WO2025179221A1

WO2025179221A1 - Compositions and methods for armored cell based therapies

Info

Publication number: WO2025179221A1
Application number: PCT/US2025/016910
Authority: WO
Inventors: Kanya Lakshmi RAJANGAM
Original assignee: Senti Biosciences Inc
Current assignee: Senti Biosciences Inc
Priority date: 2024-02-22
Filing date: 2025-02-21
Publication date: 2025-08-28
Anticipated expiration: 2026-08-22

Abstract

Described herein are NK cells engineered to express cytokines and chimeric receptors. Also described herein are nucleic acids, cells, and methods directed to the same.

Description

Compositions and Methods for Armored Cell Based Therapies

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Application No. 63/556,835, filed on February 22, 2024, the entire disclosure of which is incorporated by reference herein in its entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on XXXX , is named STB-053WO and is XXXX bytes in size.

BACKGROUND

Cell-based therapy platforms provide promising avenues for treating a variety of diseases. One such promising platform is CAR-T based therapies in the treatment of cancer. Given their promise, improvements in cell-based therapies are needed. An active area of exploration is engineering cell-based therapies to produce and/or secrete effector molecules such as cytokines, a process referred to as armoring, that enhance the cell-based therapy. For example, unarmored CAR-T therapies have poor efficacy in solid tumors and armoring can impact the entire cancer immunity cycle and boost the activity of CAR-T. However, uncontrolled or unregulated armoring strategies can have negative impacts on treatment, such as off-target effects and toxicity in subjects.

For example, GPC3, a membrane-associated heparan sulfate proteoglycan, is co-opted in various solid tumors including HCC to promote cancer growth. It is minimally or not expressed in healthy cells making it an attractive target for cell therapy. However, Phase 2 HCC clinical trial with GPC3 targeting monoclonal antibody showed no PFS difference compared to placebo. Furthermore, GPC3 CAR-T cell trials to date have shown activity but limited by toxicities, thus precluding multiple dosing and limiting durability.

SUMMARY

Provided herein, in some embodiments, is a cell-based therapy platform involving regulated armoring of the cell-based therapy, such as regulated secretion of payload effector molecules. Also provided herein, in some embodiments, is a cell-based immunotherapy involving regulated armoring for the targeted treatment of cancer, such as ovarian cancer, breast cancer, colon cancer, lung cancer, and pancreatic cancer.

The therapy provided herein, however, can limit systemic toxicity of armoring. For example, the immunotherapy provided herein can be tumor- specific and effective while limiting systemic toxicity and/or other off-target effects due to armoring. These therapies deliver proteins of interest, such as immunomodulatory effector molecules, in a regulated manner, including regulation of secretion kinetics, cell state specificity, and cell or tissue specificity. The design of the delivery vehicle is optimized to improve overall function in cell-based therapies, such as cancer therapy, including, but not limited to, optimization of the membrane-cleavage sites, promoters, linkers, signal peptides, delivery methods, combination, regulation, and order of the immunomodulatory effector molecules.

Provided herein are methods of stimulating a cell-mediated immune response to a tumor, reducing tumor volume, or providing an anti-tumor immunity in a human subject in need thereof. Generally, the methods comprise: (a) administering to the subject a first dose of engineered NK cells; (b) about seven days (e.g., 6-8 days) following (a), administering to the subject a second dose of engineered NK cells; and (c) about seven days (e.g., 6-8 days) following (b), administering to the subject a third dose of engineered NK cells.

In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells can each comprise at least about 500 million engineered NK cells, at least about 1 billion engineered NK cells, at least about 1.5 billion engineered NK cells, or at least about 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 500 million engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 1 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 1.5 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 2 billion engineered NK cells.

In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise about 500 million engineered NK cells, about 1 billion engineered NK cells, about 1.5 billion engineered NK cells, or about 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise about 500 million NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise about 1 billion NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise about 1.5 billion NK cells.

In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least 500 million engineered NK cells, at least 1 billion engineered NK cells, at least 1.5 billion engineered NK cells, or at least 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least 500 million engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least 1 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least 1.5 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least 2 billion engineered NK cells.

In some embodiment, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 500 million engineered NK cells, 1 billion engineered NK cells, 1.5 billion engineered NK cells, or 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 500 million engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 1 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 1.5 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 2 billion engineered NK cells.

