereof. In some embodiments, the additional polypeptide comprises IL- 18 or a functional fragment thereof, wherein the IL- 18 or the functional fragment thereof comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 104.

In some embodiments, the additional polypeptide comprises IL-21 or a functional fragment thereof. In some embodiments, the additional polypeptide is a transmembrane protein comprising IL-21 or a functional fragment thereof. In some embodiments, the additional polypeptide comprises IL-21 or a functional fragment thereof, wherein the IL-21 or the functional fragment thereof comprises an amino acid sequence that is at least 80%, 85%. 90%, 91%, 92%, 93%, 94%. 95%, 96%. 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 105.

Table 3 provides the amino acid sequences of exemplary additional polypeptides.

Table 3. Exemplary Additional Polypeptides

B. TGF-P Dominant Negative Receptors

In some embodiments, the additional polypeptide comprises a TGF-p dominant negative receptor. TGF-P dominant negative receptors are known in the art and are described in, for example, Burga et al., 2019, Clin Cancer Res.', 25(14):4400-4412; and International PCT Publication No. WO 2021/010951; each of which is incorporated in its entirety herein by reference. Without wishing to be bound by theory, inclusion of a TGF-P dominant negative receptor in the immune cells described herein may allow for the immune cells to have enhanced ability to overcome the tumor microenvironment in vivo. In some embodiments, the additional polypeptide comprises a TGFBR2 dominant negative receptor. In some embodiments, the additional polypeptide comprises a TGFBR1 dominant negative receptor.

In some embodiments, the additional polypeptide comprises a TGF-p dominant negative receptor comprising (a) the extracellular domain of a TGF-p receptor (e.g., the extracellular domain of human TGF-P receptor 1 (TGFBR1) or the extracellular domain of a TGF-p receptor 2 (TGFBR2)) and (b) a transmembrane domain {e.g., a transmembrane domain of a human TGF-p receptor (e.g., the transmembrane domain of TGFBR1 or TGFBR2). In some embodiments, the TGF-p dominant negative receptor includes a transmembrane domain of human TGFBR1 (e.g., AAVIAGPVCFVCLSLMLMVYI (SEQ ID NO: 199)). In some embodiments, the TGF-p dominant negative receptor includes a transmembrane domain of human TGFBR2 (e.g., VTGISLLPPLGVAISVIIIFY (SEQ ID NO: 200)). In some embodiments, the additional polypeptide is a TGF-P dominant negative receptor comprising (a) the extracellular domain of a TGFBR1 or the extracellular domain of a TGFBR2 and (b) a heterologous transmembrane domain (e.g., any of the transmembrane domains provided herein (e.g., a CD28 transmembrane domain)).

1. TGBR1 Dominant Negative Receptors

In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor.

An exemplary amino acid sequence of a reference TGFBR1 polypeptide is provided as SEQ ID NO: 106, below (signal peptide at amino acid residues 1-33 is underlined): MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATAL QCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHN SMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVELAAVIAGPVCF VCISLMLMVYICHNRTVIHHRVPNEEDPSLDRPFISEGTTLKDLIYDMTTSGSGSGLPLLVQRTIART IVLQESIGKGRFGEVWRGKWRGEEVAVKIFSSREERSWFREAEIYQTVMLRHENILGFIAADNKDNGT WTQLWLVSDYHEHGSLFDYLNRYTVTVEGMIKLALSTASGLAHLHMEIVGTQGKPAIAHRDLKSKNIL VKKNGTCCIADLGLAVRHDSATDTIDIAPNHRVGTKRYMAPEVLDDSINMKHFESFKRADIYAMGLVF WEIARRCSIGGIHEDYQLPYYDLVPSDPSVEEMRKVVCEQKLRPNIPNRWQSCEALRVMAKIMRECWY ANGAARLTALRIKKTLSQLSQQEGIKM (SEQ ID NO: 106)

In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%', 90%, 91%. 92%, 93%, 94%, 95%, 96%. 97%, 98%. 99% or 100% identical to amino acid residues 26-147 of SEQ ID NO: 106. In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor consisting of amino acid residues 34-147 of SEQ ID NO: 106.

In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%. 92%, 93%, 94%, 95%, 96%. 97%, 98%. or 99% to amino acid residues 34-503 of SEQ ID NO: 106, wherein the amino acid residue at position 376 is a glutamate (e.g., comprises a K376E amino acid substitution as compared to SEQ ID NO: 106). In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%', 98%, or 99% to amino acid residues 34-503 of SEQ ID NO: 106, wherein the amino acid residue at position 232 is an arginine (e.g., comprises a K232R amino acid substitution as compared to SEQ ID NO: 106). In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 34-503 of SEQ ID NO: 106, wherein the amino acid residue at position 200 is a isoleucine (e.g., compri ses a T200I amino acid substitution as compared to SEQ ID NO: 106). In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 34-503 of SEQ ID NO: 106, wherein the amino acid residue at position 232 is a glutamate (e.g., comprises a K232R amino acid substitution as compared to SEQ ID NO: 106). In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%', 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 34-503 of SEQ ID NO: 106, wherein the amino acid residue at position 241 is a leucine (e.g., comprises a S241L amino acid substitution as compared to SEQ ID NO: 106). In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%', 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 34-503 of SEQ ID NO: 106, wherein the amino acid residue at position 318 is an arginine (e.g., comprises a M318R amino acid substitution as compared to SEQ ID NO: 106). In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%', 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%. 98%, or 99% identical to amino acid residues 34-503 of SEQ ID NO: 106, wherein the amino acid residue at position 353 is a valine (e.g., comprises a G353V amino acid substitution as compared to SEQ ID NO: 106). In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 34-503 of SEQ ID NO: 106, wherein the amino acid residue at position 400 is a glycine (e.g., comprises a D400G amino acid substitution as compared to SEQ ID NO: 106). In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 /%, 98%, or 99% identical to amino acid residues 34-503 of SEQ ID NO: 106, wherein the amino acid residue at position 478 is a proline (e.g., comprises a R478P amino acid substitution as compared to of SEQ ID NO: 106).

In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising or consisting of the extracellular domain of a TGF-p type I receptor and a transmembrane domain. In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 201:

LQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMC IAE IDLIPRDRPFVCAP S SKTGSVTTTYCCNQDHCNK IELPTTVKS SPGLGPVEL (SEQ ID NO: 201).

In some embodiments, the additional polypeptide is a TGFBR1 dominant negative receptor comprising or consisting of an amino acid sequence having at least 80%, 95%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 202:

LQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMC I?,E IDLIPRDRPFVCAP S SKTGSVTTTYCCNQDHCNK

IELPTTVKSSPGLGPVELAAVIAGPVCFVCI SLMLMVYI (SEQ ID NO: 202)

2, TGBR2 Dominant Negative Receptors

In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor.

An exemplary amino acid sequence sequence of a reference TGFBR2 polypeptide is provided in SEQ ID NO: 107, below (signal peptide at amino acid residues 1-22 is underlined):

MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCM SNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMC SCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVII IFYCYRVNRQQKLSSTWETG KTRKLMEFSEHCAI ILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQFE TVAVKIFPYEEYASWKTEKDIFSDINLKHENILQFLTAEERKTELGKQYWLITAFHAKGNLQEYLTRH VISWEDLRKLGSSLARGIAHLHSDHTPCGRPKMP IVHRDLKSSNILVKNDLTCCLCDFGLSLRLDPTL SVDDLANSGQVGTARYMAPEVLESRMNLENVESFKQTDVYSMALVLWEMTSRCNAVGEVKDYEPPFGS KVREHPCVESMKDNVLRDRGRPEIPSFWLNHQGIQMVCETLTECWDHDPEARLTAQCVAERFSELEHL DRLSGRSCSEEKIPEDGSLNTTK (SEQ ID NO: 107)

In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%', 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% identical to amino acid residues 23-194 of SEQ ID NO: 107. In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor consists of amino acid residues 23-194 of SEQ ID NO: 106.

In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%', 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acid residues 23-187 of SEQ ID NO: 107. In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor consists of amino acid residues 23-187 of SEQ ID NO: 107. In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to amino acid residues 23-567 of SEQ ID NO: 107, wherein (a) the amino acid residue at position 528 is a cysteine; (b) the amino acid residue at position 528 is a histidine; the amino acid residue at position 460 is a cysteine; the amino acid residue at position 460 is a histidine; and/or the amino acid residue at position 537 is a histidine. In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%' to amino acid residues 23- 567 of SEQ ID NO: 107, wherein the amino acid residue at position 528 is a cysteine (e.g., comprises a R528C amino acid substitution as compared to SEQ ID NO: 107). In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%', 94%, 95%, 96%, 97%, 98%, or 99% to amino acid residues 23-567 of SEQ ID NO: 107, wherein the amino acid residue at position 528 is a histidine (e.g., comprises a R528H amino acid substitution as compared to SEQ ID NO: 107). In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to amino acid residues 23-567 of SEQ ID NO: 107, wherein the amino acid residue at position 460 is a cysteine (e.g., comprises a R460C amino acid substitution as compared to SEQ ID NO: 107). In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91%', 92%, 93%, 94%, 95%, 96%, 97%, 98%', or 99% to amino acid residues 23-567 of SEQ ID NO: 107, wherein the amino acid residue at position 460 is a histidine (e.g., comprises a R460H amino acid substitution as compared to SEQ ID NO: 107). In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to amino acid residues 23-567 of SEQ ID NO: 107, wherein the amino acid residue at position 537 is a histidine (e.g., comprises a R537H amino acid substitution as compared to SEQ ID NO: 107).

In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of the extracellular domain of a TGFBR2 and a transmembrane domain .

In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 108, below: TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVC VAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDN IIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSS (SEQ ID NO: 108)

In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 109, below: MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCD NQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK EKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVI I IF YCYRVNRQQKLSS (SEQ ID NO: 109)

In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 110, below: TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVC VAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDN I IFSEEYNTSNPDLLLVIFQFWVLVWGGVLACYSLLVTVAFI IFWVCYRVNRQQKLSS (SEQ ID NO: 110)

In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 111, below: MGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCD NQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMK EKKKPGETFFMCSCSSDECNDNI IFSEEYNTSNPDLLLVIFQFWVLVWGGVLACYSLLVTV AFI IFWVCYRVNRQQKLSS (SEQ ID NO: 111)

In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 112, below

TIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEK PQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSD ECNDNIIFSEEYNTSNPDLLLVIFQ (SEQ ID NO: 112)

In some embodiments, the additional polypeptide is a TGFBR2 dominant negative receptor comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 113, below: MGRGLLRGLWPLHIVLWTRIATIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDN QKSCMSNCSITS I CEKPQEVCVAVWRKNDENI TLETVCHDPKLP YHDF I LEDAASPKC IMKE KKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQ (SEQ ID NO: 113)

C. Linkers

Any of the polypeptides provided herein may comprise one or more linkers. For example, a linker may be positioned between two amino acid sequences (e.g., two domains). In some embodiments, the linker comprises or consists of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more amino acid residues in length. In some embodiments, the linker comprises or consists of between about 5 and about 25 amino acid residues in length, between about 5 and about 20 amino acid residues in length, between about 10 and about 25 amino acid residues in length, or between about 10 and about 20 amino acid residues in length. In some embodiments, the linker comprises or consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues in length. In some embodiments, the linker is non -immunogenic in a subject to whom a polypeptide comprising the linker is adminstered.

In some embodiments, the linker is selected from an amino acid sequence presented in Table 4. In some embodiments, the linker comprises or consists of the amino acid sequence (GGGGS)B (SEQ ID NO: 114), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In some embodiments, the linker comprises or consists of the amino acid sequence of any one of SEQ ID NO: 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and 128.

In some embodiments, the disclosure provides nucleic acid molecules comprising a coding region, wherein the coding region comprises: a nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a chimeric antigen receptor (CAR); a nucleic acid sequence encoding a second polypeptide, wherein the second polypeptide comprises a TGFBR2 dominant negative receptor; and a nucleic acid sequence encoding a third polypeptide, wherein the third polypeptide comprises a cytokine (e.g., IL- 15), or a functional fragment thereof.

In some embodiments, the coding region further comprises a nucleic acid sequence encoding a fourth polypeptide. In some embodiments, the fourth polypeptide comprises IL- 15Ra or a fragment thereof that specifically binds to IL- 15.

In some embodiments, the coding region further comprises at least one nucleic acid sequence encoding a self-cleaving peptide (e.g., disposed between nucleic acid sequences encoding polypeptides in the coding region). Self-cleaving peptide sequences could be used to co-express genes by linking open reading frames to form a single cistron. 2A self-cleaving peptide sequences can be used to create linked- or co-expressed proteins from a nucleic acid molecule of the present disclosure. In some embodiments, the self-cleaving peptide is selected from the group consisting of E2A, T2A, P2A and F2A. In some embodiments, the self-cleaving peptide is E2A. In some embodiments, the self-cleaving peptide is T2A. In some embodiments, the self-cleaving peptide is P2A. In some embodiments, the self-cleaving peptide is F2A. In some embodiments, the self-cleaving peptide is T2A comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 129. In some embodiments, the self-cleaving peptide is P2A comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 130. In some embodiments, the self- cleaving peptide is E2A comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 131. In some embodiments, the self-cleaving peptide is F2A comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%;, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 132. In some embodiments, the at least one nucleic acid sequence encoding a self-cleaving peptide is disposed between one or more of: (a) the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the second polypeptide: (b) the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the third polypeptide; and (c) the nucleic acid sequence encoding the second polypeptide and the nucleic acid sequence encoding the third polypeptide.

In some embodiments, the at least one nucleic acid sequence encoding a self-cleaving peptide is disposed between one or more of: (d) the nucleic acid sequence encoding the fourth polypeptide and the nucleic acid sequence encoding the first polypeptide; (e) the nucleic acid sequence encoding the fourth polypeptide and the nucleic acid sequence encoding the second polypeptide; and (f) the nucleic acid sequence encoding the fourth polypeptide and the nucleic acid sequence encoding the third polypeptide.

Table 4 provides exemplary amino acid sequences of linkers and self-cleaving peptides described herein. Table 4. Exemplary linkers and self-cleaving peptides

In some embodiments, the disclosure provides nucleic acid molecules comprising a coding region, wherein the coding region encodes a polycistronic mRNA. In some embodiments, the coding region described herein comprises at least one nucleic acid sequence encoding an internal ribosomal entry site (IRES). IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites, and are used to create polycistronic mRNAs. Multiple open reading frames can be transcribed together, each separated by an IRES, creating a polycistronic mRNA. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. In some embodiments, multiple genes can be efficiently expressed using a single promo ter/enhancer to transcribe a single message.

In some embodiments, the IRES comprises or consists of a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of SEQ ID NO: 145, provided below:

In some embodiments, the at least one nucleic acid sequence encoding an IRES is disposed between one or more of: (a) the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the second polypeptide; (b) the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the third polypeptide; and (c) the nucleic acid sequence encoding the second polypeptide and the nucleic acid sequence encoding the third polypeptide.

In some embodiments, the at least one nucleic acid sequence encoding an IRES is disposed between one or more of: (d) the nucleic acid sequence encoding the fourth polypeptide and the nucleic acid sequence encoding the first polypeptide; (e) the nucleic acid sequence encoding the fourth polypeptide and the nucleic acid sequence encoding the second polypeptide; and (f) the nucleic acid sequence encoding the fourth polypeptide and the nucleic acid sequence encoding the third polypeptide. D. Orientation

In some embodiments, the disclosure provides nucleic acid molecules comprising a coding region, wherein the coding region comprises: a nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a chimeric antigen receptor (CAR); a nucleic acid sequence encoding a second polypeptide, wherein the second polypeptide comprises a TGF-P dominant negative receptor (e.g., a TGFBR2 dominant negative receptor); and a nucleic acid sequence encoding a third polypeptide, wherein the third polypeptide comprises a cytokine (e.g., IL- 15), or a functional fragment thereof.

In some embodiments, the nucleic acid sequence encoding the first polypeptide is 5’ upstream of both the nucleic acid sequence encoding the second polypeptide and the nucleic acid sequence encoding the third polypeptide, and wherein the nucleic acid sequence encoding the second polypeptide i s 5’ upstream of the nucleic acid sequence encoding the third polypeptide.

Without wishing to be bound by theory, it is believed that, in some embodiments, the orientation of the nucleic acid sequences encoding the first polypeptide, the second polypeptide and the third polypeptide, and optionally a fourth polypeptide, within the coding region of the nucleic acid molecule impacts the expression levels of each polypeptide within an immune cell (e.g., by affecting the transcription of the coding region, the stability of the resulting mRNA, and/or the translation of the mRNA). Thus, the orientation of the nucleic acid sequences encoding each of the first polypeptide, the second polypeptide, and the third polypeptide, and optionally a fourth polypeptiode, within the coding region may affect the biological activity that the first polypeptide, the second polypeptide, and the third polypeptide confer to an immune cell expressing the polypeptides. Biological activities include, e.g., cytotoxicity, cytokine production (e.g., IL-15 production and/or secretion), immune cell persistence and/or survival (e.g., in vivo or in vitro).

In some embodiments, the nucleic acid sequence encoding the first polypeptide is 5’ upstream of both the nucleic acid sequence encoding the second polypeptide and the nucleic acid sequence encoding the third polypeptide, and wherein the nucleic acid sequence encoding the third polypeptide is 5’ upstream of the nucleic acid sequence encoding the second polypeptide.

In some embodiments, the nucleic acid sequence encoding the second polypeptide is 5’ upstream of both the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the third polypeptide, and wherein the nucleic acid sequence encoding the third polypeptide is 5’ upstream of the nucleic acid sequence encoding the first polypeptide.

In some embodiments, the nucleic acid sequence encoding the third polypeptide is 5’ upstream of both the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the second polypeptide, and wherein the nucleic acid sequence encoding the first polypeptide is 5’ upstream of the nucleic acid sequence encoding the second polypeptide.

In some embodiments, the nucleic acid sequence encoding the third polypeptide is 5’ upstream of both the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the second polypeptide, and wherein the nucleic acid sequence encoding the second polypeptide is 5’ upstream of the nucleic acid sequence encoding the first polypeptide.

Exemplary orientations of nucleic acid molecules comprising a coding region, wherein the coding region comprises: a nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a chimeric antigen receptor (CAR); a nucleic acid sequence encoding a second polypeptide, wherein the second polypeptide comprises a TGF-P dominant negative receptor (DNR) (e.g., a TGFBR2 DNR); and a nucleic acid sequence encoding a third polypeptide, wherein the third polypeptide comprises a cytokine (e.g., IE- 15), or a functional fragment thereof, are described in Table 5.

Table 5. Exemplary Orientation of Nucleic Acid Molecules

In some embodiments, the disclosure provides nucleic acid molecules comprising a coding region, wherein the coding region comprises or consists of a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence of SEQ ID NOs: 203-214, below. In some embodiments, the disclosure provides an immune cell (e.g., an NK cell, a T ceil or an NKT cell) comprising a nucleic acid molecule comprising a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least

99.5% identical, or 100% to the nucleic acid sequence of any one of SEQ ID NOs: 203-214.

Exemplary nucleic acid molecules comprising a coding region encoding polypeptides comprising a HER2 specific CAR and one or more additional polypeptides are described in Tables 6 and 7. Exemplary amino acid sequences produced as a product of the translation of these coding regions (without self cleavage by the encoded 2A peptides) are described in Table 8.

Table 6. Exemplary Coding Regions Encoding from 5’ to 3’ a HER2-Specific CAR, a

TGFBR2 dominant negative receptor (DNR) and a cytokine (IL-15).

Table 7. Exemplary Nucleic Acid Molecules Encoding from 5’ to 3’ a TGFBR2 dominant negative receptor (DNR), a HER2-Specific CAR, and a cytokine (IL- 15).

Table 8. Exemplary amino acid sequences of polypeptides encoded by the coding regions provided in Tables 6 and 7.

In some embodiments, the disclosure provides a nucleic acid molecule encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99% , at least 99.5% identical, or 100% to the amino acid sequence of any one of SEQ ID NOs: 133-144. In some embodiments, the disclosure provides an immune cell (e.g. , an NK cell, a T cell or an NKT cell) comprising a nucleic acid molecule encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%', at least 98.5%, at least 99%, at least 99.5% identical, or 100% to the amino acid sequence of any one of SEQ ID NOs: 133-144.

In some embodiments, the disclosure provides a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%', at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 133-144. In some embodiments, the disclosure provides an immune cell (e.g., an NK cell, a T cell or an NKT cell) comprising (e.g., expressing) a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 133-144.

4. Safety Switch Polypeptides

In some embodiments, the immune cells of the present disclosure may comprise one or more safety switch polypeptide or kill-switch genes (e.g., caspase-9, inducible FAS (iFAS), and inducible caspase-9 (iCASP9)). In some embodiments, the presence of a safety switch polypeptide or kill-switch gene may be used to ablate the immune cells in vivo following administration to a subject. In some instances, the safety switch protein expression is conditionally regulated. This conditional regulation could be variable and be regulated using small molecule-mediated post-translational activation and tissue-specific and/or temporal transcriptional regulation. The safety switch could mediate induction of apoptosis, inhibition of protein synthesis or DNA replication, growth arrest, transcriptional and post- transcriptional genetic regulation and/or antibody-mediated depletion in the immune cell. In some embodiments, the safety switch polypeptide is activated by an exogenous molecule, e.g., a prodrug.

Kill switch genes are generally genes which, upon administration of a prodrug, effects transition of a gene product to a compound which induced cell death in the cell containing the gene. Examples of kill switch gene/prodrug combinations which may be used include, but are not limited to inducible caspase 9 (iCASP9) and rimiducid; RQR8 and rituximab; truncated version of EGFR variant III (EGFRv3) and cetuximab; herpes simplex virus-thymidine kinase (HSV-tk) and ganciclovir, acyclovir, or FIAU; oxidoreductase and cycloheximide; cytosine deaminase and 5-fluorocytosine; thymidine kinase thymidilate kinase (Tdk::Tmk) and AZT; and deoxycytidine kinase and cytosine arabinoside; and Escherichia coll purine nucleoside phosphorylase and purine 6-methylpurine.

III. IMMUNE CELL COMPOSITIONS

In another aspect, the disclosure provides immune cells (e.g., NK cells, T cells or NKT cells) comprising a CAR provided herein. In some embodiments, the CAR comprises an amino acid sequence which is at least 98.5% identical to the entire amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 7; or is at least 92% identical to the entire amino acid sequence of any one of SEQ ID NOs: 2-6, SEQ ID NOs: 13-18, SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

1. Immune Cells

The immune cells of the disclosure may be natural killer (NK) cells, NKT cells (e.g., invariant NKT cells), T cells (e.g., regulatory T cells, CD4⁺ T cells, CD8⁺ T cells, or gamma- delta T cells), and stem cells (e.g., mesenchymal stem cells (MSCs) or induced pluripotent stem (iPSC) cells). In some embodiments, the immune cell is an NK cell. In some embodiments, the immune cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils. Also provided herein are methods of producing and engineering the immune cells as well as methods of using and administering the cells for adoptive cell therapy, in which case the cells may be autologous or allogeneic. Thus, the immune cells may be used as immunotherapy, such as to target cancer cells.

In some embodiments, the immune cells comprising a CAR described herein are T cells (e.g., alpha beta T cells and gamma delta T cells). In some embodiments, the T cells are one or more of CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, CD25⁺ and CD45RO⁺. In some embodiments, the T cells are isolated tumor infiltrating lymphocytes (TIL). In some embodiments, the T cells are CD4⁺ T cells. In some embodiments, the T cells are CD8⁺ T cells. In some embodiments, the T cells are regulatory T cell (e.g., a CD4⁺, CD25⁺, CD62L^hl, GITR⁺ and FoxP3⁺ T cells). In some embodiments, the T cells are memory T cells (Tew) (e.g., CD62L⁺, CCR7⁺, CD45RO’ and CD45RA ). In some embodiments, the T cells are stem cell memory T cells. In some embodiments, the T cells are naive T cells. In some embodiments, the T cells are a mixed population of CD4⁺ T cells, CD8⁺ T cells, stem cell memory T cells and naive T cells.

In some embodiments, the immune cells comprising a protein described herein (e.g., a CAR) are natural killer T (NKT) cells. NKT cells recognize glycolipid antigen presented by a molecule called CD Id.

In some embodiments, the immune cells comprising a protein described herein (e.g., a CAR) are NK cells. NK cells constitute about 10% of the lymphocytes in human peripheral blood. Human NK cells generally differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. In some embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMC), unstimulated leukapheresis products (PBSC), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), bone marrow, CD34⁺ cells or umbilical cord blood (CB) by methods well known in the art. In some embodiments, NK cells are isolated from PBMCs. In some embodiments, the NK cells are derived from umbilical CB. The NK cells may be NK cell lines, such as, but not limited to, the NK-92, NK101, KHYG-1, YT, NK-YS, YTS, HANK-1, NKL, and NK3.3 cell lines.

The immune cells may be isolated from subjects, particularly human subjects. The immune cells can be obtained from a subject of interest, such as a subject suspected of having a particular disease or condition, a subject suspected of having a predisposition to a particular disease or condition, or a subject who is undergoing therapy for a particular disease or condition. The immune ceils may be enriched/purified from any tissue where they reside including, but not limited to, blood (including blood collected by blood banks or cord blood banks), spleen, bone marrow, tissues removed and/or exposed during surgical procedures, and tissues obtained via biopsy procedures. Tissues/organs from which the immune cells are enriched, isolated, and/or purified may be isolated from both living and non-living subjects, wherein the non-living subjects are organ donors. The isolated immune cells may be used directly, or they can be stored for a period of time, such as by freezing. In some embodiments, the immune cells are isolated from blood, such as peripheral blood or cord blood. In some aspects, immune cells isolated from cord blood have enhanced immunomodulation capacity, such as measured by CD4- or CD8-positive T cell suppression. In specific aspects, the immune cells are isolated from pooled blood, particularly pooled cord blood, for enhanced immunomodulation capacity. The pooled blood may be from 2 or more sources, such as 3, 4, 5, 6, 7, 8, 9, 10 or more sources (e.g., donor subjects).

The population of immune cells can be obtained from a subject in need of therapy or suffering from a disease associated with reduced immune cell activity. Thus, the cells will be autologous to the subject in need of therapy. Alternatively, the population of immune cells can be obtained from a donor. The immune cell population can be harvested from the peripheral blood, cord blood, bone marrow, spleen, or any other organ/tissue in which immune cells reside in said subject or donor. The immune cells can be isolated from a pool of subjects and/or donors, such as from pooled cord blood.

When the population of immune cells is obtained from a donor distinct from the subject, the donor is preferably allogeneic, provided the cells obtained are subject-compatible in that they can be introduced into the subject. AHogcncic donor cells are may or may not be human-leukocyte-antigen (HLA)-compatible. To be rendered subject-compatible, allogeneic cells can be treated to reduce immunogenicity.

In some embodiments, the immune cells of the present disclosure may be generated from stem cells, such as induced pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs), or hematopoietic stem cells (HSCs). The pluripotent stem cells used herein may be induced pluripotent stem (IPS) cells, commonly abbreviated as iPS cells, iPscs or iPSCs. The use of iPSCs circumvents most of the ethical and practical problems associated with large- scale clinical use of ES cells, and patients with iPSC-derived autologous transplants may not require lifelong immunosuppressive treatments to prevent graft rejection.

Somatic cells can be reprogrammed to produce iPS cells using methods known to one of skill in the art (see for example, U.S. Patent Publication No.s 2009/0246875;

2010/0210014; 2012/0276636; U.S. Patent No.s 8,058,065; U.S. Patent No. 8,129,187; PCT Publication No. WO 2007/069666 Al, U.S. Patent No. 8,268,620; U.S. Patent No. 8,546,140; U.S. Patent No. 9,175,268; U.S. Patent No. 8,741,648; U.S. Patent Publication No.

2011/0104125, and U.S. Patent No. 8,691,574; each of which is incorporated in its entirety herein by reference). Generally, nuclear reprogramming factors are used to produce pluripotent stem cells from a somatic cell. In some embodiments, at least three, or at least four, of Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3/4, Sox2, c-Myc and Klf4 are utilized or Oct3/4, Sox2, Nanog, and Lin28.

In some embodiments, the immune cell is an NK cell. NK cells are a subpopulation of lymphocytes that have spontaneous cytotoxicity against a variety of tumor cells, virus- infected cells, and some normal cells in the bone marrow and thymus. NK cells can be detected by specific surface markers, such as CD16, CD56, and CD8 in humans. Stimulation of NK cells is achieved through a cross-talk of signals derived from cell surface activating and inhibitory receptors. The activation status of NK cells is regulated by a balance of intracellular’ signals received from an array of germ-line-encoded activating and inhibitory receptors. When NK cells encounter an abnormal cell (e.g., tumor or virus -infected cell) and activating signals predominate, the NK cells can rapidly induce apoptosis of the target cell through directed secretion of cytolytic granules containing perforin and granzymes or engagement of death domain-containing receptors. Activated NK cells can also secrete type I cytokines, such as interferon-y, tumor necrosis factor-a and granulocyte-macrophage colony- stimulating factor (GM-CSF), which activate both innate and adaptive NK cells as well as other cytokines and chemokines. Production of these soluble factors by NK cells in early innate immune responses significantly influences the recruitment and function of other hematopoietic cells. Also, through physical contacts and production of cytokines, NK cells are central players in a regulatory crosstalk network with dendritic cells and neutrophils to promote or restrain immune responses.

Expansion ofNK cells

NK cells can be expanded by various methods known in the art. In some instances, NK cells can be expanded or enriched from large volumes of peripheral blood, such as an apheresis products (e.g., mobilized PBSCs or unmobilized PBSCs). In other instances, NK cells can be expanded or enriched from a smaller number of blood or stem cells. Expansion of NK cells from apheresis products are described, for example, in Lapteva et al. Crit. Rev. Oncog. 19:121-132, 2014; Miller et al. Blood 105(8):3051-7, 2005; Lapteva et al. Cytotherapy 14(9): 1131-43, 2012; Spanholtz et al. PLoS One 6(6):e20740, 2011; Knorr et al. Stem Cells Transl. Med. 2(4):274-83, 2013; Pfeiffer et al. Leukemia 26(l l):2435-9, 2012; Shi et al. Br. J. Haematol. 143(5):641— 53, 2008; Passweg et al. Leukemia 18(11): 1835-8, 2004; Koehl et al. Klin. Padiatr. 217(6):345-50, 2005; and Klingemann et al. Transfusion 53(2):412-8, 2013; each of which is incorporated in its entirety herein by reference. NK cells may be expanded or enriched from apheresis products, buffy coats, cord blood (CB), and embryonic stem cells. In some instances, NK cells in peripheral blood and apheresis products can be detected by flow cytometry as CD45⁺CD56⁺CD3“ cells. In some instances, NK cells can be enriched from apheresis products by one or two rounds of depletion of CD3⁺ T cells using magnetic beads (e.g., CLINIMACS magnetic beads) coated with anti-CD3 antibody (e.g., CLINIMACS CD3 reagent) with or without overnight activation using IL-2 or IL- 15. Alternatively, NK cells can be enriched by isolating CD56⁺ cells using an anti-CD56 monoclonal antibody (e.g., CLINIMACS CD56 reagent) with or without CD3⁺ T cell depletion.

In some instances, NK cells can be expanded using feeder cell-based technology. Such methods are described, for example, in Berg et al. Cytotherapy 11 (3) :341- 55, 2009; Lapteva et al. 2012, supra-, and Lapteva et al. Crit. Rev. Oncog. 19:121-132, 2014; each of which is incorporated in its entirety herein by reference. Feeder-cell methods generally require cytokines as well as irradiated feeder cells, such as EBV-LCLs or genetically modified K562 cells, to produce large numbers of CD3 CD56⁺ NK cells from peripheral blood mononuclear cells (PBMCs). CD3-depleted, CD56-enriched PBMCs can be cultured in the presence of EBV-LCL feeders and X-VIVO 20 medium supplemented with 10% heat inactivated human AB serum, 500 U mL ¹ IL-2 and 2mM L-alanyl-L-glutamine over, e.g., 21 days of culture (see, e.g., Berg et al. Cytotherapy, 11(3): 341-55, 2009; incorporated in its entirety herein by reference). In some instances, the feeder cells are irradiated feeder cells. In some instances, the feeder cells are inactivated mitomycin-C.

In some instances, NK cells can be expanded using a genetically modified feeder cell expansion system, as described, for example, in Yang et al. (Mol. Therapy 18:428-445, 2020; incorporated in its entirety herein by reference). In such expansion methods, human primary NK cells can be expanded directly from PBMCs and CB, as well as tumor tissue, using an irradiated, genetically engineered feeder cells (e.g., the 721.221 cell line) that expresses membrane-bound interleukin 21 (IL-21) (221-mIL-21) (see e.g., Ojo et al. Sci. Rep. 9:14916, 2019; incorporated in its entirety herein by reference). In some instances, the NK cells are expanded in the presence of recombinant cytokines (e.g., IL-15 and IL-2).

Differentiation ofNK cells from Stem Cells

NK cells can be differentiated from stem cells by various methods known in the art. In some instances, NK cells can be differentiated from induced pluripotent stem cells (iPSCs), human embryonic stem cells (hESCs), mesenchymal stem cells (MSCs), or hematopoietic stem cells (HSCs). Protocols for the differentiation of NK cells from iPSCs and hESCs are described, for example, in Bock et al. J. Vis. Exp. (74):e50337, 2013; Knorr et al. Stem Cells Transl. Med. 2(4):274-83, 2013; Ni et al. Methods Mol. Biol. 1029:33-41, 2013; Zhu and Kaufman (Methods Mol. Biol. 2048:107-19, 2019); each of which is incorporated in its entirety herein by reference. For example, to differentiate iPSCs to CD34⁺CD45⁺ HPCs, embryonic bodies (EB) can be generated using different approaches, such as spinning of single cell iPSCs in round-shaped wells (spin EBs), culture on murine stroma cells, or direct induction of iPSC monolayer fragments in media with cytokines inducing differentiation towards the hematopoietic lineage. HPCs can be enriched by cell sorting or cell separation of CD34⁺ and/or CD45⁺ cells, and subsequently placed on feeder cells (e.g., AFT024, OP9, MS- 5, EL08-1D2) in medium containing IL-3 (during the first week), IL-7, IL- 15, SCF, IL-2, and Flt3L. NK cells can also be differentiated without usage of xenogeneic stromal feeder cells (see e.g., Knorr et al. Stem Cells Transl. Med. 2(4): 274-83, 2013; incorporated in its entirety herein by reference).

Additionally, CD56⁺CD16⁺CD3“ NK cells can be differentiated from human iPSCs and NK cell development can be characterized by surface expression of NK lineage markers (see, e.g., Euchner et al. Front. Immunol. 12:640672, 2021; incorporated in its entirety herein by reference). Hematopoietic priming of human iPSCs can result in CD34⁺CD45⁺ hematopoietic progenitor cells (HPCs) that do not require enrichment for NK lymphocyte propagation. HPCs can be further differentiated into NK cells on OP9-DL1 feeder cells resulting in high purity of CD56^bnghtCD16“ and CD56^bnghtCD16⁺ NK cells. The output of generated NK cells can be increased by inactivating OP9-DL1 feeder cells with MMC.

In some instances, CD3“CD56⁺ NK cells can be differentiated from CD34⁺ hematopoietic progenitors cells (HPCs) (see, e.g., Cichocki et al. Front Immunol, 10: 2078, 2019; incorporated in its entirety herein by reference). CD3“CD56⁺ NK cells with cytotoxic function can be differentiated in vitro after long-term culture of CD34⁺ cells isolated from cord blood, bone marrow, fetal liver, thymus, or secondary lymphoid tissue with IL-2 or IL- 15, (see, e.g., Mrozek et al. Blood 87:2632-40, 1996; Jaleco et al. J. Immunol. 159:694-702, 1997; Sanchez et al. J. Exp. Med. 178:1857-66, 1993; and Freud et al. Immunity 22:295-304, 2005; each of which is incorporated in its entirety herein by reference).

2. NK Cell Activity

In some embodiments, the disclosure provides a population of engineered NK cells that exhibit one or more NK cell effector functions. In some embodiments, the population of NK cells is cytotoxic to HER2-expressing cells. In some embodiments, the population of NK cells exhibits directed secretion of cytolytic granules or engagement of death domain- containing receptors. In some embodiments, the cytolytic granules comprise perforin and/or granzymes.

In some embodiments, the one or more NK cell effector functions comprise degranulation (e.g., CD107a expression), activation (e.g., CD69 production), cytokine production (e.g., TNFalpha or IFNgamma production), target cell line killing or anti-tumor efficacy in mouse models. Illustrative assays for measuring NK cell cytotoxicity and CD107a (granule release) are provided in Li et al., 2018, Cell Stem Cell 23, 181—192; which is incorporated in its entirety herein by reference.

In some embodiments, the population of NK cells exhibits the one or more NK cell effector functions at a level that is at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2- fold, at least 2.5-fold, at least 3-fold, at least 5-fold, or at least 10-fold higher relative to a population of cells that do not comprise NK cells expressing a CAR described herein. In some embodiments, the population of NK cells exhibits the one or more NK cell effector functions at a level that is at least 0.5-fold higher relative to a population of cells that do not comprise NK cells expressing a CAR described herein. In some embodiments, the population of NK cells exhibits the one or more NK cell effector functions at a level that is at least 1-fold higher relative to a population of cells that do not comprise NK ceils expressing a CAR described herein. In some embodiments, the population of NK cells exhibits the one or more NK cell effector functions at a level that is at least 1.5-fold higher relative to a population of cells that do not comprise NK cells expressing a CAR described herein. In some embodiments, the population of NK cells exhibits the one or more NK cell effector functions at a level that is at least 2-fold higher relative to a population of cells that do not comprise NK cells expressing a CAR described herein. In some embodiments, the population of NK cells exhibits the one or more NK cell effector functions at a level that is at least 2.5-fold higher relative to a population of cells that do not comprise NK cells expressing a CAR described herein. In some embodiments, the population of NK cells exhibits the one or more NK cell effector functions at a level that is at least 3 -fold higher relative to a population of cells that do not comprise NK cells expressing a CAR described herein. In some embodiments, the population of NK cells exhibits the one or more NK cell effector functions at a level that is at least 5-fold higher relative to a population of cells that do not comprise NK cells expressing a CAR described herein. In some embodiments, the population of NK cells exhibits the one or more NK cell effector functions at a level that is at least 10-fold higher relative to a population of cells that do not comprise NK cells expressing a CAR described herein. In some embodiments, the population of NK cells exhibits HER2- dependent cytotoxicity at a level that is at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3 -fold, at least 5-fold, or at least 10-fold higher relative to a population of cells that do not comprise NK cells expressing a CAR described herein.

3. Expression Levels

In some embodiments, the present disclosure provides a population of engineered immune cells (e.g., NK cells), wherein a plurality of the engineered immune cells (e.g., NK cells) of the population comprise one or more of the chimeric stimulatory receptors (CARs) described herein. The present disclosure also provides a composition comprising a population of immune cells (e.g., NK cells), wherein a plurality of the immune cells (e.g., NK cells) of the population comprise one or more CARs described herein. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population of immune cells (e.g., NK cells) comprises the CAR. In some embodiments, the CAR polypeptide is expressed at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 copies per cell. In some embodiments, the nucleic acid encoding the C AR is integrated into the genome at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or 30 copies per cell.

In some embodiments, the immune cells (e.g., NK cells) expressing a CAR are further engineered to express at least one cytokine and/or TGF-P DNR described herein. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the population of immune cells (e.g., NK cells) comprises the cytokine and/or DNR. In some embodiments, the cytokine and/or DNR, is expressed at a copy number of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90 or 100 copies of polypeptide per cell. In some embodiments, the nucleic acid encoding the cytokine and/or DNR, is integrated into the genome at a copy number of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or 30 copies per cell. IV. METHODS OF GENE DELIVERY AND IMMUNE CELL MODIFICATION

One of skill in the art would be well-equipped to construct a vector through standard recombinant techniques (see, for example, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 2001 ; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994) for the expression of one or more chimeric antigen receptors of the present disclosure. In some embodiments, the immune ceils provided herein are genetically engineered to express a CAR provided herein. In some embodiments, the immune cells comprise one or more exogenous nucleic acids introduced via genetic engineering that encode one or more proteins (e.g., a CAR). Suitable methods for genetically engineering immune cells are known in the art (see, e.g., Sambrook et al. 2001; and Ausubel et al., 1994). In some embodiments, the immune cells are engineered with a nucleic acid that has been codon-optimized for expression in a mammalian cell (e.g., a human cell).

Vectors include but are not limited to, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g., derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g. derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenoviral (Ad) vectors including replication competent, replication deficient and gutless forms thereof, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, yeast-based vectors, bovine papilloma virus (BPV)-based vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, polio virus vectors, vesicular stomatitis virus vectors, maraba virus vectors and group B adenovirus enadenotucirev vectors.

1. Transposition

In some embodiments, a method for gene delivery described herein comprises a transposon system. DNA transposons can translocate via a non-replicative “cut-and-paste” mechanism. This mechanism requires recognition of the two inverted terminal repeats (ITRs) by a catalytic enzyme, i.e., a transposase, which can cleave its target and consequently release the DNA transposon from its donor template. Upon excision, the DNA transposons may subsequently integrate into the acceptor DNA that is cleaved by the same transposase. In some of their natural configurations, DNA transposons are flanked by at least two ITRs, and may contain a gene encoding a transposase that catalyzes transposition.

Transposon systems offer many advantages for nucleic acid integration, e.g., as compared to viral vectors. For example, transposons can carry larger cargos, which can be advantageous for delivering one or more of the CARs, DNRs, and/or cytokines disclosed herein, to an immune cell (e.g., NK cell). Further, transposons may comprise, for example, CRISPR tools (e.g., along with cargo), and thereby allow multiplex engineering of an immune cell (e.g., an NK cell).

A transposon system comprises (i) a plasmid backbone with inverted terminal repeats (ITRs) and (ii) a transposase enzyme that recognizes the ITRs.

ITRs as described herein may contain one or more direct repeat (DR) sequences. These DR sequences usually are embedded in the ITRs. The compositions and methods of the present disclosure comprise, in various embodiments, one or more artificially engineered transposons. An engineered transposon may comprise ITRs, a cargo nucleotide sequence, or a cargo cassette. A transposon-related “cargo cassette” as used herein refers to a nucleotide sequence comprising a left ITR at the 5’ end (also referred to as a 5’ ITR) and a right ITR at the 3’ end (also referred to as a 3’ ITR), and a nucleotide sequence positioned between the left and right ITRs. The nucleotide sequence flanked by the ITRs is a nucleotide sequence intended for integration into acceptor DNA. The cargo cassette can, in some embodiments, be comprised in a vector, such as plasmid. A “cargo nucleotide sequence” refers to a nucleotide sequence (e.g., a nucleotide sequence intended for integration into acceptor DNA), flanked by an ITR at each end, wherein the ITRs are heterologous to the nucleotide sequence. A cargo cassette can be artificially engineered.

Transposons and Transposase Binding Sites

Exemplary transposon systems for use as described in the disclosure include, but are not limited to, piggyBac, hyperactive piggyBac, Sleeping Beauty (SB), hyperactive Sleeping Beauty (SBIOOx), SB 11, SB 110, Tn7, TcBuster, hyperactive TcBuster, Frog Prince, IS5, Tn3, Tn5, Tn7, TnlO, Tn903, SPIN, hAT, Hermes, Hobo, AeBusterl, AeBuster2, AeBuster3, BtBusterl , BtBuster2, CfBusterl , CfBuster2, Tol2, mini-Tol2, Tc3, Mosl, MuA, Himar I, Helitron and engineered versions of transposase family enzymes (Zhang et al., PLoS Genet. 5:el000689, 2009; Wilson et al., J. Microbiol. Methods 71:332-5, 2007; each of which is incorporated in its entirety herein by reference). Exemplary transposons also include the transposons described in Arensburger et al. (Genetics 188(l):45-57, 2011; incorporated in its entirety herein by reference), or a SPACE INVADERS (SPIN) transposon (see, e.g., Pace et al., Proc. Natl. Acad. Sci. U.S.A. 105(44): 17023-17028, 2008; each of which is incorporated in its entirety herein by reference).

In some embodiments, the gene transfer system can be delivered to the cell encoded in DNA, encoded in mRNA, as a protein, or as a nucleoprotein complex. Alternatively, the gene transfer system can be integrated into the genome of a host cell using, for example, a retro-transposon, random plasmid integration, recombinase-mediated integration, homologous recombination mediated integration, or non-homologous end joining mediated integration. More examples of transposition systems that can be used with certain embodiments of the compositions and methods provided herein include Staphylococcus aureus Tn552 (Colegio et al., J. Bacterial. 183:2384-8, 2001; Kirby et al., Mol. Microbiol. 43:173-86, 2002), Tyl (Devine & Boeke, Nucleic Acids Res. 22:3765-72, 1994 and International Publication WO 95/23875), Transposon Tn7 (Craig, Science 271:1512, 1996; Craig, Review in: Curr. Top. Microbiol. Immunol. 204:27-48, 1996), Tn/O and IS 10 (Kleckner et al. , Curr. Top. Microbiol. Immunol. 204:49-82, 1996), Mariner transposase (Lampe et al., EMBO J. 15:5470-9, 1996), Tel (Plasterk, Curr. Topics Microbiol. Immunol. 204:125-43, 1996), P Element (Gloor, Methods Mol. Biol. 260:97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J. Biol. Chem. 265:18829-32, 1990), bacterial insertion sequences (Ohtsubo & Sekine, Curr. Top. Microbiol. Immunol. 204:1-26, 1996), retroviruses (Brown et al., Proc. Natl. Acad. Sci. U.S.A. 86:2525-9, 1989), and retrotransposon of yeast (Boeke & Corces, Ann. Rev. Microbiol. 43:403-34, 1989). The entire contents of each of the foregoing references are incorporated by reference herein.

Any suitable transposase and transposon binding site may be used as provided herein. The transposase may be from a virus (e.g., a phage or a retrovirus), a prokaryote, or a eukaryote (e.g., a fungus (e.g., yeast) or a mammal). Exemplary transposases that may be used include, but are not limited to, a TcBuster transposase, a piggyBac transposase, a Sleeping Beauty transposase, a Tn3 transposase, a Tn5 transposase, a Tn7 transposase, a TnlO transposase, a MuA transposase, a Frog Prince transposase, an IS5 transposase, a TnlO transposase, a Tn903 transposase, a SPIN transposase, a hAT transposase, a Hermes transposase, a Hobo transposase, an AeBuster transposase, a BtBuster transposase, a CfBuster transposase, a Tol2 transposase, a Tc3 transposase, a Mosl transposase, a Himar I transposase, and a Helitron transposase, or biologically active variants thereof. In some embodiments, the biologically active variant of a transposase may be naturally-occurring or engineered, and may include one or more modifications relative to a reference transposase (e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more) amino acid substitutions, insertions, and/or deletions), which may affect the activity (e.g., transposition activity), binding (e.g., binding specificity or affinity), or other properties of the transposase. In some embodiments, the biologically active variant of a transposase may be a hyperactive variant, which may have increased transposition activity (e.g., in vitro or in vivo.