In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise about 500 million to about 2 billion engineered NK cells, about 500 million to about 1.5 billion engineered NK cells, about 500 million to about 1 billion engineered NK cells, about 1 billion to about 2 billion engineered NK cells, about 1 billion to about 1.5 billion engineered NK cells, or about 1.5 billion to about 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 500 million to 2 billion engineered NK cells, 500 million to 1.5 billion engineered NK cells, 500 million to 1 billion engineered NK cells, 1 billion to 2 billion engineered NK cells, 1 billion to 1.5 billion engineered NK cells, or 1.5 billion to 2 billion engineered NK cells.

In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 3xlO^A7 cells/kg body weight, 1.5xlO^A7 cells/kg body weight, or 4.5xlO^A7 cells/kg body weight.

The first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells comprise an engineered nucleic acid comprising: a first promoter operably linked to a first exogenous polynucleotide sequence and a second exogenous polynucleotide sequence, wherein the first exogenous polynucleotide sequence encodes a chimeric antigen receptor (CAR) that binds to GPC3, and wherein the second exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N- terminal to C-terminal, having the formula:

S - C - MT or MT - C - S wherein

S comprises a secretable effector molecule that is or comprises IL15,

C comprises a protease cleavage site, and

MT comprises a cell membrane tethering domain, and wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.

In some embodiments, steps (a), (b), and (c) are comprised in a first dosing cycle, and the method further comprises repeating the dosing cycle for a total of 2, 3, 4, 5, or 6 dosing cycles. In some embodiments, each dosing cycle has a duration of 28 days, with (a) occurring on day 0, (b) occurring on day 7, and (c) occurring on day 14 of the 28 day duration. In some embodiments, the method further comprises administering to the subject an anti-histamine and an anti-pyretic agent 30-60 minutes prior to the 1st dose, the second dose, and/or the third dose, and then every 8 hours for 24 hours.

In some embodiments, the subject has, prior to (a), been administered one or more lymphodepletion agents. In some embodiments, the one or more lymphodepletion agents comprises fludarabine and cyclophosphamide. In some embodiments, the subject has been administered, in the five to three days prior to (a), fludarabine at about 30 mg/m2/day over 30 minutes daily, and cyclophosphamide at about 500 mg/m2/day over 30-60 minutes daily. In some embodiments, the method comprises, in the three to five days prior to (a), administering fludarabine at about 30 mg/m2/day over 30 minutes daily, and cyclophosphamide at about 500 mg/m2/day over 30-60 minutes daily. In some embodiments, the method comprises administering oral and IV hydration to the subject prior to the cyclophosphamide administration.

In some embodiments, the CAR comprises an antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of KNAMN, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY , and wherein the VL comprises: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of WASSRES, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT.

In some embodiments, the VH region comprises an amino acid sequence with 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% identity to the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIRNKTNN YATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFAYWGQGTLVT VSA.

In some embodiments, the VH region comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIRNKTNN YATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFAYWGQGTLVT VSA, optionally wherein the VH region comprises the amino acid sequence MEVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIRNKT NNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFAYWGQGTL VTVSA.

In some embodiments, the VL region comprises an amino acid sequence with 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% identity to the amino acid sequence of: DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASS RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEIK. In some embodiments, the VL region comprises the amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASS RES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEIK, optionally wherein the VE region comprises the amino acid sequence DIVMTQSPDSEAVSEGERATINCKSSQSEEYSSNQKNYEAWYQQKPGQPPKEEIYWASS RESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEIKS.

In some embodiments, the antigen-binding domain comprises a single chain variable fragment (scFv).

In some embodiments, the VH region and the VE region are separated by a peptide linker (L). In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH. In some embodiments, the peptide linker (L) comprises a glycine-serine (GS) linker. In some embodiments, the GS linker comprises the amino acid sequence of (GGGGS)3.

In some embodiments, the CAR comprises a hinge domain, optionally wherein the hinge domain is derived from CD8, optionally wherein the hinge domain comprises the sequence GALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACD.

In some embodiments, the CAR comprises a transmembrane domain. In some embodiments, the transmembrane domain is derived from CD28 or CD8. In some embodiments, the transmembrane domain is derived from CD8. In some embodiments, the transmembrane domain comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYCNHR.