Suitable transposase binding sites that may be selectively targeted by a transposase can be used in the compositions of the disclosure (e.g., a nucleic acid molecule provided herein). The transposase binding sites may be a naturally occurring transposase binding sites (e.g., derived from a naturally occurring transposon) or a biologically active variant thereof. Biologically active variants of a transposase binding site may be naturally occurring or engineered, and may include insertions, deletions, and/or substitutions relative to a reference transposase binding site. The biologically active variant transposase binding site may affect the activity (e.g., transposition activity), binding (e.g., binding specificity or affinity), or other properties of the transposase(s) that bind to the transposase binding site. In some embodiments, the transposase binding site may be or include a sequence that does not exist in nature but still permits transposition by a transposase. In some embodiments, the transposase binding site includes an inverted terminal repeat (ITR) (e.g., two ITRs). In other embodiments, the transposase binding site may lack an ITR. Exemplary transposases and transposases that may be used as provided herein are further described below.

TcBuster™ Transposases

In some embodiments, the transposon system of the disclosure is a TcBuster family transposon system. Exemplary TcBuster family transposons of the disclosure include, but are not limited to, the following transposons (wherein the corresponding accession numbers for the appropriate database are shown in parenthesis): (GENBANK database, sequences available on the World Wide Web at ncbi.nlm.nih.gov): Ac-like (AAC46515), Ac (CAA29005), AeBusterl (ABF20543), AeBuster2 (ABF20544), AmBusterl (EFB22616), AmBuster2 (EFB25016), AmBuster3 (EFB20710), AmBuster4 (EFB22020), BtBusterl (ABF22695), BtBuster2 (ABF22700), BtBuster3 (ABF22697), CfBusterl (ABF22696), CfBuster2 (ABF22701), CfBuster3 (XP_854762), CfBuster4 (XP_545451), CsBuster (ABF20548), Daysleeper (CAB68118), DrBusterl (ABF20549), DrBuster2 (ABF20550), EcBusterl (XP_001504971), EcBuster3 (XP_001503499), EcBuster4 (XP_001504928), Hermes (AAC37217), hermit (LCU22467), Herves (AAS21248), hobo (A39652), Homer (AAD03082), hopper- we (AAL93203), HsBusterl (AAF18454), HsBuster2 (ABF22698), HsBuster3 (NP_071373), HsBuster4 (AAS01734), IpTiplOO (BAA36225), MamBuster2 (XP_001108973), MamBuster3 (XP_001084430), MamBuster3 (XP_001084430), MamBuster4 (XP_001101327), MmBuster2 (AAF18453), PtBuster2 (ABF22699), PtBuster3 (XP_001142453), PtBuster4 (XP_527300), Restless (CAA93759), RnBuster2 (NP_001102151), SpBusterl (ABF20546), SpBuster2 (ABF20547), SsBuster4 (XP_001929194), Tam3 (CAA38906), TcBuster (ABF20545), Tol2 (BAA87039), tramp (CAA76545), and XtBuster (ABF20551); (ENSEMBL database, sequences available on the World Wide Web at ensembl.org): PtBusterl (ENSPTRG00000003364): (REPBASE database, sequences available on the World Wide Web at girinst.org): Ac-like2 (hAT-7_DR), Ac-likel (hAT-6_DR), hAT-5_DR (hAT-5_DR), MIBusterl (hAT-4_ML), Myotis-hATl (Myotis-hATl), SPIN_Et (SPIN_Et), SPIN_M1 (SPIN_M1), and SPIN-Og (SPIN-Og), (TEFam database, sequences available on the World Wide Web at tefam.biochem.vt.edu): AeHermesl (TF0013337), AeBuster3 (TF001186), AeBuster4 (TF001187), AeBuster5 (TFOO1188), AeBuster7 (TF001336), AeHermes2 (TFOO13338), AeTiplOO-2 (TF000910), Cx-Kink2 (TF001637), Cx-Kink3 (TF001638), Cx-Kink4 (TF001639), Cx-Kink5 (TF001640), Cx-Kink7 (TF001636), and Cx-Kink8 (TF001635).

Compositions and methods of the disclosure may comprise a TcBuster transposase and/or a TcBuster hyperactive transposase. In some embodiments, compositions and methods of the disclosure comprise a TcBuster transposase, a TcBuster transposon, or a TcBuster transposase and TcBuster transposon. In some embodiments, compositions and methods of the disclosure comprise a hyperactive TcBuster transposase, a TcBuster transposon, or a hyperactive TcBuster transposase and TcBuster transposon. In some embodiments, a hyperactive TcBuster transposase demonstrates an increased excision and/or increased insertion frequency when compared to an excision and/or insertion frequency of a wild type TcBuster transposase.

In some embodiments, a hyperactive TcBuster transposase demonstrates an increased transposition frequency when compared to a transposition frequency of a wild type TcBuster transposase. In some embodiments, a TcBuster transposase may comprise any of the mutations disclosed in WO 2019/246486, which is incorporated herein by reference in its entirety. In some embodiments, the transposase is a TcBuster™ transposase (see, e.g.,

GenBank Accession No. ABF20545) or a biologically active variant thereof. The amino acid sequence of an exemplaryTcBuster transposase is provided below:

In some embodiments, a TcBuster transposase comprises or consists of an amino acid sequence having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, or any percentage identity in between the foregoing values, or 100% identity, to a TcBuster transposase comprising or consisting of the amino acid sequence of SEQ ID NO: 146.

In some embodiments, a TcBuster Transposase is encoded by a nucleic acid sequence comprising or consisting of a sequence having at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, or any percentage identity in between the foregoing values, or 100% identity, to a wild type TcBuster transposase encoded by a nucleic acid sequence comprising or consisting of GENBANK Accession No. DQ481197, as provided below:

In some embodiments, an immune cell, e.g., NK cell produced by transposition-based methods may comprise sequences flanking the nucleotide sequence incorporated into the immune cell’s genome by transposition. Illustrative examples of such flanking sequences (also known as excision footprints) are provided in Woodard et al., (2012) PLoS ONE 7(11): e42666; incorporated in its entirety herein by reference.

In some embodiments, the transposase is a mutant TcBuster transposase. Typically, a wild-type TcBuster transposase can be regarded as comprising, from N terminus to C terminus, a ZnF-BED domain (amino acids 76-98), a DNA Binding and Oligomerization domain (amino acids 112-213), a first Catalytic domain (amino acids 213-312), an Insertion domain (amino acids 312-543), and a second Catalytic domain (amino acids 583-620), as well as at least four inter-domain regions in between these annotated domains. Unless indicated otherwise, numerical references to amino acids of a TcBuster transposase, as used herein, are all in accordance to SEQ ID NO: 146. A mutant TcBuster transposase of the disclosure comprises one or more amino acid substitutions in any one of these domains, or any combination thereof. In some embodiments, a mutant TcBuster transposase comprises one or more amino acid substitutions in a ZnF- BED domain, a DNA Binding and Oligomerization domain, a first Catalytic domain, an insertion domain, or a combination thereof. In some embodiments, a mutant TcBuster transposase comprises one or more amino acid substitutions in at least one of the two catalytic domains.

In some embodiments, a mutant TcBuster transposase comprises one or more amino acid substitutions in comparison to a wild-type TcBuster transposase (SEQ ID NO: 146). In some embodiments, the mutant TcBuster transposase comprises an amino acid sequence having at least 70% sequence identity to the full-length sequence of a wild-type TcBuster transposase (SEQ ID NO: 146). In some embodiments, the mutant TcBuster transposase comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to full length sequence of a wild-type TcBuster transposase (SEQ ID NO: 146). In some embodiments, the mutant TcBuster transposase comprises an amino acid sequence having at least 98%, at least 98.5%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to full length sequence of a wild-type TcBuster transposase (SEQ ID NO: 146). In some embodiments, the mutant TcBuster transposase comprises an amino acid sequence having at least one amino acid that is different from the full-length sequence of a wild-type TcBuster transposase (SEQ ID NO: 146). In some embodiments, the mutant TcBuster transposase comprises an amino acid sequence having at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 or more amino acids that are different from the full-length sequence of a wild-type TcBuster transposase (SEQ ID NO: 146). In some embodiments, the mutant TcBuster transposase comprises an amino acid sequence having at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, or at least 250 amino acids that are different from the full-length sequence of a wild-type TcBuster transposase (SEQ ID NO: 146). In some embodiments, the mutant TcBuster transposase comprises an amino acid sequence having at most 3, at most 6, at most 12, at most 25, at most 35, at most 45, at most 55, at most 65, at most 75, at most 85, at most 95, at most 150, or at most 250 amino acids that are different from the full-length sequence of a wild-type TcBuster transposase (SEQ ID NO: 146).

Biologically active variants of a TcBuster transposases, including hyperactive TcBuster™ transposases, are known in the art and may be used as described herein (see, e.g., US Pat. No. 10,227,574 B2, which is incorporated in its entirety herein by reference). For example, in some embodiments, a biologically active variant of a TcBuster™ transposase includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more) of the following amino acid substitutions: Q82E, N85S, D99A, D132A, Q151S, Q151A, E153K, E153R, A154P, Y155H, E159A, T171K, T171R, K177E, D183K, D183R, D189A, E263A, E263K, E263R, E274K, E274R, S277K, N281E, L282K, L282R, K292P, V297K, K299S, A3O3T, H322E, A332S, A358E, A358K, A358S, D376A, V377T, L38ON, I398D, I398S, I398K, F400L, V431E, S447E, N450K, N450R, I452F, E469K, K469K, P510D, P510N, E519R, R536S, V553S, P554T, P559D, P559S, P559K, K573E, E578E, K590T, Y595E, V596A, T598I, K599A, Q615A, T618K, T618R, D622K, D622R, E274K, V549P, R574K, E570V, G558T, P554T, D555M, G556P, E539F, E538Q, E534A, I532E, E564C, T554N, D555S, T556D, T557A, K635P, D607I, Y595A, S591I, V583P, E578E, K573R, T544N, D545S, T546D, T547A, Y59F, G75P, E76Q, S87E, 14624D, D132K, D133E, C172V, D189N, T190N, T190D, Y201D, V206Q, N209E, T219S, A229S, A229D, I233Q, F237Y, M250F, A255P, P257E, E268T, K275E, S277G, S277K, Y284I, H285G, K292N, C318I, H322Q, and H322A (relative to the amino acid sequence of SEQ ID NO: 146).

In some embodiments, a biologically active variant of a TcBuster™ transposase includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more) of the following amino acid substitutions or combinations of amino acid substitutions selected from: V377T/E469K; V377T/E469K/R536S; A332S; V553S/P554T; E519R; K299S; Q615A/T618K; S277K; A3O3T; P510D; P510N; N281S; N281E; K590T; E274K; Q258T; E247K; S447E; N85S; V297K; A358K; I452F; V377T/E469K/D189A; K573E/E578L; I452F/V377T/E469K/D 189A; A358K/V377T/E469K/D 189A; K573E/E578L/V377T/E469K/D189A; T171R; D183R; S193R; P257K; E263R; L282K; T618K; D622R; E153K; N450K; T171K; D183K; S193K; P257R; E263K; L282R; T618R; D622K; E153R; N450R; and E247K/E274K/V297K/A358K (relative to the amino acid sequence of SEQ ID NO: 146). In some embodiments, a biologically active variant of a TcBuster™ transposase includes the following amino acid substitutions: D189A, V377T, and E469K (relative to the amino acid sequence of SEQ ID NO: 146). In some embodiments, a biologically active variant of a TcBuster™ transposase includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more) of the following amino acid substitutions or combinations of amino acid substitutions selected from: V377T/E469K;

V377T/E469K/R536S; A332S; V553S/P554T; E519R; K299S; Q615A/T618K; S277K; A3O3T; P510D; P510N; N281S; N281E; K590T; E274K; Q258T; E247K; S447E; N85S; V297K; A358K; I452F; V377T/E469K/D189A; and K573E/E578L (relative to the amino acid sequence of SEQ ID NO: 146).

In some embodiments, the TcBuster™ transposase binds to a transposase binding site including a nucleic acid sequence selected from: 5’- cagtgttcttcaac-3’ (SEQ ID NO: 148), 5’- cagtgttcttcaacctttgccatccggcggaaccctttgtcgagatatttttttttatggaacccttcatttagtaatacacccagatgagatttt agggacagctgcgttgacttgttacgaacaaggtgagcccgtgctttggtaataaaaactctaaataagatttaaatttgcatttatttaaac aaactttaaacaaaaagataaatattccaaataaaataatatataaaataaaaaataaaaatta-3’ (SEQ ID NO: 149), 5’- gttgaagaacactg-3’ (SEQ ID NO: 150), and 5’- atttctgaacgattctaggttaggatcaaacaaaatacaatttattttaaaactgtaagttaacttacctttgcttgtctaaacctaaaacaaca acaaaactacgaccacaagtacagttacatatttttgaaaattaaggttaagtgcagtgtaagtcaactatgcgaatggataacatgtttca acatgaaactccgattgacgcatgtgcattctgaagagcggcgcggccgacgtctctcgaattgaagcaatgactcgcggaaccccg aaagcctttgggtggaaccctagggttccgcggaacacaggttgaagaacactg-3’ (SEQ ID NO: 151), or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes at least one (e.g., 1, 2, or 3) transposase binding sites including an ITR, wherein the ITR comprises the nucleic acid sequence of any one of SEQ ID NO: 148-150, or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes: a 5’ transposase binding site including a nucleic acid sequence of SEQ ID NO: 148 or SEQ ID NO: 149, or a biologically active variant thereof, and/or a 3’ transposase binding site including a nucleic acid sequence of SEQ ID NO: 150 or SEQ ID NO: 151, or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes: a 5’ transposase binding site including an ITR, wherein the ITR includes a nucleic acid sequence of SEQ ID NO: 148, or a biologically active variant thereof, and/or a 3’ transposase binding site including an ITR, wherein the ITR includes a nucleic acid sequence of SEQ ID NO: 150, or a biologically active variant thereof. piggyBac™ transposase

In some embodiments, the transposase is a piggyBac™ transposase or a biologically active variant thereof. PiggyBac™ transposase have been identified in several species, including Trichoplusia ni (see, e.g., UniProt Accession No. Q27026). The amino acid sequence of a wild-type piggyBac™ transposase of Trichoplusia ni is provided below:

Biologically active variants of piggyBac™ transposases are known in the art and may be used as described herein (see, e.g., Yusa et al., Proc. Natl. Acad. Sci. USA 108(4): 1531-6, 2011, which is incorporated herein by reference).

In some embodiments, the piggyBac transposase binds to a transposase binding site having an ITR comprising a nucleic acid sequence selected from: 5’- ccctagaaagatagtctgcgtaaaattgacgcatgcattcttgaaatattgctctctctttctaaatagcgcgaatccgtcgctgtgcattta ggacatctcagtcgccgcttggagctcccgtgaggcgtgcttgtcaatgcggtaagtgtcactgattttgaactataacgaccgcgtga gtcaaaatgacgcatgattatcttttacgtgacttttaagatttaactcatacgataattatattgttatttcatgttctacttacgtgataacttatt atatatatattttcttgttatagatatc-3’ (SEQ ID NO: 160), 5’-ccctagaaagatagtctgcgtaaaattgacgcatg-3’ (SEQ ID NO: 161), 5’- tttgttactttatagaagaaattttgagtttttgtttttttttaataaataaataaacataaataaattgtttgttgaatttattattagtatgtaagtgta aatataataaaacttaatatctattcaaattaataaataaacctcgatatacagaccgataaaacacatgcgtcaattttacgcatgattatctt taacgtacgtcacaatatgattatctttctaggg-3’ (SEQ ID NO: 162), 5’- catgcgtcaattttacgcatgattatctttaacgtacgtcacaatatgattatctttctaggg-3’ (SEQ ID NO: 163) or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes at least one (e.g., 1, 2, or 3) transposase binding site including an ITR, wherein the ITR comprises the nucleic acid sequence of any one of SEQ ID NOs: 160-163, or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes: a 5’ transposase binding site including at least one ITR, wherein the ITR comprises the nucleic acid sequence of SEQ ID NO: 160 or SEQ ID NO: 161, or a biologically active variant thereof, and/or a 3’ transposase binding site including at least one ITR, wherein the ITR comprises the nucleic acid sequence of SEQ ID NO: 162 or SEQ ID NO: 163, or a biologically active variant thereof. Sleeping, Beauty transposases

In some embodiments, the transposase is a Sleeping Beauty transposase (e.g., SB 10) or a biologically active variant thereof. Several Sleeping Beauty transposases have been described to date. The amino acid sequence of a reference Sleeping Beauty transposase is provided below:

Q Q ( Q )

Biologically active variants of a Sleeping Beauty transposases are known in the art, including SB 11, SB 12, HSB1, HSB2, HSB3, HSB4, HSB5, HSB13, HSB14, HSB15, HSB16, HSB17, SB100X, and SB150X, and may be used as described herein (see, e.g., Geurts et al. Mol Ther. 8: 108-17, 2003; Zayed H et al. Mol. Ther. 9: 292-304, 2004; Yant et al. Mol. Cell Biol. 24: 9239-47, 2004; Baus et al. Mol Ther. 12:1148-56, 2005; Mates et al. Nat. Genet. 41: 753-61, 2009; and Voigt et al. Mol. Ther. 20: 1852-62, 2012, each of which are incorporated herein by reference. For example, in some embodiments, a biologically active variant of a Sleeping Beauty transposase includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18) of the following amino acid substitutions or combinations of amino acid substitutions: K13A; K14R; K13D; K30R; K33A; T83A; 1100L; R115H; R143L; R147E; A205K/H207V/K208R/D210E; H207V/K208R/D210E; R214D/K215A/E216V/N217Q; M243H; M243Q; E267D; T314N; and G317E (e.g., relative to the amino acid sequence of SEQ ID NO: 164)

In some embodiments, the piggyBac transposase binds to a transposase binding site having an ITR comprising a nucleic acid sequence selected from: 5’- cagttgaagtcggaagtttacatacacttaagttggagtcattaaaactcgtttttcaactactccacaaatttcttgttaacaaacaatagttt tggcaagtcagttaggacatctactttgtgcatgacacaagtcatttttccaacaattgtttacagacagattatttcacttataattcactgtat cacaattccagtgggtcagaagtttacatacactaagt-3’ (SEQ ID NO: 165), 5’- attgagtgtatgtaaacttctgacccactgggaatgtgatgaaagaaataaaagctgaaatgaatcattctctctactattattctgatatttc acattcttaaaataaagtggtgatcctaactgacctaagacagggaatttttactaggattaaatgtcaggaattgtgaaaaagtgagttta aatgtatttggctaaggtgtatgtaaacttccgacttcaactg-3’ (SEQ ID NO: 166), or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes at least one (e.g., 1, 2, or 3) transposase binding sites including an ITR, wherein the ITR comprises the nucleic acid sequence of any one of SEQ ID NO: 165 or SEQ ID NO: 166, or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes: a 5’ transposase binding site including at least one ITR, wherein the ITR comprises the nucleic acid sequence of SEQ ID NO: 165, or a biologically active variant thereof, and/or a 3’ transposase binding site including at least one ITR, wherein the ITR comprises the nucleic acid sequence of SEQ ID NO: 166, or a biologically active variant thereof.

Tn3 transposase

In some embodiments, the transposase is a Tn3 transposase (see, e.g., UniProt Accession No. PO3OO8) or a biologically active variant thereof. The amino acid sequence of a wild-type Tn3 transposase is provided below: MLKKPSGREADMPVDFLTTEQVESYGRFTGEPDELQLARYFHLDEADKEFIGKSRGDHNRLG IALQIGCVRFLGTFLTDMNHIPSGVRHFTARQLGIRDITVLAEYGQRENTRREHAALIRQHY QYREFAWPWTFRLTRLLYTRSWISNERPGLLFDLATGWLMQHRI ILPGATTLTRLISEVREK ATLRLWNKLALIPSAEQRSQLEMLLGPTDCSRLSLLESLKKGPVTISGPAFNEAIERWKTLN DFGLHAENLSTLPAVRLKNLARYAGMTSVFNIARMSPQKRMAVLVAFVLAWETLALDDALEV LDAMLAVI IRDARKIGQKKRLRSLKDLDKSALALASACSYLLKEETPDESIRAEVFSYIPRQ KLAEI ITLVREIARPSDDNFHDEMVEQYGRVRRFLPHLLNTVKFSSAPAGVTTLNACDYLSR EFSSRRQFFDDAPTEI ISQSWKRLVINKEKHITRRGYTLCFLSKLQDSLRRRDVYVTGSNRW GDPRARLLQGADWQANRIKVYRSLGHPTDPQEAIKSLGHQLDSRYRQVAARLGENEAVELDV SGPKPRLTISPLASLDEPDSLKRLSKMISDLLPPVDLTELLLEINAHTGFADEFFHASEASA RVDDLPVSISAVLMAEACNIGLEPLIRSNVPALTRHRLNWTKANYLRAETITSANARLVDFQ ATLPLAQIWGGGEVASADGMRFVTPVRTINAGPNRKYFGNNRGITWYNFVSDQYSGFHGFHG IVIPGTLRDSIFVLEGLLEQETGLNPTEIMTDTAGASDLVFGLFWLLGYQFSPRLADAGASV FWRMDHDADYGVLNDIARGQSDPRKIVLQWDEMIRTAGSLKLGKVQASVLVRSLLKSERPSG LTQAI IEVGRINKTLYLLNYIDDEDYRRRILTQLNRGESRHAVARAICHGQKGEIRKRYTDG QEDQLGALGLVTNAWLWNTI YMQAALDHLRAQGETLNDEDIARLSPLCHGHINMLGHYSFT LAELVTKGHLRPLKEASEAENVA (SEQ ID NO: 167)

In some embodiments, the Tn3 transposase binds to a transposase binding site having an ITR comprising a nucleic acid sequence selected from: 5’- GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG-3’ (SEQ ID NO: 168) and 5’-CTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC-3’ (SEQ ID NO: 169), or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes at least one (e.g., 1, 2, or 3) transposase binding sites including an ITR, wherein the ITR comprises the nucleic acid sequence of SEQ ID NO: 168 or SEQ ID NO: 169, or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes: a 5’ transposase binding site including at least one ITR, wherein the ITR includes the nucleic acid sequence of SEQ ID NO: 168 or a biologically active variant thereof, and/or a 3’ transposase binding site including at least one ITR, wherein the ITR includes the nucleic acid sequence of SEQ ID NO: 169 or a biologically active variant thereof.

Tn5 transposase

In some embodiments, the transposase is Tn5 (also referred to as TnpA; see, e.g., UniProt Accession No. Q46731) or a biologically active variant thereof. The amino acid sequence of a wild-type Tn5 transposase is provided below: MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSEAMQEGAYR FIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAEELGKLGSIQDKSRGW WVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLRMGSMMSNVIAV CDREADIHAYLQDKLAHNERFWRSKHPRKDVESGLYLYDHLKNQPELGGYQISIPQKGWD KRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLA QALRVIDI YTHRWRIEEFHKAWKTGAGAERQRMEEPDNLERMVSILSFVAVRLLQLRESFTL PQALRAQGLLKEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGG FMDSKRTGIASWGALW EGWEALQSKLDGFLAAKDLMAQGIKI (SEQ ID NO: 170)

Biologically active variants of Tn5 are known in the art, including hyperactive variants of Tn5 (see, e.g., U.S. Pat. Nos. 5,965,443; 5,925,545; and 6,159,736, each of which is incorporated herein by reference). For example, in some embodiments, a biologically active variant of Tn5 transposase includes one or more (e.g., 1, 2 or 3) of the following amino acid substitutions: E54K, L372P, and M56A (e.g., relative to the amino acid sequence of SEQ ID NO: 170).

In some embodiments, the Tn5 transposase binds to a transposase binding site including at least one ITR (e.g., two ITRs) including a nucleic acid sequence selected from: 5’-CTGACTCTTATACACAAGT-3’ (SEQ ID NO: 171), 5’-CTGTCTCTTGATCAGATCT- 3’ (SEQ ID NO: 172), 5’-CTGTCTCTTATACACATCT-3’ (SEQ ID NO: 173), and 5’- CTGTCTCTTATACAGATCT-3’ (SEQ ID NO: 174), or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes a transposase binding site including at least one ITR including a nucleic acid sequence of any one of SEQ ID NOs: 171-174, or a biologically active variant thereof.

Tn7 transposase

In some embodiments, the transposase is a Tn7 transposase. In some embodiments, the Tn7 transposon comprises TnsA (Uniprot Accession No. P13988) and/or TnsB (Uniprot Accession No. P13989), or biologically active variants thereof. TnsA and TnsB are believed to form a heteromeric transposase. In some embodiments, TnsA and TnsB are used in combination with one or more accessory proteins selected from TnsC, TnsD, and TnsE. The amino acid sequence of a wild-type TnsA is provided below:

MAKANSSFSEVQIARRIKEGRGQGHGKDYIPWLTVQEVPSSGRSHRI YSHKTGRVHHLLSDL ELAVFLSLEWESSVLDIREQFPLLPSDTRQIAIDSGIKHPVIRGVDQVMSTDFLVDCKDGPF EQFAIQVKPAAALQDERTLEKLELERRYWQQKQIPWFIFTDKEINPWKENIEWLYSVKTEE VSAELLAQLSPLAHILQEKGDENI INVCKQVDIAYDLELGKTLSEIRALTANGFIKFNI YKS FR ANKCADLCISQWNMEELRYVAN (SEQ ID NO: 175)

The amino acid sequence of wild-type TnsB is provided below:

MWQINEWLFDNDPYRILAIEDGQWWMQISADKGVPQARAELLLMQYLDEGRLVRTDDPYV HLDLEEPSVDSVSFQKREEDYRKILPI INSKDRFDPKVRSELVEHWQEHKVTKATVYKLLR RYWQRGQTPNALIPDYKNSGAPGERRSATGTAKIGRAREYGKGEGTKVTPEIERLFRLTIEK HLLNQKGTKTTVAYRRFVDLFAQYFPRIPQEDYPTLRQFRYFYDREYPKAQRLKSRVKAGVY KKDVRPLSSTATSQALGPGSRYEIDATIADI YLVDHHDRQKI IGRPTLYIVIDVFSRMITGF YI GFENP S YWAMQAFVNACSDKTAI CAQHD I E I S S SDWP CVGLPDVLLADRGELMSHQVEA LVSSFNVRVESAPPRRGDAKGIVESTFRTLQAEFKSFAPGIVEGSRIKSHGETDYRLDASLS VFEFTQI ILRTILFRNNHLVMDKYDRDADFPTDLPSIPVQLWQWGMQHRTGSLRAVEQEQLR VALLPRRKVSISSFGVNLWGLYYSGSEILREGWLQRSTDIARPQHLEAAYDPVLVDTIYLFP QVGSRVFWRCNLTERSRQFKGLSFWEVWDIQAQEKHNKANAKQDELTKRRELEAFIQQTIQK ANKLTPSTTEPKSTRIKQIKTNKKEAVTSERKKRAEHLKPSSSGDEAKVIPFNAVEADDQED YSLPTYVPELFQDPPEKDES (SEQ ID NO: 176)

Biologically active variants of Tn7 transposase are known in the art (see, e.g., Lu and Craig EMBO J. 19(13): 3446-57, 2000, incorporated herein by reference) and may be used as provided herein. For example, in some embodiments, a biologically active variant of TnsA includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) of the following amino acid substitutions: S69N, E73K, A65V, E185K, Q261Z, G239S, G239D, E185K, and Q261Z

(e.g., relative to the amino acid sequence of SEQ ID NO: 175). In some embodiments, a biologically active variant of TnsB includes one or more (e.g., 1, 2, or 3) of the following amino acid substitutions: M3661, A325T, and A325V (e.g., relative to the amino acid sequence of SEQ ID NO: 176).

In some embodiments, the Tn7 transposase (e.g., TnsA or TnsB) binds to a transposase binding site having a nucleic acid sequence comprising a consensus sequence of TGAYAATAAAGTTGATTATACT, wherein Y denotes a pyrimidine (C or T) (SEQ ID NO: 177), or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) transposase binding sites, each including the consensus sequence of SEQ ID NO: 177, or a biologically active variant thereof. TnlO transposase

In some embodiments, the transposase is a TnlO transposase (see, e.g., UniProt Accession No. Q70BL4) or a biologically active variant thereof. The amino acid sequence of a wild-type TnlO transposase is provided below: MCELDILHDSLYQFCPELHLKRLNSLTLACHALLDCKTLTLTELGRNLPTKARTKHNIKRID RLLGNRHLHKERLAVYRWHASFICSGNTMPIVLVDWSDIREQKRLMVLRASVALHGRSVTLY EKAFPLSEQCSKKAHDQFLADLASILPSNTTPLIVSDAGFKVPWYKSVEKLGWYWLSRVRGK VQYADLGAENWKP I SNLHDMSSSHSKTLGYKRLTKSNP I SCQI LLYKSRSKGRKNQRSTRTH CHHPSPKI YSASAKEPWVLATNLPVEIRTPKQLVNI YSKRMQIEETFRDLKSPAYGLGLRHS RTSSSERFDIMLLIALMLQLTCWLAGVHAQKQGWDKHFQANTVRNRNVLSTVRLGMEVLRHS GYTITREDLLVAATLLAQNLFTHGYALGKL (SEQ ID NO: 178)

Biologically active variants of TnlO transposase are known in the art (see, e.g., Way et al. Gene 32(3): 369-79, 1984, incorporated herein by reference) and may be used as provided herein.

In some embodiments, the TnlO transposase binds to a transposase binding site including at least one ITR. In some embodiments, the TnlO transposase binds to a transposase binding site having an ITR including a nucleic acid sequence comprising a consensus sequence of CTGAKRRATCCCCTMATRATTTY, wherein Y denotes a pyrimidine (C or T) (SEQ ID NO: 179), R denotes a purine (G or A), M denotes A or G, and K denotes G or T; or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) transposase binding site including a consensus sequence of SEQ ID NO: 179, or a biologically active variant thereof.

MuA transposase

In some embodiments, the transposase is a MuA transposase (see, e.g., UniProt

Accession No. P07636) or a biologically active variant thereof. The amino acid sequence of a wild-type MuA transposase is provided below:

MELWVSPKECANLPGLPKTSAGVI YVAKKQGWQNRTRAGVKGGKAIEYNANSLPVEAKAALL LRQGEIETSLGYFEIARPTLEAHDYDREALWSKWDNASDSQRRLAEKWLPAVQAADEMLNQG ISTKTAFATVAGHYQVSASTLRDKYYQVQKFAKPDWAAALVDGRGASRRNVHKSEFDEDAWQ FLIADYLRPEKPAFRKCYERLELAAREHGWSIPSRATAFRRIQQLDEAMWACREGEHALMH LIPAQQRTVEHLDAMQWINGDGYLHNVFVRWFNGDVIRPKTWFWQDVKTRKILGWRCDVSEN IDSIRLSFMDWTRYGIPEDFHITIDNTRGAANKWLTGGAPNRYRFKVKEDDPKGLFLLMGA KMHWTSWAGKGWGQAKPVERAFGVGGLEEYVDKHPALAGAYTGPNPQAKPDNYGDRAVDAE LFLKTLAEGVAMFNARTGRETEMCGGKLSFDDVFEREYARTIVRKPTEEQKRMLLLPAEAVN VSRKGEFTLKVGGSLKGAKNVYYNMALMNAGVKKVWRFDPQQLHSTVYCYTLDGRFICEAE CLAPVAFNDAAAGREYRRRQKQLKSATKAAIKAQKQMDALEVAELLPQIAEPAAPESRIVGI

FRP SGNTERVKNQERDDEYETERDEYLNHSLD I LEQNRRKKAI (SEQ ID NO: 180)

Biologically active variants of MuA transposases are known in the art, including truncated variants (e.g., MUA77-663) and hyperactive mutants are known in the art (see, e.g., U.S. Pat. No. 9,234,190, incorporated herein by reference). For example, in some embodiments, a biologically active variant of a MuA transposase includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26, or 27) of the following amino acid substitutions: A59V, D97G, W160R, E179V, E233K, E233V, Q254R, E258G, G302D, I335T, G340S, W345C, W345R, M374V, F447S, F464Y, R478H, R478C, E482K, E483G, E483V, M4871, V495A, V507A, Q539H, Q539R, and I617T (e.g., relative to the exemplary wild-type Tn5 transposase sequence of SEQ ID NO: 180). In some embodiment, a biologically active variant of a MuA transposase includes the amino acid substitution E223V (e.g., relative to the exemplary wild-type Tn5 transposase sequence of SEQ ID NO: 180). In some embodiments, a biologically active variant of MuA transposase includes the amino acid substitutions W160R, E233K, and W345R (e.g., relative to the amino acid sequence of SEQ ID NO: 180).

In some embodiments, the MuA transposase binds to a transposase binding site including at least one (e.g., 1, 2, or 3) Mu binding site selected from LI (TGTATTGATTCACTGAAGTACGAAAA (SEQ ID NO: 181)), L2 (CCTTAATCAATGAAACGCGAAAG, SEQ ID NO: 182), L3 (TTGTTTCATTGAAAATACGAAAA, SEQ ID NO: 183), or a biologically active variant thereof. In some embodiments, the MuA transposase binds to a transposase binding site including at least one (e.g., 1, 2, or 3) Mu binding site selected from R1 (TGAAGCGGCGCACGAAAAATGCGAAAA, SEQ ID NO: 184), R2 (GCGTTTCACGATAAATGCGAAAA, SEQ ID NO: 185), and R3 (CCGTTTCATTTGAAGCGCGAAAA, SEQ ID NO: 186), or a biologically active variant of any of the foregoing. In some embodiments, a nucleic acid molecule provided herein includes a transposase binding site including at least one Mu binding site including a nucleic acid sequence of any one of SEQ ID NOs: 181-186, or a biologically active variant thereof. In some embodiments, a nucleic acid molecule provided herein includes a 5’ transposase binding site including at least one (e.g., 1, 2 or 3) Mu binding site selected from LI (SEQ ID NO: 181), L2 (SEQ ID NO: 182) and L3 (SEQ ID NO: 183). In some embodiments, a nucleic acid molecule provided herein includes a 3’ transposase binding site including at least one (e.g., 1, 2 or 3) Mu binding site selected from R1 (SEQ ID NO: 184), R2 (SEQ ID NO: 185) and R3 (SEQ ID NO: 186).

Inverted Terminal Repeats (ITRs)

A transposon generally comprises two ITR nucleotide sequences. A transposon described herein may be engineered to comprise a cargo cassette comprising two ITR sequences. In some embodiments, at least one of the ITRs contains at least one direct repeat. In some embodiments, the transposase is one or more of the TcBuster transposases (e.g., mutant TcBuster transposases) disclosed herein, and the TcBuster transposase recognizes one or more ITRs of SEQ ID NOs: 148-151. The terms “left” and “right”, as used herein with respect to inverted terminal repeats, can refer to the 5’ and 3’ sides or ends of the cargo cassette on the sense strand of the double strand transposon, respectively. In some embodiments, a left or a 5’ inverted terminal repeat can comprise a nucleic acid sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 148. In some embodiments, a left or a 5’ inverted terminal repeat can comprise a nucleic acid sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 149. In other embodiments, a right or a 3’ inverted terminal repeat can comprise a sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 150. In some embodiments, a right or a 3’ inverted terminal repeat can comprise a sequence at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 151.

A transposon may comprise a cargo cassette comprising two inverted terminal repeats that have different nucleotide sequences, or a combination of the various sequences known to one skilled in the art. In some embodiments, at least one of the two inverted terminal repeats of a transposon provided herein may contain a nucleic acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 148-151.

The choice of inverted terminal repeat sequences may vary depending on the expected transposition efficiency, the type of cell to be modified, the transposase to use, and many other factors. In some embodiments, minimally sized transposon vector inverted terminal repeats that conserve genomic space may be used. The ITRs of hAT family transposons diverge greatly with differences in right-hand and left-hand ITRs. In some embodiments, smaller ITRs consisting of just 100-200 nucleotides are as active as the longer native ITRs in hAT transposon vectors. These sequences may be consistently reduced while mediating hAT family transposition. These shorter ITRs can conserve genomic space within hAT transposon vectors.

The inverted terminal repeats of a transposon provided herein can be about 50 to 2000 nucleotides, about 50 to 1000 nucleotides, about 50 to 800 nucleotides, about 50 to 600 nucleotides, about 50 to 500 nucleotides, about 50 to 400 nucleotides, about 50 to 350 nucleotides, about 50 to 300 nucleotides, about 50 to 250 nucleotides, about 50 to 200 nucleotides, about 50 to 180 nucleotides, about 50 to 160 nucleotides, about 50 to 140 nucleotides, about 50 to 120 nucleotides, about 50 to 110 nucleotides, about 50 to 100 nucleotides, about 50 to 90 nucleotides, about 50 to 80 nucleotides, about 50 to 70 nucleotides, about 50 to 60 nucleotides, about 75 to 750 nucleotides, about 75 to 450 nucleotides, about 75 to 325 nucleotides, about 75 to 250 nucleotides, about 75 to 150 nucleotides, about 75 to 95 nucleotides, about 100 to 500 nucleotides, about 100 to 400 nucleotides, about 100 to 350 nucleotides, about 100 to 300 nucleotides, about 100 to 250 nucleotides, about 100 to 220 nucleotides, or about 100 to 200 nucleotides in length, or any range having upper and lower values derived from any of the foregoing recited values, e.g., from about 50 to 75 nucleotides.

Transposon Cargo Nucleotide Sequences and Cargo Cassetes

In some embodiments, the disclosure provides a nucleic acid molecule comprising a cargo nucleotide sequence encoding a CAR described herein and optionally a cytokine and/or a DNR.

In some embodiments, the nucleic acid molecule is operably linked to a promoter capable of expressing an exogenous sequence in a human immune cell (e.g., NK cell). In some embodiments, the nucleic acid molecule is operably linked to at least one (e.g., one, two, three, or four) promoters. In some embodiments, the promoter is selected from the group consisting of an MND promoter, an EF-la promoter, an EFS promoter, a MSCV promoter, a CMV promoter, a PGK promoter, a CAG promoter, a SFFV promoter, a CBH promoter, a SV40 promoter, a UBC promoter, or a RPBSA promoter.

In some embodiments, the MND promoter is an MND5 promoter. In some embodiments, the MND promoter is an MND5 promoter comprising a nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence of: CCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCG

GGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGA

ATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAAC

AACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCG

GCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTG

AGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAA

GGATGCCCAGAAGG I ACCCCAT I G 1 Al GGGATCT GA1 CIGGGGCCTCGGT GC AC A TGC I T 1 A

CATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTC

CTTTGAAAAACACGATGATAATATGGCCACAACC(SEQ ID NO: 154)

The cargo nucleotide sequence may comprise any nucleotide sequence described herein, e.g., a nucleotide sequence intended for integration into acceptor DNA and/or a nucleotide sequence encoding for one or more polypeptides intended to be expressed or produced in an immune cell, e.g., an NK cell. In some embodiments, the cargo nucleotide sequence comprises a nucleotide sequence that encodes for a CAR, a cytokine, and/or a DNR described herein. The disclosure further provides a nucleic acid molecule comprising a cargo nucleotide sequence comprising any nucleotide sequence described herein, e.g., a nucleotide sequence intended for integration into acceptor DNA and/or a nucleotide sequence encoding for one or more polypeptides intended to be expressed or produced in immune cell, e.g., an NK cell.

In some embodiments, the disclosure provides a transposon comprising a nucleic acid molecule encoding a 5’ ITR, a promoter sequence, a Kozak sequence, a cargo nucleotide sequence described herein, a polyA sequence, and/or a 3’ ITR. In some embodiments, the 5’ ITR is present in a first position, the promoter sequence is present in a second position, the Kozak sequence is present in a third position, the cargo nucleotide sequence is present in a fourth position, the polyA sequence is present in a fifth position and the 3’ ITR is present in a sixth position in a 5’ to 3’ orientation in the nucleic acid molecule, wherein the first, second, third, fourth, fifth and sixth positions are oriented from 5’ to 3’.

In some embodiments, the Kozak sequence comprises an nucleic acid sequence of GCCACC (SEQ ID NO: 158). In some embodiments, the promoter comprises an nucleic acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 154. In some embodiments, the cargo nucleic acid sequence further encodes an additional polypeptide, wherein the sequence encoding the additional polypeptide is located downstream of the nucleic acid sequence encoding the CAR. In some embodiments, the additional polypeptide comprises a cytokine (e.g., IL- 15 or a functional fragment thereof, IL-15Ra or a fragment thereof that specifically binds to a IL- 15), and/or a TGFBR2 DNR.

In some embodiments, the disclosure provides a transposon-related nucleic acid molecule, wherein the nucleic acid molecule is engineered to comprise a cargo cassette comprising ITRs flanking a cargo nucleotide sequence. The transposase and related ITR nucleotide sequences may be from any transposon/transposase system described herein.

The disclosure further provides a nucleic acid molecule comprising a nucleotide sequence of a first ITR, a nucleotide sequence of a second ITR, and a cargo nucleotide sequence, i.e., a nucleotide sequence encoding for one or more polypeptides intended to be expressed or produced in an immune cell, e.g., an NK cell. In some embodiments, the polypeptide is a CAR, a cytokine, and/or a TGF-P DNR described herein. In some embodiments, the first and second ITRs are any two of the ITR nucleotide sequences described herein. In some embodiments, the first and second ITRs are IRDR-L-Seq3 and IRDR-R-Seq3, respectively. In some embodiments, the first and second ITRs flank the cargo nucleotide sequence.

Polynucleotides encoding the transposase system

One aspect of the present disclosure provides a polynucleotide comprising a nucleotide sequence that encodes for a transposase described herein. In some embodiments, the polynucleotide further comprises a nucleotide sequence of a transposon (e.g., an engineered transposon) recognizable by the transposase.

In some embodiments, the polynucleotide is comprised in an expression vector. In some embodiments, the expression vector is a DNA plasmid. In some embodiments, the expression vector is a mini-circle vector. In some embodiments, the expression vector is a nanoplasmid.

The term “mini-circle vector” as used herein can refer to a small circular plasmid derivative that is free of most, if not all, prokaryotic vector parts (e.g., control sequences or non-functional sequences of prokaryotic origin).

For genome editing applications with transposons, in some embodiments, it may be desirable to use a binary system based on two distinct plasmids, whereby the nucleic acid sequence encoding for the transposase is physically separated from the transposon nucleic acid sequence containing the gene of interest flanked by the inverted terminal repeats. Co- delivery of the transposon and transposase-encoding plasmids into the target cells enables transposition via a conventional cut-and-paste mechanism. In some other embodiments, a transposon based system as described herein may comprise a polynucleotide comprising both a nucleic acid sequence encoding a transposase as described herein, and a nucleic acid sequence of a transposon as described herein,

wherein the nucleic acid encoding for the transposase and the transposon nucleic acid are present in the same plasmid.

In some embodiments, a vector useful in various aspects of the disclosure is a nanoplasmid vector. The term “nanoplasmid vector” as used herein, refers to a vector combining an RNA selectable marker with a R6K, ColE2 or ColE2 related replication origin. Nanoplasmid vectors can be selected from the nanoplasmid vectors disclosed in any of International PCT Publication No. WO2014/035457, International PCT Publication No. WO2014/077866, and International PCT Publication No. WO2019/183248, each of which is incorporated in its entirety herein by reference.

In some embodiments, a vector useful in the present disclosure is selected from the vectors NTC8385, NTC8485 and NTC8685. NTC8385, NTC8485 and NTC8685 are antibiotic-free pUC origin vectors, which are precursors to nanoplasmid vectors, and contain a short RNA (RNA-OUT) selectable marker instead of an antibiotic resistance marker such as kanR. The creation and application of these RNA-OUT based antibiotic-free vectors is described in International PCT Publication No. WO2008/153733 and US Publication No. 2010/0184158, each of which is incorporated in its entirety herein by reference.

2. Viral Vectors

Viral vectors encoding a CAR, a cytokine and/or a TGF-P DNR may be provided in certain aspects of the methods of the present disclosure. Non-limiting examples of virus vectors that may be used to deliver a nucleic acid of certain aspects of the present disclosure are described below.

An engineered virus vector may comprise long terminal repeats (LTRs), a cargo nucleotide sequence, or a cargo cassette. A viral vector-related “cargo cassette” as used herein refers to a nucleotide sequence comprising a left LTR at the 5’ end and a right LTR at the 3’ end, and a nucleotide sequence positioned between the left and right LTRs. The nucleotide sequence flanked by the LTRs is a nucleotide sequence intended for integration into acceptor DNA. A “cargo nucleotide sequence” refers to a nucleotide sequence (e.g., a nucleotide sequence intended for integration into acceptor DNA), flanked by an LTR at each end, wherein the LTRs are heterologous to the nucleotide sequence. A cargo cassette can be artificially engineered.

In some embodiments, the methods of the disclosure comprise introducing a nucleic acid sequence and/or a genomic editing construct into an immune cell (e.g. , an NK cell) ex vivo, in vivo, in vitro, or in situ by use of a viral vector. In some embodiments, the viral vector is a non-integrating non-chromosomal vector. Exemplary non-integrating non- chromosomal vectors include, but are not limited to, adeno-associated virus (AAV), adenovirus, and herpes viruses. In some embodiments, the viral vector is an integrating chromosomal vector. Integrating chromosomal vectors include, but are not limited to, adeno- associated vectors (AAV) and retroviral vectors (e.g., lentiviral vectors, and gamma-retroviral vectors). Retroviral vectors (e.g., lentiviral vectors) are well known in the art (see, for example, U.S. Patents 6,013,516 and 5,994,136; each of which is incorporated in its entirety herein by reference). Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, e.g., in Maetzig et al. Viruses 3(6):677-713, 2011; incorporated in its entirety herein by reference.

In some embodiments, introducing a nucleic acid sequence and/or a genomic editing construct into an immune cell (e.g., an NK cell) cell ex vivo, in vivo, in vitro, or in situ comprises use of a combination of vectors. Exemplary, non-limiting combination of vectors include: a combination of viral and non-viral vectors, a plurality of non-viral vectors, a plurality of viral vectors, a combination of a DNA-derived and an RNA-derived vector, a combination of an RNA and a reverse transcriptase, a combination of a transposon and a transposase, a combination of a non-viral vector and an endonuclease, and a combination of a viral vector and an endonuclease.

In some embodiments, the methods of the disclosure comprise genome modification comprising introducing a nucleic acid sequence and/or a genomic editing construct into an immune cell (e.g., an NK cell) ex vivo, in vivo, in vitro, or in situ to stably integrate a nucleic acid sequence (e.g., a transgene), transiently integrate a nucleic acid sequence, produce site- specific integration of a nucleic acid sequence, or produce a biased integration of a nucleic acid sequence. In some embodiments, genome modification comprises introducing a nucleic acid sequence and/or a genomic editing construct into an immune cell (e.g., an NK cell) ex vivo, in vivo, in vitro, or in situ to stably integrate a nucleic acid sequence. In some embodiments, the stable integration of a nucleic acid sequence can be a random integration, a site-specific integration, or a biased integration. In some embodiments, the site-specific integration can be non-assisted or assisted. In some embodiments, the assisted site-specific integration is co- delivered with a site-directed nuclease. In some embodiments, the site-directed nuclease comprises a transgene with 5’ and 3’ nucleotide sequence extensions that contain a percentage homology to upstream and downstream regions of the site of genomic integration. In some embodiments, the transgene with homologous nucleotide extensions enable genomic integration by homologous recombination, microhomology-mediated end joining, or nonhomologous end-joining.

In some embodiments, the site-specific integration occurs at a safe harbor site. Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene and the site of the human ortholog of the mouse Rosa26 locus.