In some embodiments, the CAR comprises one or more intracellular signaling domains. In some embodiments, at least one of the one or more intracellular signaling domains is derived from CD28. In some embodiments, the intracellular signaling domain derived from CD28 comprises the sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.

In some embodiments, the CAR comprises an intracellular signaling domain derived from CD28 and an intracellular signaling domain derived from CD3zeta. In some embodiments, the ICD derived from CD3zeta comprises the amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

In some embodiments, the CAR further comprises a signal sequence. In some embodiments, the signal sequence comprised in the CAR is derived from GM-CSF-Ra, optionally wherein the signal sequence comprises the amino acid sequence MLLLVTSLLLCELPHPAFLLIP, optionally wherein the signal sequence operably linked to the CAR comprises the amino acid sequence MLLLVTSLLLCELPHPAFLLIPH. In some embodiments, the CAR comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence: MLLLVTSLLLCELPHPAFLLIPHMEVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMN WVRQAPGKGLEWVGRIRNKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTED TAVYYCVAGNSFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGE RATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTD FTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEIKSGALSNSIMYFSHFVPVFLPAKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYCNHRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

In some embodiments, the IL 15 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical, or 100% identical to NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

In some embodiments, the secretable effector molecule further comprises a signal sequence. In some embodiments, the signal sequence is derived from IgE. In some embodiments, the signal sequence comprises the amino acid sequence MDWTWILFLVAAATRVHS

In some embodiments, the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI.

In some embodiments, the protease cleavage site is cleavable by a protease, optionally wherein the protease cleavage site is cleavable by an ADAM 17 protease. In some embodiments, the protease is endogenously expressed by at least a portion of the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells.

In some embodiments of S-C-MT or MT-C-S, C further comprises one or more glycineserine linker motifs, optionally wherein the glycine-serine linker motif comprises (GGGS) or (GGGGS). In some embodiments, C comprises the amino acid sequence SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ.

In some embodiments, the cell membrane tethering domain is derived from B7-1. In some embodiments, the cell membrane tethering domain comprises the amino acid sequence LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV.

In some embodiments, the first exogenous polynucleotide sequence and the second exogenous polynucleotide sequence are separated by a linker polynucleotide sequence encoding an E2A/T2A ribosome skipping element. In some embodiments, the E2A/T2a ribosome skipping element comprises the amino acid sequence GSGQCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP.

In some embodiments, the tumor is a GPC3 expressing tumor. In some embodiments, the GPC3 expressing tumor is selected from the group consisting of: hepatocellular carcinoma (HCC), lung squamous cell cancer, esophageal squamous cell cancer, pancreatic cancer, papillary thyroid cancer, lung large cell cancer, AFP producing gastric cancer, follicular thyroid cancer, medullary thyroid cancer, ovarian clear cell carcinoma, melanoma, hepatoblastoma, nephroblastoma (Wilms tumor), hepatoblastoma, and yolk sac tumor. In some embodiments, the tumor is HCC, optionally wherein the tumor is unresectable, recurrent, and/or metastatic HCC. In some embodiments, over 50% of the subject’s liver is not occupied by the HCC tumor.

In some embodiments, the subject had received at least one prior treatment and have been exposed to a checkpoint inhibitor and a tyrosine kinase inhibitor. In some embodiments, the subject had received a prior systemic treatment for the tumor. In some embodiments, the prior systemic treatment comprises a chemotherapy per respective NCCN guidelines for the indication, an immune checkpoint inhibitor approved for commercial use for the indication, and/or an agent that targets a tumor-associated mutation.

In some embodiments, the subject does not have a history of organ transplantation and is not on a waiting list for organ transplantation, including liver transplantation.

In some embodiments, tumor thrombus is not present in the portal vein, mesenteric vein, or inferior vena of the subject based on imaging.

In some embodiments, the subject is not or has not been diagnosed with brain or leptomeningeal metastases.

In some embodiments, the subject has not been previously administered a prior adoptive cell therapy, a prior GPC3 targeted anti-cancer therapy.

In some embodiments, the subject has not been previously administered an investigational therapy within 14 days prior to (a).

In some embodiments, the subject has not been administered an anti-cancer chemotherapeutic or targeted small molecule drug within 14 days or 5 half-lives (whichever is shorter), or an anti-cancer biologic within 28 days prior to (a).