In some embodiments, the site-specific transgene integration occurs at a site that disrupts expression of a target gene. In some embodiments, disruption of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. In some embodiments, exemplary target genes targeted by site-specific integration include but are not limited to PD1, any immunosuppressive gene, and genes involved in allo-rejection.

In some embodiments, the site-specific transgene integration occurs at a site that results in enhanced expression of a target gene. In some embodiments, enhancement of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. A. Regulatory Elements

Expression cassettes included in vectors useful in the present disclosure in particular contain (in a 5'-to-3' direction) a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, splice signals including intervening sequences, and a transcriptional termination/poly adenylation sequence .

(i) Promoter/Enhancers

The expression constructs provided herein comprise a promoter to drive expression of the chimeric antigen receptor of the disclosure. To bring a coding sequence “under the control” of a promoter, the 5' end of the transcription initiation site of the transcriptional reading frame is positioned “downstream” of (z.e., 3' of) the selected promoter. The “upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis- acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not “naturally occurring,” z.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. For example, promoters that are most commonly used in recombinant DNA construction include the lactamase (penicillinase), lactose and tryptophan (trp-) promoter systems. Furthermore, it is contemplated that the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high-level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

Additionally, any promoter/enhancer combination (as per, for example, the Eukaryotic Promoter Data Base EPDB, through world wide web at epd.isb-sib.ch/) could also be used to drive expression. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment. Non-limiting examples of promoters include early or late viral promoters, such as, SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoters, Rous Sarcoma Virus (RSV) early promoters; eukaryotic cell promoters, such as, e.g., beta actin promoter, GADPH promoter, metallothionein promoter; and concatenated response element promoters, such as cyclic AMP response element promoters (ere), serum response element promoter (sre), phorbol ester promoter (TPA) and response element promoters (tre) near a minimal TATA box. It is also possible to use human growth hormone promoter sequences (e.g., the human growth hormone minimal promoter described at GENBANK, accession no. X05244, nucleotide 283-341) or a mouse mammary tumor promoter (available from the ATCC, Cat. No. ATCC 45007). In some embodiments, the promoter is EFl, EFlalpha, MND, CMV IE, dectin-1, dectin-2, human CD1 1c, F4/80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter, U6 promoter or Hl promoter, however any other promoter that is useful to drive expression of the therapeutic gene is applicable to the practice of the present disclosure.

(ii) Initiation Signals and Linked Expression

A specific initiation signal also may be used in the expression constructs provided in the present disclosure for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. It is well known that the initiation codon must be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription functional effector elements. In some embodiments, internal ribosome entry sites (IRES) elements described herein can be used to create multigene, or polycistronic, messages. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic mRNAs. Additionally, one or more 2A sequences described herein could be used to create linked- or co-expression of genes in the constructs provided in the present disclosure.

(Hi) Origins of Replication

To propagate a vector in a host cell, it may contain one or more origins of replication sites (“ori”), for example, a nucleic acid sequence corresponding to oriP of EBV or a genetically engineered oriP with a similar or elevated function in programming, which is a specific nucleic acid sequence at which replication is initiated. Alternatively, a replication origin of other extra-chromosomally replicating virus or an autonomously replicating sequence (ARS) can be employed.

B. Selection and Screenable Markers

In some embodiments, immune cells (e.g., NK cells) comprising a nucleic acid molecule of the present disclosure may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of immune cells (e.g., NK cells) containing the expression vector. Generally, a selection marker is one that confers a property that allows for selection. A positive selection marker is one in which the presence of the marker allows for its selection, while a negative selection marker is one in which presence of the marker prevents its selection. An example of a positive selection marker is a drug resistance marker (e.g., genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol). Other types of markers including screenable markers such as GFP are also contemplated. Alternatively, screenable enzymes as negative selection markers such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. Methods to employ immunologic markers, possibly in conjunction with FACS analysis would be apparent to one of skill in the art. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with a nucleic acid encoding a gene product described herein. Further examples of selection and screenable markers are well known to one of skill in the art. 3. Methods for Modification of Immune Cells

In addition to viral delivery of the nucleic acid molecules encoding a chimeric antigen receptor of the disclosure, the following are additional methods of recombinant gene delivery to a given immune cell (e.g., NK cell).

Introduction of a nucleic acid, such as DNA or RNA, into the immune cells (e.g., NK cells) of the disclosure may use any suitable methods for nucleic acid delivery for transformation of an immune cell (e.g., NK cell), as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection; by injection (including microinjection); by electroporation; by calcium phosphate precipitation; by using DEAE-dextran followed by polyethylene glycol; by direct sonic loading; by liposome mediated transfection and receptor- mediated transfection; by microprojectile bombardment; by agitation with silicon carbide fibers; by Agrobacterium-mediated transformation; by desiccation/inhibition-mediated DNA uptake, and any combination thereof. Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.

A. Other Methods of Modification

In some embodiments, an immune cell (e.g., NK cell) of the disclosure may be produced by introducing a transgene into the immune cell (e.g., NK cell). The introducing step may comprise delivery of a nucleic acid sequence and/or a genomic editing construct via a non-transposition delivery system.

In some embodiments, introducing a nucleic acid sequence and/or a genomic editing construct into an immune cell (e.g., NK cell) ex vivo, in vivo, in vitro or in situ comprises one or more of topical delivery, adsorption, absorption, electroporation, spin-fection, co-culture, transfection, mechanical delivery, sonic delivery, vibrational delivery, magnetofection or nanoparticle-mediated delivery. In some embodiments, introducing a nucleic acid sequence and/or a genomic editing construct into an immune cell (e.g., NK cell) ex vivo, in vivo, in vitro or in situ comprises liposomal transfection, calcium phosphate transfection, fugene transfection, and dendrimer-mediated transfection. In some embodiments, introducing a nucleic acid sequence and/or a genomic editing construct into an immune cell (e.g., NK cell) ex vivo, in vivo, in vitro or in situ by mechanical transfection comprises cell squeezing, cell bombardment, or gene gun techniques. In some embodiments, introducing a nucleic acid sequence and/or a genomic editing construct into an immune cell (e.g., NK cell) ex vivo, in vivo, in vitro or in situ by nanoparticle-mediated transfection comprises liposomal delivery, delivery by micelles, and/or delivery by polymero somes.

In some embodiments, introducing a nucleic acid sequence and/or a genomic editing construct into an immune cell (e.g., NK cell) ex vivo, in vivo, in vitro or in situ comprises a non-viral vector. In some embodiments, the non-viral vector comprises a nucleic acid. In some embodiments, the non-viral vector comprises plasmid DNA, linear double-stranded DNA (dsDNA), linear single-stranded DNA (ssDNA), DoggyBone™ DNA, nanoplasmids, minicircle DNA, single- stranded oligodeoxynucleotides (ssODN), DDNA oligonucleotides, single-stranded mRNA (ssRNA), and double-stranded mRNA (dsRNA). In some embodiments, the non-viral vector comprises a transposon of the disclosure.

In some embodiments, enzymes may be used to create strand breaks in the host genome to facilitate delivery or integration of the transgene. In some embodiments, enzymes create single-strand breaks. In some embodiments, enzymes create double-strand breaks. In some embodiments, examples of break-inducing enzymes include but are not limited to: transposases, integrases, endonucleases, meganucleases, megaTALs, CRISPR-Cas9, CRISPR-CasX, transcription activator-like effector nucleases (TALEN) and/or zinc finger nucleases (ZFN). Other editing or break- inducing enzymes may include, without limitation, nucleases such as Casl2a (includes MAD7), Casl2b, Casl2c, Casl3 etc.

In some embodiments, break-inducing enzymes can be delivered to the cell encoded in DNA, encoded in mRNA, as a protein, and/or as a nucleoprotein complex with a guide RNA (gRNA).

In some embodiments, the site-specific transgene integration is controlled by a vector- mediated integration site bias. In some embodiments vector- mediated integration site bias is controlled by the chosen lentiviral vector. In some embodiments vector-mediated integration site bias is controlled by the chosen gamma-retroviral vector.

In some embodiments, the site-specific transgene integration site is a non-stable chromosomal insertion. In some embodiments, the integrated transgene may become silenced, removed, excised, or further modified.

In some embodiments, the genome modification is a non-stable integration of a transgene. In some embodiments, the non-stable integration can be a transient non- chromosomal integration, a semi-stable non chromosomal integration, a semi-persistent non- chromosomal insertion, or a non-stable chromosomal insertion. In some embodiments, the transient non-chromosomal insertion can be epi-chromosomal or cytoplasmic. In some embodiments, the transient non-chromosomal insertion of a transgene does not integrate into a chromosome and the modified genetic material is not replicated during cell division.

In some embodiments, the genome modification is a semi-stable or persistent non- chromosomal integration of a transgene. In some embodiments, a DNA vector encodes a Scaffold/matrix attachment region (S-MAR) module that binds to nuclear matrix proteins for episomal retention of a non-viral vector allowing for autonomous replication in the nucleus of dividing cells.

4. Methods of Imm une Cell Expansion

In some embodiments, artificial antigen presenting cells (aAPCs) may be developed, expressing the HER2 antigen along with costimulatory molecules, such as 4-1BBL, CD28, membrane bound IL-15 (mbIL-15) and/or membrane-bound IL-21 (mbIL-21), to select for immune cells (e.g., NK cells) in vitro that are capable of sustained CAR-mediated propagation. This powerful technology allows the manufacture of clinically relevant numbers (up to IO¹⁰) of CAR* immune cells (e.g., NK cells) suitable for human application. As needed, additional stimulation cycles can be undertaken to generate larger numbers of genetically modified immune cells (e.g., NK cells). For example, at least 90% of the propagated immune cells (e.g., NK cells) express a CAR described herein and can be cryopreserved for infusion. Furthermore, this approach can be harnessed to generate immune cells (e.g., NK cells) to diverse tumor types by pairing the specificity of the introduced CAR with expression of the HER2 antigen recognized by the CAR on the aAPC.

Following genetic modification, the immune cells (e.g., NK cells) may be immediately infused or may be stored. In certain aspects, following genetic modification, the immune cells (e.g., NK cells) may be propagated for days, weeks, or months ex vivo as a bulk population within about 1, 2, 3, 4, 5 days or more following gene transfer into the immune cells (e.g., NK cells). In a further aspect, the transfectants are cloned and a clone demonstrating presence of a single integrated or episomally maintained expression cassette or plasmid, and expression of the chimeric receptor is expanded ex vivo. In some embodiments, the clone is expanded at least 1,000-fold in culture. In some embodiments, the cells are expanded in the absence of feeder cells. In some embodiments, the immune cells (e.g., NK cells) are expanded in the presence of feeder cells. The clone selected for expansion demonstrates the capacity to specifically recognize and lyse HER2 expressing target cells. In some embodiments, the immune cells (e.g., NK cells) are expanded in the presence of a cytokine. The immune cells (e.g., NK cells) may be expanded by stimulation with IL-2, or other cytokines that bind the common gamma-chain (e.g., IL-7, IL-12, IL-15, IL-21, and others). The immune cells (e.g., NK cells) may be expanded by stimulation with artificial antigen presenting (aAPC) cells. In some embodiments, the immune cells (e.g., NK cells) may be cryopreserved.

5. Methods of Immune cell Cry opreservation

In some embodiments of the present disclosure, the immune cells (e.g., NK cells) described herein are modified at a point-of-care site. In some cases, immune cells (e.g., NK cells) are also referred to as engineered immune cells (e.g., engineered NK cells). In some cases, the point-of-care site is at a hospital or at a facility (e.g., a medical facility) near a subject in need of treatment. The subject undergoes apheresis and peripheral blood mononuclear cells (PBMCs) or a sub population of PBMC can be enriched for example, by elutriation or Ficoll separation. Enriched PBMC or a subpopulation of PBMC can be cryopreserved in any appropriate cryopreservation solution prior to further processing. In one instance, the elutriation process is performed using a buffer solution containing human serum albumin. Immune ceils (e.g., NK cells) can be isolated by selection methods described herein. In one instance, the selection method for NK cells includes beads specific for CD56 on NK cells. In one case, the beads can be paramagnetic beads. The harvested immune effector cells can be cryopreserved in any appropriate cryopreservation solution prior to modification. The immune cells (e.g., NK cells) can be thawed up to 24 hours, 36 hours, 48 hours. 72 hours or 96 hours ahead of infusion. The thawed cells can be placed in cell culture buffer, for example in cell culture buffer (e.g., RPMI) supplemented with fetal bovine serum (FBS) or human serum AB or placed in a buffer that includes cytokines such as IL-2 and IL-21, prior to modification. In another aspect, the harvested immune cells (e.g., NK cells) can be modified immediately without the need for cryopreservation.

In some embodiments, the population of immune cells (e.g., NK cells) is immediately infused into a subject. In another aspect, the population of immune cells (e.g., NK cells) is placed in a cytokine bath prior to infusion into a subject. In a further aspect, the population of immune cells (e.g., NK cells) is cultured and/or stimulated for no more than 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35 42 days, 49, 56, 63 or 70 days. In some embodiments, the population of immune cells (e.g., NK cells) is stimulated for not more than: IX stimulation, 2X stimulation, 3X stimulation, 4X stimulation, 5X stimulation, 5X stimulation, 6X stimulation, 7X stimulation, 8X stimulation, 9X stimulation or 10X stimulation. In some instances, the immune cells (e.g., NK cells) are not cultured ex vivo in the presence of aAPCs. In some specific instances, the method of the embodiment further comprises enriching the cell population of CAR-expressing immune cells {e.g., NK cells) after the transfection and/or culturing step. The enriching can comprise fluorescence-activated cell sorting (FACS) to sort for CAR-expressing cells. The enriching can comprise use of a resin (e.g. magnetic bead) to sort for CAR-expressing cells. In a further aspect, the sorting for CAR-expressing cells comprises use of a CAR-binding antibody. The enriching can also comprise depletion of CD56+ cells. In some embodiments, the method further comprises cryopreserving a sample of the population of immune cells (e.g., NK cells).

In some cases, the immune cells {e.g., NK cells) of the disclosure do not undergo a propagation and activation step. In some cases, the immune cells (e.g., NK cells) do not undergo an incubation or culturing step {e.g., ex vivo propagation). In certain cases, the immune cells (e.g., NK cells) are placed in a buffer that includes IL-2 and IL21 prior to infusion. In other instances, the immune cells (e.g., NK cells) are placed or rested in cell culture buffer, for example in cell culture buffer {e.g., RPMI) supplemented with fetal bovine serum (FBS) prior to infusion. Prior to infusion, the immune cells (e.g., NK cells) can be harvested, washed and formulated in saline buffer in preparation for infusion into the subject.

In some embodiments, a population of cryopreserved NK cells provided herein exhibits greater than about 60%, about 65%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% viability post-thaw. In some embodiments, a population of cryopreserved NK cells provided herein exhibits greater than about 60%, about 65%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of an activity (e.g., CAR-dependent cytotoxicity and/or cytokine production or secretion) post-thaw, e.g., as compared to the activity of the population of NK cells prior to cryopreservation. 6. Modification of Gene Expression

In some embodiments, the immune cells (e.g., NK cells) of the present disclosure are modified to have altered expression of certain genes such as glucocorticoid receptor, TGF beta receptor (e.g., TGFBR1 or TGFBR2), PD1, SOCS1, SOCS2, SOCS3, and, ''or CISH.

In some embodiments, the altered gene expression is carried out by effecting a disruption in the gene, such as a knock-out, insertion, mis sense or frameshift mutation, such as biallelic frameshift mutation, deletion of all or part of the gene, e.g., one or more exon or portion therefore, and/or knock-in. For example, the altered gene expression can be effected by sequence- specific or targeted nucleases, including DNA-binding targeted nucleases such as zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases such as a CRLSPR-associated nuclease (Cas), specifically designed to be targeted to the sequence of the gene or a portion thereof.

In some embodiments, the alteration of the expression, acti vity, and/or function of the gene is carried out by disrupting the gene. In some embodiments, the gene is modified so that its expression is reduced by at least at or about 20, 30, or 40%, generally at least at or about 50, 60, 70, 80, 90, or 95% as compared to the expression in the absence of the gene modification or in the absence of the components introduced to effect the modification.

In some embodiments, the alteration is transient or reversible, such that expression of the gene is restored at a later time. In other embodiments, the alteration is not reversible or transient, e.g., is permanent.

In some embodiments, gene alteration is carried out by induction of one or more double- stranded breaks and/or one or more single- stranded breaks in the gene, typically in a targeted manner. In some embodiments, the double- stranded or single- stranded breaks are made by a nuclease, e.g. an endonuclease, such as a gene-targeted nuclease. In some embodiments, the breaks are induced in the coding region of the gene, e.g. in an exon. For example, in some embodiments, the induction occurs near the N-terminal portion of the coding region, e.g. in the first exon, in the second exon, or in a subsequent exon.

In some aspects, the double- stranded or single- stranded breaks undergo repair via a cellular repair process, such as by non-homologous end-joining (NHEJ) or homology- directed repair (HDR). In some aspects, the repair process is error-prone and results in disruption of the gene, such as a frameshift mutation, e.g., biallelic frameshift mutation, which can result in complete knockout of the gene. For example, in some aspects, the disruption comprises inducing a deletion, mutation, and/or insertion. In some embodiments, the disruption results in the presence of an early stop codon. In some aspects, the presence of an insertion, deletion, translocation, frameshift mutation, and/or a premature stop codon results in disruption of the expression, activity, and/or function of the gene.

In some embodiments, alteration in gene expression is achieved using antisense techniques, such as by RNA interference (RNAi), short interfering RNA (siRNA), short hairpin (shRNA), and/or ribozymes to selecti vely suppress or repress expression of the gene. In some aspects, the siRNA is comprised in a polycistronic construct.

V. METHODS OF USE

In some embodiments, the present disclosure provides methods for immunotherapy comprising administering an effective amount of the immune cells, such as NK cells, of the present disclosure. In some embodiments, the immune cells (e.g., NK cells) comprise a CAR described in Table 2.

In some embodiments, a medical disease or disorder in a subject in need thereof is treated by administering to the subject an immune cell (e.g., NK cell) population that elicits an immune response. In some embodiments, the disease or disorder is a cancer. Provided herein are methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of the immune cells (e.g., NK cells) described herein.

In some embodiments, the cancer comprises a solid tumor or a hematological tumor. Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like.

Further examples of cancers that may be treated using one or more of the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, various types of head and neck cancer, and melanoma. The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; testicular seminoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia: thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant: Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; uveal melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma: mesenchymoma, malignant: brenner tumor, malignant: phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing’s sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin’s disease; hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; B-cell lymphoma; low grade/follicular non-Hodgkin's lymphoma. (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblaslic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); myelodysplastic syndrome (MDS); chronic myeloblasts leukemia; diffuse large B-cell lymphoma (DLBCL); peripheral T-cell lymphoma (PTCL); or anaplastic large cell lymphoma (ALCL).

In some embodiments, the cancer is selected from the group consisting of breast cancer, gastric cancer, esophageal cancer, esophagogastric junction (GEJ) cancer, ovarian cancer, medulloblastoma, osteosarcoma, non-small cell lung carcinoma, colorectal rectal cancer, bladder cancer and prostate cancer. In some embodiments, the cancer is a HER2- positive cancer.

1. Pharmaceutical Compositions

The disclosure further provides pharmaceutical compositions and formulations comprising one or more immune cells (e.g., NK cells) described herein and optionally a pharmaceutically acceptable carrier. The pharmaceutical compositions can include a combination of one or more physiologically acceptable carriers. The pharmaceutical compositions may include buffers (e.g., neutral buffered saline, phosphate buffered saline and the like), antioxidants, carbohydrates, amino acids, antioxidants; chelating agents, and preservatives. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters. In some embodiments, the pharmaceutical compositions are formulated for intravenous or intraperitoneal administration. In some embodiments, the pharmaceutical composition includes cryopreserved engineered immune cells.

2. Combination Therapies

In some embodiments, the methods of the disclosure comprise administering to a subject in need thereof a therapeutically effective amount of an immune cell (e.g., NK cell) or a pharmaceutical composition described herein. In some embodiments, the methods further comprise administering an additional therapeutic agent to the subject. The additional therapeutic agent may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapeutic agent may be in the form of adjuvant or neoadjuvant therapy.

In some embodiments, the additional therapeutic agent comprises administration of small molecule enzymatic inhibitors and/or anti-metastatic agents. In some embodiments, the additional therapeutic agent comprises the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc. ). In some embodiments, the additional therapeutic agent is radiation therapy. In some embodiments, the additional therapeutic agent is surgery. In some embodiments, additional therapeutic agent is a combination of radiation therapy and surgery. In some embodiments, the additional therapeutic agent is gamma irradiation. In some embodiments, additional therapeutic agent is selected from the group consisting of an agent targeting PBK/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and a chemopreventative agent. The additional therapeutic agent may be one or more of the chemotherapeutic agents known in the art. In some embodiments, a growth factor that promotes the growth and activation of the immune cells (e.g., NK cells) is administered to the subject either concomitantly with the immune cells or subsequently to the immune cells. The growth factor can be any suitable growth factor that promotes the growth and activation of the immune cells. In some embodiments, the growth factor is a cytokine. In some embodiments, the cytokine comprises at least one chemokine, interferon, interleukin, lymphokine, tumor necrosis factor, or variant or combination thereof. In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is IL-15, IL-21, IL-2, IL-12, IL-18, IL-21, IL-1, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-13, IL-14, IL-15, IL-16, IL-17, IL-19, IL-20, IL- 22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, functional fragments thereof or combinations thereof.

In some embodiments, one or more CARs described herein are administered to a subject with one or more additional therapeutic agents including but not limited to p40, LIGHT, CD40L, FLT3L, 41 BBL. FASL, and haparanese.

Combination therapies can further include, but are not limited to, use of one or more anti-microbial agents (for example, antibiotics, anti-viral agents and anti-fungal agents), anti- tumor agents (for example, fluorouracil, methotrexate, paclitaxel, fludarabine, etoposide, doxorubicin, or vincristine), immune-depleting agents (for example, fludarabine, etoposide, doxorubicin, or vincristine), immunosuppressive agents (for example, azathioprine, or glucocorticoids, such as dexamethasone or prednisone), anti-inflammatory agents (for example, glucocorticoids such as hydrocortisone, dexamethasone or prednisone, or non- steroidal anti-inflammatory agents such as acetyls alicylic acid, ibuprofen or naproxen sodium), cytokine antagonists (for example, anti-TNF and anti-IL-6), cytokines (for example, interleukin- 10 or transforming growth factor-beta), hormones (for example, estrogen), or a vaccine. In addition, immunosuppressive or tolerogenic agents including but not limited to calcineurin inhibitors (e.g., cyclosporin and tacrolimus); mTOR inhibitors (e.g., Rapamycin); mycophenolate mofetil, antibodies (e.g., recognizing CD3, CD4, CD40, CD154, CD45, IVIG, or B cells); chemotherapeutic agents (e.g., Methotrexate, Treosulfan, Busulfan); irradiation; or chemokines, interleukins or their inhibitors (e.g., BAFF, IL-2, anti-IL-2R, II.,- 4, JAK kinase inhibitors) can be administered. Such additional pharmaceutical agents can be administered before, during, or after administration of the immune cells (e.g., NK cells), depending on the desired effect. An immune cell (e.g., NK cell) described herein may be administered before, during, after, or in various combinations relative to an additional cancer therapy, such as immune checkpoint therapy.

A. Chemotherapy

In some embodiments, the additional therapeutic agent comprises chemotherapy. A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlomaphazine, choiophosphamide, estramustine, ifosfamide, mechlorethamine, mechloreth amine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemu stine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, camiinomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti- metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6- mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, decitabine, di deoxy uridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid: aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine;

PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; niitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT- 11); topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, famesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above. B. Radiotherapy

In some embodiments, the additional therapeutic agent comprises radiotherapy.

Factors that cause DNA damage and have been used extensively include what are commonly known as y-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation, and UV -irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

C. Immunotherapy

In some embodiments, the additional therapeutic agent comprises immunotherapy. The skilled artisan will understand that additional immunotherapies may be used in combination or in conjunction with methods of the disclosure.

In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent.

Antibody-drug conjugates have emerged as a breakthrough approach to the development of cancer therapeutics. Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) that are covalently linked to cell -killing drugs. This approach combines the high specificity of MAbs against their antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in decreased toxicity and improved therapeutic index. Two criteria for ADC targets are upregulated/high levels of expression in tumor cells and robust internalization. In some embodiments, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, FLAP, laminin receptor, erb B, and p!55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL- 12, GM-CSF, gamma- IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand. In some embodiments, the additional therapeutic agent is a therapeutic agent that targets HER2. Examples of therapeutic agents that target HER2 include, but are not limited to Trastuzumab, Pertuzumab, Margetuximab, Trastuzumab-DM- 1, Lapatinib, Neratinib and Tucatinib.

Examples of immunotherapies comprise use of immune adjuvants, e.g., Mycobacterium bovisdinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169: Hui and Hashimoto, 1998: Christodoulides et al., 1998; each of which is incorporated in its entirety herein by reference); cytokine therapy, e.g., interferons a, p, and y, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998; each of which is incorporated in its entirety herein by reference); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945; each of which is incorporated in its entirety herein by reference); and monoclonal antibodies, e.g., anti-CD20, anti- ganglioside GM2, and anti-pl85 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent 5,824,311 ; each of which is incorporated in its entirety herein by reference).

In some embodiments, the immunotherapy comprises use of an immune checkpoint inhibitor. Immune checkpoints either turn up a signal (e.g., co-stimulatory molecules) or turn down a signal. Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD 152), indoleamine 2,3-dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-1 axis and/or CTLA- 4. The immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies (e.g., International PCT Publication No. WO 2015016718: Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; each of which is incorporated in its entirety herein byreference) . Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used. As the skilled person will know, alternative and/or equivalent names may be in use for certain antibodies mentioned in the present disclosure. Such alternative and/or equivalent names are interchangeable in the context of the present disclosure. For example, it is known that lanibrolizumab is also known under the alternative and equivalent names MK-3475 and pembroliz umab .

Examples of immunotherapies for use in treatment of kidney cancer or renal cell cancer include, but are not limited to Afinitor (Everolimus), Afinitor Disperz (Everolimus), Aldesleukin, Avastin (Bevacizumab), Avelumab, Axitinib, Bavencio (Avelumab), Bevacizumab, Cabometyx (Cabozantinib-S-Malate), Cabozantinib-S-Malate, Everolimus, IL- 2 (Aldesleukin), Inlyta (Axitinib), Interleukin-2 (Aldesleukin), Ipilimumab, Keytruda (Pembrolizumab), Lenvatinib Mesylate, Lenvinia (Lenvatinib Mesylate), Mvasi (Bevacizumab), Nexavar (Sorafenib Tosylate), Nivolumab, Opdivo (Nivolumab), Pazopanib, Hydrochloride, Pembrolizumab, Proleukin (Aldesleukin), Sorafenib Tosylate, Sunitinib Malate, Sutent (Sunitinib Malate), Temsirolimus, Torisel (Temsirolimus), Votrient (Pazopanib Hydrochloride), and/or Yervoy (Ipilimumab).

Examples of immunotherapies for use in treatment of Acute Myeloid Leukemia (AML) include, but are not limited to Azacitidine, Arsenic Trioxide, Cerubidine (Daunorubicin Hydrochloride), Cyclophosphamide, Cytarabine, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Daurismo (Glasdegib Maleate), Dexamethasone, Doxorubicin Hydrochloride, Enasidenib Mesylate, Gemtuzumab Ozogamicin, Gilteritinib Fumarate, Glasdegib Maleate, Idamycin PFS (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idhifa (Enasidenib Mesylate), Ivosidenib, Midostaurin, Mitoxantrone Hydrochloride, Mylotarg (Gemtuzumab Ozogamicin), Rubidomycin (Daunorubicin Hydrochloride), Rydapt (Midostaurin), Tabloid (Thioguanine), Thioguanine, Tibsovo (Ivosidenib), Trisenox (Arsenic Trioxide), Venclexta (Venetoclax), Venetoclax, Vincristine Sulfate, Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposome), and/or Xospata (Gilteritinib Fumarate). 3. Surgery

In some embodiments, the additional therapeutic agent comprises surgery. Approximately 60% of patients with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies described herein. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment bysurgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs' surgery).

Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months.

4. Other Agents

It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with one or more therapies described herein to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy. VI. ADMINISTRATION REGIMENS

In some embodiments, immune cells (e.g., NK cells) described herein are genetically engineered by introducing one or more nucleic acid molecules encoding a HER2 specific CAR and optionally one or more additional polypeptides described herein into the immune cells, and then infused into a subject in need thereof. In some embodiments, the immune cells (e.g., NK cells) or precursors thereof are genetically engineered and then infused within about 0 days, within about 1 day, within about 2 days, within about 3 days, within about 4 days, within about 5 days, within about 6 days or within about 7 days into the subject.

In some embodiments, a specific amount of engineered immune cells (e.g., NK cells) is administered to the subject and the amount is determined based on the efficacy and the potential of inducing a cytokine-associated toxicity. In some embodiments, the engineered immune cells are CD56⁺ NK cells.

In some embodiments, the engineered immune cells provided herein are administered to a subject via any suitable route, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection, or infusion.

In some embodiments, the engineered immune cells (e.g., NK cells) are targeted to the cancer via regional delivery directly to the tumor tissue. For example, in ovarian or renal cancer, the engineered immune cells (e.g., NK cells) can be delivered intraperitoneally (IP) to the abdomen or peritoneal cavity. Such IP delivery can be performed via a port or pre- existing port placed for delivery of chemotherapy drugs. Other methods of regional delivery of modified immune effector cells can include catheter infusion into resection cavity, ultrasound guided intratumoral injection, hepatic artery infusion or intrapleural delivery.

VII. ARTICLES OF MANUFACTURE OR KITS

In another aspect, the disclosure provides an article of manufacture or a kit comprising any of the immune cells (e.g., NK cells) or other compositions described herein. The article of manufacture or kit can further comprise a package insert, comprising instructions for using the immune cells (e.g., NK cells) or other compositions to treat or delay progression of cancer in a subject, or to enhance immune function of a subject having cancer. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or poly olefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more agents (e.g., a chemotherapeutic agent, and anti-neoplastic agent). Suitable containers for the one or more agents include, for example, bottles, vials, bags, and syringes.

All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

VIII. EXAMPLES

Example 1 - Methods of Modification of NK cells to Express HER2 Specific CARs

NK cell derivation from CAR-expressing iPSCs

The derivation of NK cells from iPSCs and CAR transfected iPSCs has been previously described (Knorr et al., 2013 (supra) ; Ng et al. Nat Protoc. 3:768-76, 2008). Briefly, 3,000 TrypLE-adapted iPSCs are seeded in 96-well round-bottom plates with APEL culture (Ng et al., 2008) containing 40 ng/ml human stem cell factor (SCF), 20 ng/ml human vascular endothelial growth factor (VEGF), and 20 ng/ml recombinant human bone morphogenetic protein 4 (BMP-4). After day 11 of hematopoietic differentiation, spin embryoid bodies (EBs) are then directly transferred into each well of uncoated 24-well plates under a condition of NK cell culture. Cells are then further differentiated into NK cells as previously reported (Bachanova et al. Blood 123(25): 3855-63 2014; Ni et al. Methods Mol. Biol. 1029: 33-41 2013) using 5 ng/mL IL-3 (first week only), 10 ng/mL IL-15, 20 ng/mL IL-7, 20 ng/mL SCF, and 10 ng/mL flt3 ligand for 28-32 days. Half-media changes are performed weekly. NK cells are harvested for irradiated mb IL-21 expressing artificial antigen presenting cells (aAPCs) expansion (Denman et al. PLoS One 7(l):e30264, 2012) with 50 units/mL of hIL-2. Isolation of NK cells from peripheral blood or cord blood

NK cells are isolated from either human peripheral blood leukapheresis samples or cord blood units. Briefly, leukapheresis samples or cord blood units are enriched for peripheral blood mononuclear cells (PBMC). One method for PBMC enrichment is separation using a Ficoll density gradient. Next, peripheral blood NK cells are isolated from PBMC samples using immunomagnetic separation beads. Beads are conjugated to a cocktail of specific immunophenotypic antibodies to enable NK cell isolation through either positive or negative selection. Isolated NK cells are activated prior to transduction. One method for NK cell activation is co-culture with irradiated artificial antigen presenting cells (aAPCs) expressing mbIL-21 and 4-1BBL for expansion in the presence of hIL-2.

Molecular Constructs

TcBuster transposon vectors are designed and reconstructed as previously described. Transgene expression is driven by the EFla promoter. Transposon vectors including a transposon encoding a CAR that specifically bind to HER2 (provided herein) a TGFB2 DNR and a cytokine comprising IL- 15 are synthesized as gBlocks gene fragment and cloned into the transposon using restriction enzyme cloning and ligation. Correct sequences are confirmed by restriction enzyme digest and sequencing analyses. Insulated TcBuster vectors are generated by PCR of CAR expression cassettes from TcBuster transposon vectors and subsequent BP Clonase reaction into pDONR221 to generate pENTR221-CAR cassette plasmids. pENTR221-CAR cassette plasmids are subsequently used for LR Clonase reaction into PB-I-DEST-I to generate final TcBuster expression vectors. The PB-I-DEST-I vector contains a 2.4kb cHS4 insulator (I) flanking the Gateway destination cassette (DEST) used for LR clonase cloning. Generation of transfected NK cells are performed as described above. To determine the copy numbers of integrated vector, genomic DNA is isolated from the engineered cells and quantitative PCR is performed using sets of primers specific for the CAR construct and for the human RNase gene. To determine absolute value, a standard curve is generated using serial dilutions of a plasmid containing the CAR construct. Reactions are carried out in triplicate in CFX384 Touch™ Real-Time PCR Detection System.

Nucleic acids/DNA/genes encoding the constructs are cloned into the multiple cloning site of retroviral gene transfer vectors: pELNS or pES.12-6(g)ps under control of one of the following promoters: EF-1, EFS, MND, MSCV, CMV, PGK or RPBSA. Retrovirus is produced in 293T cells by transfecting the cells with gene transfer vectors. Cells are placed in fresh culturing medium. The virus supernatant is collected 48-72 hours post-medium change by centrifugation at 800xg for 5 minutes. The supernatant is collected, filtered, and frozen in aliquots at -80°C.

Quantitative RT-PCR

To test the level of chimeric antigen receptor transcript expression in modified NK cells, RNA are processed from (at day 9 post-electoporation) NK cells. For gene expression analysis, transcripts are evaluated using the Human Cell Cycle RT² Profiler PCR Array (Qiagen). Transcripts are analyzed and normalized to GAPDH.

Immunoblot

To test the expression of one or more of the CAR, TGF-P DNR, and/or cytokine (e.g., IL- 15) in modified NK cells, suspension cells are lysed in RIPA lysis buffer with fresh protease inhibitor cocktail on ice for 20 min and sonicated for 2 seconds on ice. Membrane proteins are extracted using a Membrane Protein Extraction Kit. Sample proteins are measured by a standard bicinchoninic acid assay, size fractioned by polyacrylamide gel electrophoresis (PAGE), and are transferred to nitrocellulose membrane. Nonspecific binding are blocked by incubating in TBST 5% BSA plus 1% Triton X-100 solution for 1 hours, followed by incubation with primary antibodies, overnight at 4°C. Species specific IRDye- conjugated secondary antibodies (1:10, 000,) are applied to membranes for 1 hour at room temperature. Immunoreactive products are visualized in Odyssey Imaging Systems. All loading samples are normalized by staining of GAPDH.

Proliferation assays

To test the proliferation and viability of NK cells, modified NK cells or NK cells from healthy donors are labeled with Cell Proliferation Dye and placed in the in the continuous IL- 15 treatment (IL-15cont) or intermittent IL- 15 treatment (IL-15gap) conditions for 9 days (e.g., as described in Felices et al. (2018) J CI Insight 3(3): e96219). Viable NK cells (CD56⁺CD3 ) are then analyzed for dilution of dye.

Cell lysis assay

To test the ability of the modified NK cells to specifically target cells for lysis, human HER2 -positive breast caner cell lines are incubated with ⁵¹ chromium (^5/Cr) or europium for 1 hour at 37°C, washed three times, and cocultured with NK cells at the indicated effector to target (E:T) ratios. Total lysis (test release) is achieved with the use of 5% Triton-X 100. After a period of incubation, cells are harvested and analyzed. Specific ⁵¹Cr or europium/ lysis is determined following the equation: Percentage of specific lysis = 100 x (Test release - Spontaneous release)/(Maximal release - Spontaneous release). CD107a expression and IFN-y staining

CD107a (LAMP1) expression and IFN-y production by target cells are two proxys for the level of NK cell binding with said cells. NK cells are incubated with or without each of the cancer targets provided in Table 9 below at 1:2 effector to target ratios. CD107a-APC antibody is added to each well and allowed to incubate for 1 hour, following by adding GolgiStop and GolgiPlug for additional 2 hours incubation. At the completion of incubation, cells are ished with FACS buffer, are stained with CD56-PE and LIVE/DEAD Fixable Aqua Sstain (ThermoFisher Scientific). Cells are then fixed with fixation buffer for 10 minutes on ice, following by permeabilization with perm/ish buffer for 10 minutes at 4°C. Cells are washed and stained with interferon-y (IFN-y)-Pacific Blue for 30 min at 4°C, then final washed for analysis. CD 107a expression and intracellular IFN-y production are evaluated by normalization data of NK cell without target cell co-culture.

Table 9. Exemplary Cancer Cell Lines. The BT20, MDA-MB-231, and MDA-MB-468 breast cancer cell lines are HER2 negative. The remaining breast cancer cell lines are HER2 positive.

Metabolic studies

NK cells engineered to express HER2 specific CARs are harvested at day 9 of culture and resuspended in Seahorse XF Assay Medium (Agilent Technologies). One million cells/well are immobilized with Poly-L- Lysine (MilliporeSigma). The extracellular acidification rate and the oxygen consumption rate are measured (pmoles/min) in real time in an XFe24 analyzer after injection of glucose (10 mM), oligomycin (1 pM), FCCP (1 pM) plus sodium pyruvate (1 mM), and rotenone/antimycin A (0.5 pM). SRC is calculated from the change from basal oxygen consumption, after addition of glucose, to maximal oxygen consumption, after addition of FCCP. In experiments measuring the input of FAO, glucose is added to the media prior to beginning measurements. This is the followed by injection of the CPT-1 inhibitor etomoxir, injection of oligomycin, injection of FCCP plus sodium pyruvate, and final injection of rotenone/antimycin A.

HER2-positive tumor xenografts treatment with NK cells

Isolated peripheral blood NK cells are engineered to express an anti-HER2 CAR provided herein alone or in combination with IL- 15 and/or a TGFBR2 dominant negative receptor. As additional controls, NK cells are engineered to express an inert protein including a tag alone or in combination with IL- 15 and/or a TGFBR2 dominant negative receptor or not engineered to express an exogenous protein. To assess the in vivo anti-tumor activity of the engineered NK cells, a xenogeneic mouse model system bearing HER-2-positive tumors is used (see e.g., Hermanson et al. Methods Mol Biol. 1441: 277-84). Exemplary human HER2-positive cancer cell lines are provided in Table 9 above.

Briefly, tumorogenic cancer cell line cells engineered to express luciferase (tumor cells) are implanted into NOD-scid IL2ry null (NSG) mice at a dose of about 2 x 10⁵ to about IxlO⁸ cells per mouse via subcutaneous, intraperitoneal, or intravenous injection. The tumor cells and NK cells may be co-administered to the mice on the same day, or the tumor cells may be administered 1 to 14 days prior to the NK cells (e.g., to allow for engraftment of the tumor cells). Bioluminescent imaging (BLI) is used to normalize tumor engraftment burden inthe mice. NK cells are then administered to the mice in doses of 1 x 10⁶ to 1.0 x 10⁷ NK cells/mouse via intraperitoneal or intravenous injection.

Tumor growth and expansion in the mice cohorts is monitored periodically (e.g., every 2, 4, or 7 days) using caliper measurements, imaging of luminescence signal (e.g., using weekly BLI measurements with a Xenogen IVIS® Spectrum in vivo imaging system (PerkinElmer®), or frequency of tumor cells in peripheral blood and/or peritoneal fluid (e.g., measured by flow cytometry). Tumor clearance, tumor growth rate, the number of NK cells in tumor tissue (e.g., biopsied tumor tissue), NK cell persistence, and cytokine (e.g., IFN-y, TNF-a, IL-8, IP-10, MCP-1, and MIP-la/b) production levels (e.g., in vivo or in vitro, e.g., in blood, peritoneal fluid, and/or tumor tissue) is assessed using methods known in the art. For example, cytokine production in vivo is measured in blood or peritoneal fluid and detected as protein (e.g., by sandwich ELISA) or in tumor cell-containing tissue (e.g., detected as mRNA following tissue homogenization, RNA isolation, and quantitative RT-PCR). Ex vivo cytokine production and/or cytotoxicity is measured using NK cells isolated from blood, peritoneal fluid, or single cell suspensions prepared from dissociation of tumor-containing tissue. After isolation, NK cells are cultured for about 4 to about 48 hours in media without stimulation to analyze cytokine production and/or cytotoxicity using methods known in the art.

Example 2 - Allogeneic NK Cells Engineered to Express HER2 Specific CAR, Interleukin 15 And TGF-p Dominant Negative Receptor Effectively Control HER2 positive Tumors

Despite the success of HER2-targeted therapies in HER2 positive breast and gastric cancer, additional therapies are needed to address treatment-resistant metastatic disease. Adoptive immune cell therapy is a promising therapeutic modality given the remarkable clinical responses seen with autologous chimeric antigen receptor (CAR) T cells in hematological malignancies. However, success of cell therapy in solid tumors has been more limited. Three major impediments to the success of adoptive cell therapies in solid tumors are the heterogeneity of antigen expression, the immune suppressive tumor microenvironment, and the inherent challenges of manufacturing autologous cells and consequent variability of these cell products. Engineered, off-the-shelf, allogeneic NK cells described herein provide a solution to these challenges.

A novel engineered CAR-NK cell therapy for HER2 positive solid tumors was utilized. NK cells were engineered to express three transgenes: a HER2-specific CAR to effectively eliminate tumor cells, a TGF-P dominant negative receptor for resistance to TGFP-mediated immune suppression in the tumor microenvironment, and IL- 15 cytokine to enhance NK cell persistence and activity for durable response. High efficiency engineering of the large (~3.7Kb) cargo containing the CAR, IL- 15, and TGF-P dominant negative receptor in the NK cells was enabled by the non-viral TcBuster™ Transposon System. Transposon engineering resulted in high and stable expression of CAR (45% CAR at day 7 post gene delivery) without the need for post-engineering selection. NK cells expressing these three transgenes demonstrated both CAR-dependent and innate NK receptor-dependent tumor cell killing in vitro, reducing the likelihood of tumor escape through antigen loss. Further, these engineered NK cells effectively killed in vitro both high HER2-expressing SK0V3 cells, as well as lower HER2-expressing HT-29 cells; and also demonstrated resistance to TGFP- mediated immunosuppression, as evidenced by 75% reduction in TGFP-induced phosphorylation of SMAD2 as well as prevention of TGFP induced downregulation of NK cell activating receptors and restoration of NK cell cytotoxic activity.

These data demonstrate that these engineered NK cells will be protected from TGFP - mediated immune suppression in the tumor microenvironment. Finally, the addition of IL- 15 in the NK cells significantly enhanced their persistence for at least fourteen days in vitro without the need for exogenous cytokines. Moreover, administration of these engineered NK cells to NSG mice showed expansion and persistence of the transferred cell product. These engineered NK cells, therefore, address key hurdles to allogeneic cell therapy for solid tumors and provide a promising new therapeutic approach for HER2 expressing breast, gastric and other cancers.

A. Peripheral blood NK cells engineered using a transposon system express a HER2 specific CAR, a TGFBR2 dominant negative receptor, and IL-15

Peripheral blood NK cells were isolated from peripheral blood and activated and electroporated with a mixture of TcBuster™ transposase-encoding mRNA and a plasmid comprising a transposon including an MND promoter sequence, a 5’ transposase binding site specifically recognized by the TcBuster transposase, a coding region comprising a nucleic acid sequence encoding a HER2-specific CAR (comprising the amino acid sequence of a HER2-specific CAR described in Table 2), TGFBR2 DNR (comprising the amino acid sequence of a TGFBR2 DNR selected from the group consisting of SEQ ID NOs: 108-113), and IL- 15 (comprising the amino acid sequence of an IL- 15 polypeptide described in Table 3) (FIG. 1A), and a 3’ transposase binding site specifically recognized by the TcBuster transposase. Subsequently, the engineered NK cells were expanded with feeder cells expressing membrane bound IL-21 and 4-1BBL, using methods previously described (see, e.g., Pomeroy et al. bioRxiv 2021.08.02.454772). As control, unmodified, mock electroporated, peripheral blood NK cells were used.

The expression of HER2-specific CAR, TGFBR2 DNR and IL- 15 in the engineered NK cells was assessed 14 days post-electroporation. The expression of HER2- specific CAR and TGFBR2 DNR was detected by staining with biotinylated human HER2 protein (amino acid residues 23-652; Biotinylated Human Her2/ErbB2 Protein, His, Avitag™; ACRO BIOSYSTEMS; Cat No. #HE2-H82E2) and Alexa Fluor® 488 dye-labeled streptavidin; and an anti-TGFBR2 antibody (allophycocyanin (APC)-labeled anti-human TGF-P Receptor II antibody, clone W17055E; BIOLEGEND), respectively, and flow cytometry. As shown in FIG. IB, greater than 40% of NK cells (about 45%) in the cell population stained positive for the HER2-specific CAR and the TGFBR2 DNR. Expression of IL- 15 by the engineered NK cells was assessed by co-culturing 1.6 x 10⁵ NK cells with SKOV3 tumor cells for 24 hours and analyzing the level of IL- 15 in recovered supernatants via ELISA (V-PLEX Human IL- 15 Kit (MESO SCALE DIAGNOSTICS; Cat. No. K151RDD-1) (FIG. 1C). Together, these data demonstrate that transposon-based engineering of NK cells using a transposon encoding a HER2-specific CAR, TGFBR2 DNR, and IL- 15 results in high efficiency engineering of a large (~3.7 Kb) cargo, and results in the stable expression of CAR (45% CAR at day 14 post- gene delivery) without the need for post-engineering selection.

B. Engineered NK cells expressing HER2-specific CAR, TGFBR2 DNR and IL-15 kill HER2⁺ SK0V3 cells in vitro and produce interferon gamma

To assess the in vitro CAR-dependent cytotoxic activity of the engineered NK cells expressing HER-2 specific CAR, TGFBR2 DNR and IL- 15, cytotoxicity assays were performed. Briefly, engineered NK cells or unmodified, mock electroporated, NK cells (control), were incubated for 1 hour in AIM V™ medium (GIBCO) including 5% (v/v) serum replacement (THERMO FISHER SCIENTIFIC), 5% Penicillin-Streptomycin-Glutamine 100X (GIBCO), N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES; 10 mM), and recombinant human IL-2 (100 lU/mL) (NK cell culture medium), further including an inhibitory antibody cocktail to block NK cell activating receptors (anti-NKp30 (10 pg/mL; Cat. No. 325224; BIOLEGEND); anti-NKp46 (10 pg/mL; Cat. No. 331948; BIOLEGEND); anti-NKG2D (10 pg/mL; Cat. No. 320814; BIOLEGEND); anti-DNAM-1 (10 pg/mL; Cat. No. 559786; BIOLEGEND); and anti-2B4 (10 pg/mL; Cat. No. 16-2449-81; THERMO FISHER SCIENTIFIC)). Following this incubation period, the NK cells were co-cultured with HER2⁺ SKOV3 cells engineered to express firefly luciferase (SKOV3-fluc tumor cells; (1 x 10⁴ SKOV3-fluc tumor cells/well) in flat bottom 96-well tissue culture coated plates for 24 hours at effector: target (E:T) ratios of 0.5:1, 1:1, 2:1, 4:1, 8:1, and 16:1 to determine the degree of CAR- specific killing without background innate NK cell killing. SKOV3-fluc cell lysis was detected with the Bright-Glo™ Luciferase Assay System (PROMEGA) as instructed by the manufacturer using microplate reader.