In some embodiments, the subject has recovered from toxicities related to prior treatment to < Gr 2.

In some embodiments, the subject has not been chronically administered an immunosuppressive agent or corticosteroids at >10mg/day prednisone or equivalent, optionally wherein the subject has not been chronically administered the immunosuppressive agent or corticosteroids at > 10 mg/day within 14 days prior to (a). In some embodiments, the subject does not have a history of significant cardiac or pulmonary disease or dysfunction within 12 weeks of (a).

In some embodiments, the subject does not have a history of infection selected from: (i) known active HIV infection, (ii) active or latent hepatitis B or C infection in cases wherein the tumor is not an HCC, and (iii) ongoing active infection requiring systemic anti-infectives within 7 days prior to the first dose, in cases wherein the tumor is not an HCC and the systemic anti- infectives within 7 days prior to the first dose is for use in treatment of hepatitis B or C infection.

In some embodiments, the subject does not have a history of prior malignancy, unless the prior malignancy comprises adequately treated basal cell or squamous cell skin cancer, in-situ cervical cancer, prostate cancer with stable PSA, or other prior malignancy wherein the subject has been malignancy free for 2 years prior to selection for administration of the first dose, the second dose, and the third dose.

In some embodiments, the subject is not or has not been chronically administered an immunosuppression agent.

Also provided herein are kits. In some aspects, a kit comprises: (a) a first dose of engineered NK cells, comprising at least about 500 million engineered NK cells, at least about 1 billion engineered NK cells, at least about 1.5 billion engineered NK cells, at least about 2 billion engineered NK cells, at least about 500 million to at least about 2 billion engineered NK cells, at least about 500 million to at least about 1.5 billion engineered NK cells, at least about 500 million to at least about 1 billion engineered NK cells, at least about 1 billion to at least about 2 billion engineered NK cells, at least about 1 billion to at least about 1.5 billion engineered NK cells, or at least about 1.5 billion to at least about 2 billion engineered NK cells; and (b) instructions for use in performing a method provided herein, and wherein the first dose of engineered NK cells comprises an engineered nucleic acid comprising: a first promoter operably linked to a first exogenous polynucleotide sequence and a second exogenous polynucleotide sequence, wherein the first exogenous polynucleotide sequence encodes a chimeric antigen receptor (CAR) that binds to GPC3, and wherein the second exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:

S - C - MT or MT - C - S wherein

S comprises a secretable effector molecule that is or comprises IL15, C comprises a protease cleavage site, and

In some aspects, a kit comprises: (a) a first dose of engineered NK cells, comprising about 500 million engineered NK cells, about 1 billion engineered NK cells, about 1.5 billion engineered NK cells, about 2 billion engineered NK cells, about 500 million to about 2 billion engineered NK cells, about 500 million to about 1.5 billion engineered NK cells, about 500 million to about 1 billion engineered NK cells, about 1 billion to about 2 billion engineered NK cells, about 1 billion to about 1.5 billion engineered NK cells, or about 1.5 billion to about 2 billion engineered NK cells; and (b) instructions for use in performing a method provided herein, and wherein the first dose of engineered NK cells comprises an engineered nucleic acid comprising: a first promoter operably linked to a first exogenous polynucleotide sequence and a second exogenous polynucleotide sequence, wherein the first exogenous polynucleotide sequence encodes a chimeric antigen receptor (CAR) that binds to GPC3, and wherein the second exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:

S - C - MT or MT - C - S wherein

S comprises a secretable effector molecule that is or comprises IL15,

C comprises a protease cleavage site, and

In some aspects, a kit comprises: (a) a first dose of engineered NK cells, comprising at least 500 million engineered NK cells, at least 1 billion engineered NK cells, at least 1.5 billion engineered NK cells, at least 2 billion engineered NK cells, at least 500 million to at least 2 billion engineered NK cells, at least 500 million to at least 1.5 billion engineered NK cells, at least 500 million to at least 1 billion engineered NK cells, at least 1 billion to at least 2 billion engineered NK cells, at least 1 billion to at least 1.5 billion engineered NK cells, or at least 1.5 billion to at least 2 billion engineered NK cells; and (b) instructions for use in performing a method provided herein, and wherein the first dose of engineered NK cells comprises an engineered nucleic acid comprising: a first promoter operably linked to a first exogenous polynucleotide sequence and a second exogenous polynucleotide sequence, wherein the first exogenous polynucleotide sequence encodes a chimeric antigen receptor (CAR) that binds to GPC3, and wherein the second exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:

S - C - MT or MT - C - S wherein

S comprises a secretable effector molecule that is or comprises IL15,

C comprises a protease cleavage site, and

In some aspects, a kit comprises: (a) a first dose of engineered NK cells, comprising 500 million engineered NK cells, 1 billion engineered NK cells, 1.5 billion engineered NK cells, 2 billion engineered NK cells, 500 million to 2 billion engineered NK cells, 500 million to 1.5 billion engineered NK cells, 500 million to 1 billion engineered NK cells, 1 billion to 2 billion engineered NK cells, 1 billion to 1.5 billion engineered NK cells, or 1.5 billion to 2 billion engineered NK cells; and (b) instructions for use in performing a method provided herein, and wherein the first dose of engineered NK cells comprises an engineered nucleic acid comprising: a first promoter operably linked to a first exogenous polynucleotide sequence and a second exogenous polynucleotide sequence, wherein the first exogenous polynucleotide sequence encodes a chimeric antigen receptor (CAR) that binds to GPC3, and wherein the second exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:

S - C - MT or MT - C - S wherein

S comprises a secretable effector molecule that is or comprises IL15,

C comprises a protease cleavage site, and

MT comprises a cell membrane tethering domain, and wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.In some aspects, the administering comprises systemic administration. In some aspects, the administering comprises intratumoral administration. In some aspects, the engineered NK cells are derived from the subject. In some aspects, the engineered NK cells are allogeneic with reference to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic of a cytokine-CAR bidirectional construct in head-to-head directionality (FIG. 1A), head-to-tail directionality (FIG. IB), tail-to-tail directionality (FIG. 1C), and.an exemplary anti-GPC3 CAR + IL15 bidirectional construct (FIG. ID).

FIG. 2 provides CAR expression plots assessed by flow cytometry for cells transduced with lentivirus encoding a CAR + IL 15 bidirectional construct and cells transduced with a lentivirus encoding the CAR-only (day 7).

FIG. 3 provides CAR expression plots assessed by flow cytometry for cells transduced with retrovirus encoding a CAR + IL 15 bidirectional construct and cells transduced with a retrovirus encoding the CAR-only (day 7).

FIG. 4 provides CAR expression plots assessed by flow cytometry for cells transduced with lentivirus encoding a CAR + IL 15 bidirectional construct and cells transduced with a lentivirus encoding the CAR-only (day 15).

FIG. 5 provides CAR expression plots assessed by flow cytometry for cells transduced with retrovirus encoding a CAR + IL 15 bidirectional construct and cells transduced with a retrovirus encoding the CAR-only (day 15).

FIG. 6 provides IL15 levels assessed by immunoassay for NK cells transduced with lentiviruses encoding CAR + IL 15 bidirectional construct (“Lenti”) or y-retroviruses encoding CAR + IL15 bidirectional constructs (“SinVec”).

FIG. 7 provides killing by NK cells transduced with lentiviruses encoding CAR-only or CAR + IL15 bidirectional constructs, as assessed by a co-culture killing assay.

FIG. 8 provides killing by NK cells transduced with y-retroviruses encoding CAR-only or CAR + IL15 bidirectional constructs, as assessed by a co-culture killing assay.

FIG. 9 illustrates a schematic of bidirectional construct encoding a cleavable release IL15.

FIG. 10 provides a summary of IL 15 bicistronic constructs tested and performance in functional assays.

FIG. 11A and FIG. 11B provide expression plots as assessed by flow cytometry for NK cells transduced with SB06251, SB06257, and SB06254, for GPC3 CAR and IL15. Two independent replicates are shown (FIG. 11A and FIG. 11B). FIG. 12A and FIG. 12B provides secreted IL15 levels as assessed by immunoassay for NK cells transduced with SB06251, SB06257, and SB06254. Two independent replicates are shown (FIG. 12A and FIG. 12B).

FIG. 13A and FIG. 13B provide cell growth of target cell population following coculture with NK cells transduced with SB06251, SB06257, and SB06254. Two independent replicates are shown (FIG. 13A and FIG. 13B).

FIG. 14 provides target cell counts in a serial-killing assay when co-cultured with NK cells transduced with SB06251, SB06257, and SB06254.