To assess the production of interferon-gamma (IFN-y) by the engineered NK cells in response to the HER2⁺ SKOV3-fluc cells, supernatants from the cytotoxicity assay were collected and IFN-y levels were determined using the V-PLEX Proinflammatory Panel 1 Human Kit (Cat No. K151A9H-4) and the MESO QuickPlex SQ 120 instrument (both from MESO SCALE DIAGNOSTICS), as instructed by the manufacturer.

As shown in FIG. 2A, the engineered NK cells expressing HER-2 specific CAR, TGFBR2 DNR and IL- 15 exhibited CAR-dependent and NK receptor-driven cytotoxic activity against the HER2⁺ SKOV3-fluc tumor cells. Further, as shown in FIG. 2B, the engineered NK cells also exhibited CAR-dependent and NK receptor-driven IFN-y production. Without wishing to be bound by any particular theory, these activities by the engineered NK cells reduces the likelihood of tumor escape through antigen loss when the cells are administered to a subject.

C. TGFBR2 DNR traps TGF-P and protects engineered NK cells from TGFP- mediated immunosuppression

To evaluate the ability TGFBR2 DNR to impact TGF-P mediated immunosuppression of NK cells, the following experiments were performed. NK cells were engineered using gammaretroviral transduction to express TGFBR2 DNR (comprising the amino acid sequence of a TGFBR2 DNR selected from the group consisting of SEQ ID NOs: 108-113) and a tag. To assess the effect of TFGBR2 DNR expression on TGFP-induced signaling, engineered 5 x 10⁵ NK cells expressing TGFBR2 DNR with a tag, or untransduced cells (control), were cultured for one hour in the NK cell culture medium described above, supplemented with increasing concentrations of recombinant human TGF-P 1 (PEPROTECH; up to 10,000 pg/mL). Following this incubation period, the cells were fixed, permeabilized and stained for phosphorylated SMAD2 (pSMAD2) using an anti-human pSMAD2 antibody (anti-phospho- SMAD2 (Ser465/Ser467) (E8F3R) rabbit monoclonal antibody conjugated to Alexa Fluor® 647; CELL SIGNALING TECHNOLOGY; Cat No. #68550) and analyzed by flow cytometry. The percentage of pSMAD2-positive cells was determined after gating on non- engineered NK cells. As shown in FIG. 3A, expression of the TGFBR2 DNR protected the engineered NK cells from TGFpi induced signaling. Moreover, the engineered NK cells expressing TFGBR2 DNR protected neighboring non-engineered NK cells from TFB-P induced signaling, as determined by examining the percentage of pSMAD2-positive NK cells after gating on non-engineered NK cells, which were previously labeled with CellTrace™ Violet dye (THERMO FISHER SCIENTIFIC, Cat. No. C34557), as shown in FIG. 3B.

The effect of TGFBR2 DNR expression on TGFP-induced downregulation of the NK cell activating receptor DNAX accessory molecule (DNAM-1, also known as CD226) was also evaluated. Briefly, engineered NK cells expressing TGFBR2 DNR with a tag or NK cells engineered using gammaretroviral transduction to express a fusion protein including truncated CD19 (CD191-319) and a tag (control) were treated with or without 10 ng/ml recombinant TGFpi (PEPROTECH) for 5 days in the culture media described above, with cytokine replenishment (IL-2 and TGF pi) on Days 2 and 4. Following these treatments, the cells were stained for DNAM-1 using an fluorophore-labelled anti-DNAM-1 antibody (PE/Cyanine7 anti-human CD226 (DNAM-1) antibody; BIOLEGEND; Cat. No. 338315), and analyzed by flow cytometry. The analysis was gated on live NK cells. As shown in FIG. 3C, NK cells engineered to express TGFBR2 DNR were protected against the TGFP-induced downregulation of DNAM- 1.

To assess whether TFGBR2 DNR expression can restore NK cell cytotoxicity when the cells are exposed to TGFp, the following experiment was performed. Engineered NK cells expressing TGFBR2 DNR with a tag or NK cells expressing a fusion protein including truncated CD19 (CD191-319) and a tag (control) were pre-treated with 5 ng/ml recombinant TGFpi for 5 days, or not pre-treated (as control), and then co-cultured for 3 hours with K562 cells expressing luciferase (K562-luc) at effector: target (E:T) ratios of 1:3 to evaluate NK cell-mediated cytotoxicity. K562-luc cell lysis was detected with the Steady-Gio™ Luciferase Assay System (PROMEGA, Cat. No. E2510) as instructed by the manufacturer using microplate reader. As shown in FIG. 3D, the control NK cells pre-treated with TGF-P exhibited reduced cytotoxic activity against target K562-luc cells. In contrast, engineered NK cells expressing TGFBR2 DNR pre-treated with TGF-P exhibited similar cytotoxic activity against target K562-luc cells as both engineered NK cells and control NK cells that were not- pretreated with TGF-p.

D. IL-15 supports cell survival in vitro and persistence in vivo

To evaluate the function of IL-15 in NK cells engineered to express HER2-specific CAR, TGFBR2 DNR and IL- 15, the ability of the engineered NK cells to survive in vitro in the absence of exogenous cytokine support was examined. Briefly, the engineered NK cells, or unmodified NK cells (control), were cultured for 14 days in the absence of exogenous cytokine and the number of live NK cells counted over time. As shown in FIG. 4A, NK cells engineered to express IL- 15 showed increased survival in vitro, as compared to unmodified NK cells, without the need for exogenous cytokine. To evaluate the function of IL-15 in the engineered NK cells in vivo, 1.2 x 10⁷ total NK cells/mouse that underwent engineering to express HER2-specific CAR, TGFBR2 DNR and IL- 15 as described above (of which 4 x 10⁶ engineered NK cells were CAR⁺) or the same amount of unmodified NK cells (control), were administered intravenously (IV) into NOD- scid IL2ry null (NSG) mice (n=5). NK cells counts were monitored over time in blood samples by staining for CD45⁺/CD56⁺ cells and analyzing the samples via flow cytometry. As shown in FIG. 4B, the engineered NK cells showed enhanced persistence in vivo up to 40 days, demonstrating the ability of IL- 15 to support NK cell persistence in vivo.

E. NK cells engineered to express HER2-specific CAR, TGFBR2 DNR and secreted IL-15 are efficacious in vivo against HER2⁺ tumors and enable prolonged survival of mice

To assess the in vivo anti-tumor activity of the NK cells engineered to express HER2- specific CAR, TGFBR2 DNR and IL-15, the following experiment was performed. Briefly, 1 x 10⁶ HER2⁺ SKOV3-fluc cells/mouse were implanted into NSG mice via intraperitoneal (IP) injection. On the same day (day 0), and again on days 7 and 14, 1.2 x 10⁷/mouse of either the engineered NK cells (of which 4 x 10⁶ engineered NK cells were CAR⁺) or unmodified, mock electroporated NK cells (control), or saline (control), was administered to the mice (n = 10/group) via IP injection. A schematic of the study design is depicted in FIG. 5A. Tumor growth and expansion was monitored periodically using BLI measurements with a Xenogen IVis® Spectrum in vivo imaging system (PerkinElmer®). As shown in FIG. 5B and FIG. 5C, the engineered NK cells showed potent anti-tumor activity in vivo against the xenografted HER2⁺ SKOV3-fluc ovarian cancer cell line (p<0.05 using non-parametric t-test at individual time points between mice administered the engineered NK cells and both controls) and led to a substantial survival benefit in tumor bearing mice as determined by Kaplan-Meyer analysis (FIG. 5D; 57 days extended median survival in mice receiving engineered NK cells vs. both controls).

Example 3 -NK Cells Engineered to Express TGF-p DNR, HER2-specific CAR, and IL- 15 are Cytotoxic Against HER2⁺ Cells In Vitro and Produce Effector Cytokines

To assess in vitro CAR-dependent cytotoxic activity of engineered NK cells expressing a transgene encoding TGFBR2 DNR, HER-2 specific CAR, and IL- 15, cytotoxicity assays were performed. Briefly, engineered NK cells or unmodified, mock electroporated NK cells (control), were co-cultured for up to 56 hours with HER2⁺ SKOV3 cells engineered to express green fluorescent protein (GFP) (1 x 10⁴ SKOV3-GFP tumor cells/well) in flat bottom 96-well tissue culture coated plates at effector: target (E:T) ratios of 0.625:1, 2.5:1, or 10:1, in Excellerate™ medium (R&D SYSTEMS) including 5% (v/v) human A/B serum, 5% Penicillin-Streptomycin-Glutamine 100X (GIBCO), and N-2- hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES; 10 mM) (NK cell culture medium). Target cell survival was enumerated using an Incucyte® S3 Live-Cell Analysis System (SARTORIUS).

To assess production of interferon-gamma (IFN-y) from NK cells in response to co- culture with HER2⁺ cells, either engineered NK cells, or unmodified, mock electroporated NK cells (control), were co-cultured at an E:T ratio of 2.5:1 with tumor cell lines of gastric cancer (NCI-N87), ovarian cancer (SKOV3), and two tumor cell lines of breast cancer origin (HCC1954 and SKBR3; 1 x 10⁴HER2⁺ tumor cells/well) in flat bottom 96-well tissue culture coated plates in the NK cell culture medium described above for 24 hours. Subsequently, supernatants were collected and IFN-y levels were determined using the V-PLEX Proinflammatory Panel 1 Human Kit (Cat No. K151A9H-4) and the MESO QuickPlex SQ 120 instrument (both from MESO SCALE DIAGNOSTICS), as instructed by the manufacturer.

As shown in FIG. 6A, engineered NK cells expressing TGFBR2 DNR, HER-2 specific CAR, and IL- 15 killed target HER2⁺ SKOV-3 cells in a CAR-dependent manner. Further, as shown in FIG. 6B, the engineered NK cells exhibited CAR-dependent IFN-y production when co-cultured with target HER2⁺ cell lines of gastric cancer, ovarian cancer, and breast cancer origin.

Example 4 - IL-15 Secreted by NK Cells Engineered to Express TGF-p DNR, HER2- specific CAR, and IL- 15 Supports Cell Survival In Vivo in a Dose-Dependent Manner

To assess the ability of secreted IL- 15 to support the in vivo survival of engineered NK cells expressing a transgene encoding TGFBR2 DNR, HER-2 specific CAR, and IL- 15, the following experiment was performed (see schematic in FIG. 7A). Briefly, either 6.5 x 10⁶ engineered NK cells/mouse (of which 2 x 10⁶ engineered NK cells were CAR⁺), 1.3 x 10⁷ engineered NK cells/mouse (of which 4 x 10⁶ engineered NK cells were CAR⁺) or 2.6 x 10⁷ engineered NK cells/mouse (of which 8 x 10⁶ engineered NK cells were CAR⁺), or 2.6 x 10⁷ control (unmodified, mock electroporated) NK cells/mouse were administered intravenously (IV) into NOD-scid IL2ry null (NSG) mice (n=5/group). NK cells counts in peripheral blood samples and IL- 15 in plasma were monitored over time. NK cell counts in peripheral blood samples were determined by staining for mouse CD45’ cells and human CD45⁺/CD56⁺ cells and analyzing the samples via flow cytometry. The concentration of IL- 15 in plasma was determined using the V-PLEX Human IL- 15 Kit (Cat No. K151RDD) and the MESO QuickPlex SQ 120 instrument (both from MESO SCALE DIAGNOSTICS), as instructed by the manufacturer.

As shown in FIG. 7B, the engineered NK cells in vivo initially expanded, showed enhanced persistence, and subsided after 43 days, demonstrating the ability of IL- 15 to support NK cell persistence in vivo. In contrast, control NK cells were detected at significantly lower levels and failed to persist. Further, as shown in FIG. 7C, dose-dependent IL- 15 was detected in the plasma of the mice following administration. Moreover, NK cell counts correlated with IL- 15 levels in plasma (correlation at day 29 = 0.73; p=0.0009).

Example 5 - NK Cells Engineered to Express HER2-specific CAR, IL-15 And TGF-p DNR Remain Cytolytic In Vivo Following a Prolonged Period of Time in Circulation

To assess the in vivo anti-tumor activity of the NK cells expressing a transgene encoding TGFBR2 DNR, HER-2 specific CAR, and IL- 15 following prolonged circulation in vivo, 1 x 10⁶ HER2⁺ SKOV3-fluc cells/mouse were implanted into NSG mice of Example 4 via intraperitoneal (IP) injection. A schematic of the study design is depicted in FIG. 8A. Tumor growth and expansion was monitored periodically using BLI measurements with a Xenogen IVIS® Spectrum in vivo imaging system (PerkinElmer®). As shown in FIG. 8B, the engineered NK cells showed potent anti-tumor activity in vivo against the xenografted HER2⁺ SKOV3-fluc ovarian cancer cell line. Moreover, NK cell count inversely correlated with tumor volume (correlation at day 23 post-tumor initiation = -0.74, p<0.001 using non- parametric Spearman correlation). Overall, this data indicate that the engineered NK cells maintain their cytolytic activity in vivo after months in circulation.

Example 6 - NK Cells Engineered to Express HER2-specific CAR, IL-15 And TGF-p DNR Significantly Reduce HER2⁺ Tumor Burden In Vivo Following Therapeutic Administration

To assess the in vivo anti-tumor activity of the NK cells expressing a transgene encoding TGFBR2 DNR, HER-2 specific CAR, and IL- 15, the following experiment was performed. Briefly, 1 x 10⁶ HER2⁺ SKOV3-fluc cells/mouse were implanted into NSG mice via intraperitoneal (IP) injection. On the same day (day 0), and again on days 4 and 11, 1.2 x 10⁷/mouse of either the engineered NK cells expressing a transgene encoding TGFBR2 DNR, HER-2 specific CAR, and IL- 15 (of which 4 x 10⁶ engineered NK cells were CAR⁺) or unmodified, mock electroporated NK cells (control), or saline (control), was administered to the mice (n = 10/group) via IP injection. A schematic of the study design is depicted in FIG. 9A. Tumor burden was monitored periodically using BLI measurements with a Xenogen IVis® Spectrum in vivo imaging system (PerkinElmer®). As shown in FIG. 9B and FIG. 9C, the engineered NK cells showed potent anti-tumor activity in vivo against the xenografted HER2⁺ SKOV3-fluc ovarian cancer cell line (p<0.05 using non-parametric Kruskal-Wallis test at individual time points between mice administered the engineered NK cells and both controls). Indeed, tumor signal level over time in animals from the engineered NK cell-dosed group was significantly lower than the control groups (area under the curve (AUC) comparison, p<0.001, non-parametric Mann-Whitney test). This demonstrates that the engineered NK cells were efficacious following therapeutic administration.

Example 7 - NK Cells Engineered to Express HER2-specific CAR, IL-15 and TGF-p DNR Are Efficacious In Vivo Against HER2⁺ Tumor Xenografts and Prolong the Survival of Xenografted Mice

To further assess the in vivo anti-tumor activity of the engineered NK cells expressing a transposon encoding HER-2 specific CAR, TGFBR2 DNR, and IL- 15, the following experiment was performed. Briefly, IxlO⁶ HER2⁺ N87 gastric cancer cell line cells expressing green flourescent protein per mouse were implanted into NSG mice via subcutaneous (SC) injection ten days prior to start of treatment (Day -10). Ten days later (Day 0), groups were staged and treatment administered when the mean tumor volume reached ~70mm³. 2-8 x 10⁶ (as noted below) CAR⁺ engineered NK cells expressing a transgene encoding HER2-specific CAR, TGFBR2 DNR, and IL-15, 2xl0⁷ unmodified, mock electroporated NK cells (control NKs), or saline (vehicle control), was administered to the mice (n = 10/group) via intravenous (IV) injection. A schematic of the study design is depicted in FIG. 10A. Turn or volume was monitored periodically and measured by a digital caliper. As shown in FIG. 10B, the group of mice administered the engineered NK cells expressing HER2-specific CAR, TGFBR2 DNR, and IL- 15 exhibited a significantly lower tumor burden (p<0.0001, non-parametric t-test) as compared to either control group of mice. Moreover, as shown in FIG. 10C, a Kaplan-Meyer analysis demonstrated significant (p<0.0001) prolonged survival of mice administered the engineered NK cells expressing HER2-specific CAR, TGFBR2 DNR, and IL-15 relative to both control arms.

Further, blood analysis from mice in a different arm of the study at day 60 was performed using flow cytometry to quantify the number of NK cells in the circulation. As shown in FIG. 10D, there was a significant non-linear inverse correlation (Spearman Correlation = -0.63; p<0.0001) between the number of NK cells detected in the peripheral blood of mice administered 8xl0⁶ engineered NK cells expressing HER2-specific CAR, TGFBR2 DNR, and IL-15, and the measured tumor volume (measured by caliper). This result demonstrates that circulating NK cells were generally higher in individual animals in which antitumor responses are the highest.

In a separate arm of the same study, mice administered with engineered NK cells expressing HER2-specific CAR, TGFBR2 DNR, and IL-15, were sacrificed on Day 90 to harvest several organs and tumor tissue for analyes. The collected tissues were dissociated into single cells and analyzed by flow cytometry for the presence of human CD56⁺ NK cells to examine the biodistribution of the administered NK cells. As shown in FIG. 10E, NK cells effectively infiltrated tumors as well as different organs 90 days after administration. The total number of NK cells detected in the circulation and organs suggested that the engineered NK cells expanded in vivo over 90 days.

Overall, this study demonstrated that engineered NK cells expressing HER2-specific CAR, TGFBR2 DNR, and IL- 15 persisted, expanded and showed potent anti-tumor activity in vivo against the xenografted HER2⁺ N87 gastric cancer cell line tumors. Indeed, administration of the engineered NK cells resulted in a significant survival benefit as compared to control groups.

Example 8 -NK Cells Engineered to Express TGF-p DNR, HER2-specific CAR, and IL- 15 are Cytotoxic Against HER2⁺ Cells In Vitro and Produce Effector Cytokines

Engineered NK cells expressing TGFBR2 DNR, HER2-specific CAR, and IL- 15 (DNR/CAR/IL-15) were also capable of reacting with cytokine production and cytotoxic activity across a > 100-fold range of HER expression levels. Relative HER2 surface protein expression levels of SKOV3, NCI-N87, and HT-29 cell lines, shown in FIG. 11A, was determined by staining with a HER2-specific antibody and measured by flow cytometry. SKOV3, NCI-N87, and HT-29 HER2⁺ tumor target cell lines were engineered to express GFP, seeded in 96- well tissue culture coated plates, and incubated overnight to adhere. Unmodified, mock electroporated (control) and DNR/CAR/IL-15 (engineered) NK cells were thawed and rested overnight. At the start of the assay, tumor target and NK cells were washed, and NK cells were added to the target wells at a 10 effector : 1 target ratio, and target cell survival was evaluated by GFP fluorescence every 4 hours, as described in Example 3 (FIG. 6A). After 24 hours, 50 uL of supernatant was collected and IFN-y release was measured as described in FIG. 6B. As shown in FIG. 11B, DNR/CAR/IE-15 expression enabled greater IFN-y by engineered than control NK cells across a > 100-fold range of HER2 expression levels by the tumor target cells. Similarly, DNR/CAR/IE-15 expression also enabled greater cytotoxic activity by engineered NK cells compared to control NK cells stimulated by HER-high SKOV3 cells, as shown in FIG. 11C, HER2-intermediate NCI-N87 cells, as shown in FIG. 11D, and HER2-low HT-29 cells, as shown in FIG. HE. Together these data demonstrate that NK cells expressing TGFBR2 DNR, HER2-specific CAR, and IL- 15 gain anti-tumor activity, in both cytokine-producing and cytotoxic functions, across tumor cell line targets with a > 100-fold range of HER2 protein expression. Moreover, this finding supports that DNR/CAR/IL-15 engineered NK cells will show enhanced activity regardless of the heterogeneity in HER2 expression by tumor cells.

Example 9 - NK Cells Engineered to Express HER2-specific CAR, TGF-p DNR, and IL-15 Are Efficacious In Vivo Against HER2⁺ Tumor Xenografts and Prolong the Survival of Xenografted Mice

In a separate arm of the study described above in Example 7, three intravenous administrations (20 days apart) of engineered NK cells expressing HER2-specific CAR, TGFBR2 DNR, and IL- 15 demonstrated different anti-tumor activity when harvested 10 versus 14 days post electroporation. As shown in FIG. 12A, engineered NK cells harvested on day 10 post electroporation were able to regress established subcutaneous tumors from a peak tumor volume of ~300-400mm³. Otherwise equivalent engineered NK cells harvested on day 14 post electroporation did not result in a significant reduction in tumor burden compared to the control groups (Control NKs and Saline). Furthermore, NK cells expressing HER2-specific CAR, TGFBR2 DNR, and IL- 15 in different orders demonstrated different anti-tumor activity. As shown in FIG. 12B, a single dose of 2xl0⁶ engineered NK cells with a nucleic acid sequence encoding DNR first, CAR second, and IL- 15 third (Construct 2) achieved similar strong anti-tumor activity and tumor regression as a 4-fold higher dose of 8xl0⁶ engineered NK cells with a nucleic acid sequence encoding CAR first, DNR second, and IL- 15 third (Construct 1). Together rhese results demonstrate that changes in either the engineering and expansion process (e.g., the interval of expansion after electroporation) or the transgene order (e.g., TGFBR2 DNR first, HER2-specific CAR second, versus CAR first, DNR second) can impact the function of the resulting engineered NK cells. This impact may be qualitative, e.g., causing or failing to cause tumor regression, or quantitative, e.g., changing the efficacious dose of engineered NK cells.

Example 10 - NK Cells Engineered to Express HER2-specific CAR, TGF-p DNR, and IL-15 Infiltrate, Persist, and Retain Activity Within HER2⁺ Tumor Xenografts in vivo

To further assess the durability of in vivo anti-tumor activity of the engineered NK cells expressing a transposon encoding HER2-specific CAR, TGFBR2 DNR, and IL- 15, the following experiment is performed. HER2⁺ cancer cell line cells are implanted into groups of NSG mice via subcutaneous injection and allowed to grow into a tumor mass of desired volume prior to NK cell treatment. For example, 1 x 10⁶ HER2⁺ N87 gastric cancer cell line cells are subcutaneously implanted ten days prior to start of NK cell treatment and reach ~70 mm³. Groups of mice are treated with 1-20 x 10⁶ transposon-engineered NK cells expressing a HER2-specific CAR, TGFBR2 DNR, and IL- 15 or an equal dose of control NK cells, such as unmodified mock electroporated NK cells, administered by intravenous injection. At various time points after NK cell treatment, ranging from 1 day to 6 months, the mice are sacrificed and tumor masses excised. A tissue dissociation method, a well-established technique combining mechanical and enzymatic mechanisms, is used to recover tumor- infiltrating NK cells as a single cell suspension. NK cells are counted, stained to assess viability and phenotype, and analyzed by flow cytometry. In addition, the recovered NK cells are stimulated ex vivo using chemical (e.g., phorbol myristate acetate plus ionomycin), biological (e.g., immobilized HER2 protein, agonist antibodies to NK-activating receptors, or cytokines), and/or cellular (e.g., HER2⁺ N87 cells) to assess their responsiveness. The excised tumor masses are also analyzed by histological methods, such as immunohistochemistry or immunofluorescence, to identify and characterize the NK cells.

The greater number of viable CAR/DNR/IL-15 than control NK cells within the tumor mass over time demonstrates enhanced infiltration, retention, and/or survival. The differences in phenotype show that CAR/DNR/IL-15 NK cells are more productively activated than the control NK cells by the surrounding tumor cells, such as by measuring: a higher percentage of proliferating cells (e.g., Ki67⁺), a higher percentage of non-quiescent lymphoblasts (larger cell diameter), higher levels of activation markers (e.g., CD69, CD25), higher levels of NK-activating receptors (e.g., DNAM-1, NKG2A, NCRs), lower levels of NK-inhibiting receptors (e.g., NKG2A, CD96), higher ratios of NK-activating to NK- inhibiting receptors, higher levels of perforin and granzymes, higher in situ cytokine production (e.g., intracellular IFN-yor TNF-oc without exogenous stimulation), lower levels of phosphorylated Smad2/3, CD103, and other TGF-P-induced changes to NK cells, or higher phosphorylated levels of STAT3/5, Akt, S6 kinase, Erkl/2, and other IL-15-induced changes to NK cells. These same difference phenotypic characteristics also arise, remain evident, or grow greater following ex vivo stimulation, demonstrating that CAR/DNR/IL-15 NK cells from within the tumor mass are more responsive to activating signals than control NK cells. When stimulated with HER2⁺ target cells, the CAR/DNR/IL-15 NK cells also demonstrate higher cytotoxic potency and cytokine release than control NK cells. Histological methods also show that CAR/DNR/IL-15 NK cells have a stronger activation- associated phenotype than control NK cells when analyzed in situ within the tumor mass. Together these data show that NK cells engineered to express HER2-specific CAR, TGF-P DNR, and IL- 15 gain durable advantages in their activation and function within a model tumor compared to control NK cells.

Claims

1. A nucleic acid molecule comprising a coding region flanked by a transposase binding site, wherein the coding region comprises:

(a) a nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18, or wherein the CAR comprises an amino acid sequence that is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 1,

(b) a nucleic acid sequence encoding a second polypeptide, wherein the second polypeptide comprises a transforming growth factor beta receptor 2 (TGFBR2) dominant negative receptor, and

(c) a nucleic acid sequence encoding a third polypeptide, wherein the third polypeptide comprises a cytokine, or a functional fragment thereof.

2. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the first polypeptide is 5’ upstream of both the nucleic acid sequence encoding the second polypeptide and the nucleic acid sequence encoding the third polypeptide, and wherein the nucleic acid sequence encoding the second polypeptide is 5’ upstream of the nucleic acid sequence encoding the third polypeptide.

3. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence encoding the second polypeptide is 5’ upstream of both the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the third polypeptide, and wherein the nucleic acid sequence encoding the first polypeptide is 5’ upstream of the nucleic acid sequence encoding the third polypeptide.

4. The nucleic acid molecule of any one of claims 1-3, wherein the CAR comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

5. The nucleic acid molecule of any one of claims 1-3, wherein the CAR comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-6 or SEQ ID NOs: 13-18.

6. The nucleic acid molecule of any one of claims 1-3, wherein the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 1-6 or SEQ ID NOs: 13-18.

7. The nucleic acid molecule of any one of claims 1-3, wherein the CAR consists of an amino acid sequence of any one of SEQ ID NOs: 1-6 or SEQ ID NOs: 13-18.

8. The nucleic acid molecule of any one of claims 1-3, wherein the transposase binding site comprises a 5’ transposase binding site and/or a 3’ transposase binding site that are specifically recognized by a transposase.

9. The nucleic acid molecule of claim 8, wherein the transposase is selected from the group consisting of a TcBuster transposase, a piggyBac transposase, a Sleeping Beauty transposase, a Tn3 transposase, a Tn5 transposase, a Tn7 transposase, a TnlO transposase, a Frog Prince transposase, an IS5 transposase, a TnlO transposase, a Tn903 transposase, a SPIN transposase, a hAT transposase, a Hermes transposase, a Hobo transposase, an AeBuster transposase, a BtBuster transposase, a CfBuster transposase, a Tol2 transposase, a Tc3 transposase, a Mosl transposase, a MuA transposase, a Himar I transposase, and a Helitron transposase.

10. The nucleic acid molecule of any one of claims 8-9, wherein the 5’ transposase binding site comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of any one of SEQ ID NOs: 148, 149, 160, 161, 165, 168 or 181-183.

11. The nucleic acid molecule of any one of claims 1-3, wherein the 5’ transposase binding site comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 148, 149, 160, 161, 165, 168 or 181-183.

12. The nucleic acid molecule of any one of claims 1-3, wherein the 5’ transposase binding site comprises the nucleic acid sequence of any one of SEQ ID NOs: 148, 149, 160, 161, 165, 168 or 181-183.

13. The nucleic acid molecule of any one of claims 1-3, wherein the 3’ transposase binding site comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of any one of SEQ ID NOs: 150, 151, 162, 163, 166, 169 or 184-186.

14. The nucleic acid molecule of any one of claims 1-3, wherein the 3’ transposase binding site comprises a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of any one of SEQ ID NOs: 150, 151, 162, 163, 166, 169 or 184-186.

15. The nucleic acid molecule of any one of claims 1-3, wherein the 3’ transposase binding site comprises the nucleic acid sequence of any one of SEQ ID NOs: 150, 151, 162, 163, 166, 169 or 184-186.

16. The nucleic acid molecule of any one of claims 1-3, wherein the coding region is operably linked to a promoter.

17. The nucleic acid molecule of claim 16, wherein the promoter is selected from the group consisting of an MND promoter, an EF-la promoter, an EFS promoter, a MSCV promoter, a CMV promoter, a PGK promoter, a CAG promoter, a SFFV promoter, a CBH promoter, a SV40 promoter, a UBC promoter, or a RPBSA promoter.

18. The nucleic acid molecule of any one of claims 1-3, wherein the coding region further comprises a nucleic acid sequence encoding a fourth polypeptide.

19. The nucleic acid molecule of claim 18, wherein the fourth polypeptide comprises a cytokine receptor or a fragment thereof that specifically binds to a cytokine.

20. The nucleic acid molecule of claim 18, wherein the fourth polypeptide comprises IL- 15 receptor alpha or a fragment thereof that specifically binds to IL- 15.

21. The nucleic acid molecule of claim 18, wherein the fourth polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 102-103 or 194-198.

22. The nucleic acid molecule of claim 18, wherein the nucleic acid sequence encoding the fourth polypeptide is 3’ downstream of both the nucleic acid molecule encoding the first polypeptide and the nucleic acid molecule encoding the second polypeptide.

23. The nucleic acid molecule of claim 22, wherein the nucleic acid sequence encoding the fourth polypeptide is 5’ upstream of the nucleic acid molecule encoding the third polypeptide.

24. The nucleic acid molecule of any one of claims 1-3, wherein the coding region further comprises at least one nucleic acid sequence encoding a self-cleaving peptide.

25. The nucleic acid molecule of claim 24, wherein the self-cleaving peptide is selected from the group consisting of E2A, T2A, P2A and F2A.

26. The nucleic acid molecule of any one of claims 1-3, wherein the coding region comprises at least one nucleic acid sequence encoding an internal ribosomal entry site (IRES).

27. The nucleic acid molecule of any one of claims 1-3, wherein the cytokine, or a functional fragment thereof comprises IL- 15, or a functional fragment thereof.

28. The nucleic acid molecule of claim 27, wherein the cytokine, or the functional fragment thereof, comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 100-101.

29. The nucleic acid molecule of claim 27, wherein the cytokine, or the functional fragment thereof, comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 100-101.

30. The nucleic acid molecule of claim 27, wherein the cytokine, or the functional fragment thereof, comprises the amino acid sequence of any one of SEQ ID NOs: 100-101.

31. The nucleic acid molecule of claim 27, wherein the cytokine, or the functional fragment thereof, consists of the amino acid sequence of any one of SEQ ID NOs: 100-101.

32. The nucleic acid molecule of any one of claims 1-3, wherein the third polypeptide comprises a fusion protein comprising IL- 15 or a functional fragment thereof, and IL-15RA or a fragment thereof that specifically binds to IL- 15.

33. The nucleic acid molecule of any one of claims 1-3, wherein the TGFBR2 dominant negative receptor comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 108-113.

34. The nucleic acid molecule of any one of claims 1-3, wherein the TGFBR2 dominant negative receptor comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108-113.

35. The nucleic acid molecule of any one of claims 1-3, wherein the TGFBR2 dominant negative receptor comprises the amino acid sequence of any one of SEQ ID NOs: 108-113.

36. The nucleic acid molecule of any one of claims 1-3, wherein the TGFBR2 dominant negative receptor consists of the amino acid sequence of any one of SEQ ID NOs: 108-113.

37. A nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

38. The nucleic acid molecule of claim 37, wherein the CAR comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

39. The nucleic acid molecule of claim 37, wherein the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

40. The nucleic acid molecule of claim 37, wherein the CAR consists of an amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

41. A nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 1.

42. The nucleic acid molecule of claim 41, wherein the CAR comprises an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO: 1.

43. The nucleic acid molecule of claim 41, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 1.

44. The nucleic acid molecule of claim 41, wherein the CAR consists of an amino acid sequence of SEQ ID NO: 1.

45. A nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

46. The nucleic acid molecule of claim 45, wherein the CAR comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

47. The nucleic acid molecule of claim 45, wherein the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

48. The nucleic acid molecule of claim 45, wherein the CAR consists of an amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

49. A nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 7.

50. The nucleic acid molecule of claim 49, wherein the CAR comprises an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO: 7.

51. The nucleic acid molecule of claim 49, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 7.

52. The nucleic acid molecule of claim 49, wherein the CAR consists of an amino acid sequence of SEQ ID NO: 7.

53. A nucleic acid molecule comprising:

(a) a nucleic acid sequence encoding a first polypeptide comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18 or wherein the CAR comprises an amino acid sequence that is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 1;

(b) a nucleic acid sequence encoding a second polypeptide comprising a TGFBR2 dominant negative receptor; and

(c) a nucleic acid sequence encoding a third polypeptide comprising a cytokine, or a functional fragment thereof.

54. The nucleic acid molecule of claim 53, wherein the nucleic acid sequence encoding the first polypeptide is 5’ upstream of both the nucleic acid sequence encoding the second polypeptide and the nucleic acid sequence encoding the third polypeptide, and wherein the nucleic acid sequence encoding the second polypeptide is 5’ upstream of the nucleic acid sequence encoding the third polypeptide.

55. The nucleic acid molecule of claim 53, wherein the nucleic acid sequence encoding the second polypeptide is 5’ upstream of both the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding the third polypeptide, and wherein the nucleic acid sequence encoding the first polypeptide is 5’ upstream of the nucleic acid sequence encoding the third polypeptide.

56. The nucleic acid molecule of any one of claims 53-55, wherein the CAR comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

57. The nucleic acid molecule of any one of claims 53-55, wherein the CAR comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1-6 or SEQ ID NOs: 13-18.

58. The nucleic acid molecule of any one of claims 53-55, wherein the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 1-6 or SEQ ID NOs: 13-18.

59. The nucleic acid molecule of any one of claims 53-55, wherein the CAR consists of an amino acid sequence of any one of SEQ ID NOs: 1-6 or SEQ ID NOs: 13-18.

60. The nucleic acid molecule of any one of claims 53-55, wherein the TGFBR2 dominant negative receptor comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 108-113.

61. The nucleic acid molecule of any one of claims 53-55, wherein the TGFBR2 dominant negative receptor comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of any one of SEQ ID NOs: 108-113.

62. The nucleic acid molecule of any one of claims 53-55, wherein the TGFBR2 dominant negative receptor comprises the amino acid sequence of any one of SEQ ID NOs: 108-113.

63. The nucleic acid molecule of any one of claims 53-55, wherein the TGFBR2 dominant negative receptor consists of an amino acid sequence of any one of SEQ ID NOs: 108-113.

64. The nucleic acid molecule of any one of claims 53-55, wherein the cytokine is selected from the group consisting of IL-15, IL-2, IL-12, IL-18, IL-21, LIGHT, CD40L, FLT3L, 4-1BBL and FASL, or a functional fragment thereof.

65. The nucleic acid molecule of claim 64, wherein the cytokine is IL-15, IL-2, IL- 12, IL- 18 or IL-21, and the cytokine is expressed as a fusion protein with a transmembrane domain.

66. The nucleic acid molecule of claim 64, wherein the cytokine is IL-15, or a functional fragment thereof.

67. The nucleic acid molecule of claim 66, wherein the IL- 15, or the functional fragment thereof, comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 100-101.

68. The nucleic acid molecule of claim 66, wherein the IL- 15, or the functional fragment thereof, comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 100-101.

69. The nucleic acid molecule of claim 66, wherein the IL- 15, or the functional fragment thereof, comprises the amino acid sequence of any one of SEQ ID NOs: 100-101.

70. The nucleic acid molecule of 66, wherein the IL- 15, or the functional fragment thereof, consists of the amino acid sequence of any one of SEQ ID NOs: 100-101.

71. The nucleic acid molecule of any one of claims 53-55, wherein the third polypeptide comprises a fusion protein comprising IL- 15 or a functional fragment thereof, and IL-15RA or a fragment thereof that specifically binds to IL- 15.

72. The nucleic acid molecule of any one of claims 53-55, further comprising a nucleic acid sequence encoding a fourth polypeptide.

73. The nucleic acid molecule of claim 72, wherein the fourth polypeptide comprises a cytokine receptor or a fragment thereof that specifically binds to a cytokine.

74. The nucleic acid molecule of claim 72, wherein the fourth polypeptide comprises IL- 15 receptor alpha or a fragment thereof that specifically binds to IL- 15.

75. The nucleic acid molecule of claim 74, wherein the fourth polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 102-103 or 194-198.

76. The nucleic acid molecule of claim 72, wherein the nucleic acid sequence encoding the fourth polypeptide is 3’ downstream of both the nucleic acid molecule encoding the first polypeptide and the nucleic acid molecule encoding the second polypeptide.

77. The nucleic acid molecule of claim 76, wherein the nucleic acid sequence encoding the fourth polypeptide is 5’ upstream of the nucleic acid molecule encoding the third polypeptide.

78. The nucleic acid molecule of any one of claims 53-55, further comprising at least one nucleic acid sequence encoding a self-cleaving peptide.

79. The nucleic acid molecule of claim 78, wherein the self-cleaving peptide is selected from E2A, T2A, P2A and F2A.

80. The nucleic acid molecule of any one of claims 53-55, further comprising at least one nucleic acid sequence encoding an internal ribosomal entry site (IRES).

81. A nucleic acid molecule encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 133-144.

82. A nucleic acid molecule encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 133-144.

83. A nucleic acid molecule encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 133-144.

84. The nucleic acid molecule of any one of claims 1, 37, 41, 45, 49, 53, and 81-83, wherein the nucleic acid molecule comprises an origin of replication.

85. The nucleic acid molecule of any one of claims 1, 37, 41, 45, 49, 53, and 81-83, wherein the nucleic acid molecule is a DNA molecule.

86. The nucleic acid molecule of any one of claims 1, 37, 41, 45, 49, 53, and 81-83, wherein the nucleic acid molecule is an RNA molecule.

87. The nucleic acid molecule of any one of claims 1, 37, 41, 45, 49, 53, and 81-83, wherein the nucleic acid molecule is a circular molecule.

88. An expression vector comprising the nucleic acid molecule of any one of claims 1, 37, 41, 45, 49, 53, and 81-83.

89. The nucleic acid molecule of any one of claims 1, 37, 41, 45, 49, 53, and 81-83, wherein the nucleic acid molecule is a linear nucleic acid molecule.

90. An engineered immune cell comprising the nucleic acid molecule of any one of claims 1, 37, 41, 45, 49, 53, and 81-83.

91. The engineered immune cell of claim 90, wherein the immune cell is a natural killer (NK) cell, a T cell, or a NKT cell.

92. An engineered natural killer (NK) cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

93. The engineered NK cell of claim 92, wherein the CAR comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

94. The engineered NK cell of claim 92, wherein the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

95. The engineered NK cell of claim 92, wherein the CAR consists of an amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

96. An engineered natural killer (NK) cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence that is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 1.

97. The engineered NK cell of claim 96, wherein the CAR comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 1.

98. The engineered NK cell of claim 96, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 1.

99. The engineered NK cell of claim 96, wherein the CAR consists of an amino acid sequence of SEQ ID NO: 1.

100. An engineered natural killer (NK) cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence that is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

101. The engineered NK cell of claim 100, wherein the CAR comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

102. The engineered NK cell of claim 100, wherein the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

103. The engineered NK cell of claim 100, wherein the polypeptide consists of an amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

104. An engineered natural killer (NK) cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence that is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 7.

105. The engineered NK cell of claim 104, wherein the CAR comprises an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO: 7.

106. The engineered NK cell of claim 104, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 7.

107. The engineered NK cell of claim 104, wherein the CAR consists of an amino acid sequence of SEQ ID NO: 7.

108. A population of cells comprising the engineered immune cell of claim 90, or the engineered NK cell of any one of claims 92, 96, 100, and 104.

109. The population of cells of claim 108, wherein at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the cells in the population express a CAR on the cell surface.

110. The population of cells of claim 108, wherein the population of cells comprises the engineered NK cell, and wherein the engineered NK cell exhibits an NK cell effector function.

111. The population of cells of claim 110, wherein the NK cell effector function is selected from the group consisting of HER2-dependent cytotoxicity, directed secretion of cytolytic granules, expression of CD107a, expression of CD69, production of tumor necrosis factor (TNF)-alpha, and production of interferon (IFN)-gamma.

112. The population of cells of any one of claims 108-111, wherein the population exhibits one or more NK cell effector functions at a level that is at least 0.5-fold, at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 5-fold, or at least 10-fold higher relative to a population of cells that do not comprise NK cells expressing a CAR.

113. The population of cells of any one of claims 111-112, wherein the population exhibits HER2-dependent cytotoxicity at a level that is at least 0.5-fold, at least 1-fold, at least 1.5- fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 5-fold, or at least 10-fold higher relative to a population of cells that do not comprise NK cells expressing a CAR.

114. A pharmaceutical composition comprising the engineered immune cell of claim 90 or claim 91, the engineered NK cell of any one of claims 92, 96, 100, and 104, or the population of cells of claim 108, and a pharmaceutically acceptable carrier.

115. A method for treating a HER2 -positive cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the engineered immune cell of claim 90 or claim 91, the engineered NK cell of any one of claims 92, 96, 100, and 104, the population of cells of claim 108, or the pharmaceutical composition of claim 114, thereby treating the HER2-positive cancer in the subject.

116. The method of claim 115, wherein the cancer comprises a solid tumor.

117. The method of claim 115 or claim 116, wherein the cancer is selected from the group consisting of a breast cancer, a gastric cancer, an esophageal cancer, an esophagogastric junction (GEJ) cancer, an ovarian cancer, a medulloblastoma, an osteosarcoma, a non-small cell lung carcinoma, a colorectal rectal cancer, a bladder cancer, and a prostate cancer.

118. The method of claim 115, wherein the engineered immune cell or the engineered NK cell is an allogeneic cell.

119. The method of claim 115, wherein the engineered immune cell or the engineered NK cell is an autologous cell.

120. The method of claim 115, wherein the method further comprises administering an additional therapeutic agent to the subject.

121. The method of claim 120, wherein the additional therapeutic agent is selected from the group consisting of an immune activator, a tyrosine kinase inhibitor, a metabolic inhibitor, an immune checkpoint inhibitor, a cytokine, a hypomethylating agent, and a therapeutic agent that targets HER2.

122. The method of claim 121, wherein:

(a) the additional therapeutic is the immune activator and wherein the immune activator is selected from the group consisting of 4-1BBL and OX-40;

(b) the additional therapeutic is the metabolic inhibitor, and wherein the metabolic inhibitor is selected from the group consisting of an A2AR inhibitor and an IDO inhibitor;

(c) the additional therapeutic is the checkpoint inhibitor and wherein the checkpoint inhibitor is an inhibitor of a protein selected from the group consisting of PD-1, PD-L1, PD- L2, CTLA4, B7-H3, BTLA, KIR, LAG3, TIM-3, VISTA, AHR, c-cbl, and HPK1;

(d) the additional therapeutic is the cytokine and wherein the cytokine is selected from the group consisting of IL-2, IL-15, IL-12, IL-18, IL-21, and a functional fragment thereof; and/or (e) the additional therapeutic is the therapeutic agent that targets HER2 and wherein the therapeutic agent that targets HER2 is selected from the group consisting of trastuzumab, pertuzumab, margetuximab, trastuzumab-DM-1, lapatinib, neratinib and tucatinib.

123. A method for generating an engineered natural killer (NK) cells, the method comprising:

(a) providing an NK cell or a precursor thereof;

(b) contacting the NK cell or the precursor thereof with the nucleic acid molecule of any one of claims 1, 37, 41, 45, 49, 53, and 81-83, under conditions sufficient to transfer the nucleic acid molecule across a cell membrane of the NK cell or the precursor thereof; and

(c) culturing the NK cell or the precursor thereof under conditions suitable for expression of the nucleic acid molecule, thereby generating an engineered NK cell.

124. The method of claim 123, further comprising contacting the NK cell with a transposase or a nucleic acid molecule encoding a transposase.

125. The method of claim 124, wherein the transposase is selected from the group consisting of of a TcBuster transposase, a piggyBac transposase, a Sleeping Beauty transposase, a Tn3 transposase, a Tn5 transposase, a Tn7 transposase, a TnlO transposase, a Frog Prince transposase, an IS5 transposase, a TnlO transposase, a Tn903 transposase, a SPIN transposase, a hAT transposase, a Hermes transposase, a Hobo transposase, an AeBuster transposase, a BtBuster transposase, a CfBuster transposase, a Tol2 transposase, a Tc3 transposase, a Mosl transposase, a MuA transposase, a Himar I transposase, and a Helitron transposase.

126. A polypeptide comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

127. The polypeptide of claim 126, wherein the CAR comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

128. The polypeptide of claim 126, wherein the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

129. The polypeptide of claim 126, wherein the CAR consists of an amino acid sequence of any one of SEQ ID NOs: 2-6 or SEQ ID NOs: 13-18.

130. A polypeptide comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 1.

131. The polypeptide of claim 130, wherein the CAR comprises an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO: 1.

132. The polypeptide of claim 130, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 1.

133. The polypeptide of claim 130, wherein the CAR consists of an amino acid sequence of SEQ ID NO: 1.

134. A polypeptide comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 92% identical to the amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

135. The polypeptide of claim 134, wherein the CAR comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

136. The polypeptide of claim 134, wherein the CAR comprises an amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

137. The polypeptide of claim 134, wherein the CAR consists of an amino acid sequence of any one of SEQ ID NOs: 8-12 or SEQ ID NOs: 19-24.

138. A polypeptide comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an amino acid sequence which is at least 98.5% identical to the amino acid sequence of SEQ ID NO: 7.

139. The polypeptide of claim 138, wherein the CAR comprises an amino acid sequence which is at least 99% identical to the amino acid sequence of SEQ ID NO: 7.

140. The polypeptide of claim 138, wherein the CAR comprises an amino acid sequence of SEQ ID NO: 7.

141. The polypeptide of claim 138, wherein the CAR consists of an amino acid sequence of SEQ ID NO: 7.

142. A polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 133-144.

143. The polypeptide of claim 142, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 133-144.

144. The polypeptide of claim 142, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 133-144.

145. The polypeptide of claim 142, wherein the polypeptide consists of an amino acid sequence of any one of SEQ ID NOs: 133-144.