FIG. 15A and FIG. 15B provide expression plots as assessed by flow cytometry for NK cells transduced with SB06252, SB06258, and SB06255, for GPC3 CAR and IL15. Two independent replicates are shown (FIG. 15A and FIG. 15B).

FIG. 16A and FIG. 16B provide secreted IL15 levels as assessed by immunoassay for NK cells transduced with SB06252, SB06258, and SB06255. Two independent replicates are shown (FIG. 16A and FIG. 16B).

FIG. 17A and FIG. 17B provide cell growth of target cell population following coculture with NK cells transduced with SB06252, SB06258, and SB06255. Two independent replicates are shown (FIG. 17A and FIG. 17B).

FIG. 18 provides target cell counts in a serial-killing assay when co-cultured with NK cells transduced with SB06252, SB06258, and SB06255.

FIG. 19A and FIG. 19B provide expression plots as assessed by flow cytometry for NK cells transduced with bicistronic constructs SB06261, SB6294, and SB6298, for GPC3 CAR and IL15. Two independent replicates are shown (FIG. 19A and FIG. 19B).

FIG. 20A and FIG. 20B provide secreted IL15 levels as assessed by immunoassay for NK cells transduced with SB06261, SB6294, and SB6298. Two independent replicates are shown (FIG. 20A and FIG. 20B).

FIG. 21A and FIG. 21B provide cell growth of target cell population following coculture with NK cells transduced with SB06252, SB06258, and SB06255. Two independent replicates are shown (FIG. 21 A and FIG. 21B).

FIG. 22A and FIG. 22B provide characterization of cleavable release IL 15 bicistronic constructs SB06691, SB06692, and SB06693. Expression plots as assessed by flow cytometry for NK cells transduced with SB06691, SB06692, and SB06693, for GPC3 CAR and IL15, are shown in FIG. 22A. Secreted IL15 levels as assessed by immunoassay for NK cells transduced with SB06691, SB06692, and SB06693 are shown in FIG. 22B.

FIG. 23A, FIG. 23B, and FIG. 23C provide characterization of cells transduced with different constructs expressing the GPC3 CAR and IL15. FIG. 23A shows flow cytometry plots demonstrating expression of GPC3 CAR, membrane bound IL15, and respective copy numbers on NK cells transduced with different GPC3 CAR/IL15 expression constructs. FIG. 23B shows measurement of secreted IL- 15. FIG. 23C shows cell killing of HepG2 as assessed by a serial killing assay.

FIG. 24A and FIG. 24B provide additional data of serial killing using transduced NK Cells. FIG. 24A shows serial killing of HepG2 cells. FIG. 24B shows serial killing of HuH-7 cells.

FIG. 25A and FIG. 25B provide data assessing transduced NK cell function using rapid expansion (G-Rex). FIG. 25A shows expression of GPC3 CAR, membrane bound IL 15 (mIL15), and secreted IL15 (sIL15). FIG. 25B shows serial killing of the transduced NK cells.

FIG. 26 provides results from a xenograft tumor model as measured by bioluminescence imaging, in which mice are injected with NK cells.

FIG. 27A, FIG. 27B, and FIG. 27C provide the results of a xenograft tumor model in mice that are injected with NK cells and summary. FIG. 27A provides a survival curve of mice treated with NK cells. FIG. 27B provides a summary of the median survival of mice treated with the NK cells. FIG. 27C provides survival curve and median survival from extended time points.

FIG. 28 provides results of a BLI experiment to assess tumor reduction in mice injected with NK cells.

FIG. 29 provides a quantification of each condition in terms of BLI measurements that were normalized to day 10.

FIG. 30A and FIG. 30B provide results from a xenograft tumor (HepG2) mouse model in which mice were injected three times with NK cells over the course of the study. FIG. 30A provides results of mice that were imaged using BLI. FIG. 30B provides a time course of fold change of BLI over the course of the study.

FIG. 31A and FIG. 31B provide the fold change BLI in mice injected with transduced NK cells. FIG. 31A provides results corresponding to measurements performed 13 days after tumor implantation. FIG. 31B provides results corresponding to measurements performed 20 days after tumor implantation.

FIG. 32A and FIG. 32B provide results of tumor reduction in a xenograft model. FIG. 32A shows a summary of the BLI Fold change in two different in vivo experiments. FIG. 32B shows a summary of the normalized mean BLI Fold change in two different in vivo experiments, but the treatment groups are separated, and animal are tracked individually. FIG. 33A and FIG. 33B provide results from a xenograft tumor model in which NK cells are injected intratumorally. FIG. 33A provides measurements of tumor volume. FIG. 33B shows a survival curve.

FIG. 34 depicts results from an experiment assessing effect of engineered NK cells modified with SB06258 vs. unengineered NK cells on Huh7 target cell cytotoxicity in the presence of various concentrations of soluble GPC3.

DETAILED DESCRIPTION

Provided herein are methods of stimulating a cell-mediated immune response to a tumor, reducing tumor volume, or providing an anti-tumor immunity in a human subject in need thereof. The methods may comprise administering to the subject a first dose of engineered NK cells, a second dose of engineered NK cells, and a third dose of engineered NK cells.

The second dose may be administered about seven days following the first dose. The second dose may be administered seven days following the first dose. The third dose may be administered about seven days following the second dose. The third dose may be administered seven days following the second dose.

The second dose may be administered about seven days following the first dose, and the third dose may be administered about seven days following the second dose. The second dose may be administered seven days following the first dose, and the third dose may be administered seven days following the second dose. The second dose may be administered at least seven days following the first dose, and the third dose may be administered at least seven days following the second dose.

In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 500 million engineered NK cells, at least about 1 billion engineered NK cells, at least about 1.5 billion NK cells, at least about 2 billion engineered NK cells, at least about 500 million to at least about 2 billion engineered NK cells, at least about 500 million to at least about 1.5 billion engineered NK cells, at least about 500 million to at least about 1 billion engineered NK cells, at least about 1 billion to at least about 2 billion engineered NK cells, at least about 1 billion to at least about 1.5 billion engineered NK cells, or at least about 1.5 billion to at least about 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise about 500 million engineered NK cells, about 1 billion engineered NK cells, about 1.5 billion NK cells, about 2 billion engineered NK cells, about 500 million to about 2 billion engineered NK cells, about 500 million to about 1.5 billion engineered NK cells, about 500 million to about 1 billion engineered NK cells, about 1 billion to about 2 billion engineered NK cells, about 1 billion to about 1.5 billion engineered NK cells, or about 1.5 billion to about 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least 500 million engineered NK cells, at least 1 billion engineered NK cells, at least 1.5 billion NK cells, at least 2 billion engineered NK cells, at least 500 million to at least 2 billion engineered NK cells, at least 500 million to at least 1.5 billion engineered NK cells, at least 500 million to at least 1 billion engineered NK cells, at least 1 billion to at least 2 billion engineered NK cells, at least 1 billion to at least 1.5 billion engineered NK cells, or at least 1.5 billion to at least 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 500 million engineered NK cells, 1 billion engineered NK cells, 1.5 billion NK cells, 2 billion engineered NK cells, 500 million to 2 billion engineered NK cells, 500 million to 1.5 billion engineered NK cells, 500 million to 1 billion engineered NK cells, 1 billion to 2 billion engineered NK cells, 1 billion to 1.5 billion engineered NK cells, or 1.5 billion to 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 500 million engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 1 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 1.5 billion NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 500 million to 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 500 million to 1.5 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 500 million to 1 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 1 billion to 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 1 billion to 1.5 billion engineered NK cells, or 1.5 billion to 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 3xlO^A7 cells/kg body weight, 1.5xlO^A7 cells/kg body weight, or 4.5xlO^A7 cells/kg body weight.

In some aspects, the engineered NK cells comprise an engineered nucleic acid comprising: a first promoter operably linked to a first exogenous polynucleotide sequence and a second exogenous polynucleotide sequence, wherein the first exogenous polynucleotide sequence encodes a chimeric antigen receptor (CAR) that binds to GPC3, and the second exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:

S - C - MT or MT - C - S, wherein

S comprises a secretable effector molecule that is or comprises IL15,

C comprises a protease cleavage site, and

MT comprises a cell membrane tethering domain, and

S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.

The membrane-cleavable chimeric protein is engineered such that secretion of the effector molecule can be regulated in a protease-dependent manner. Specifically, the membrane- cleavable chimeric protein is engineered such that secretion of the effector molecule can be regulated as part of a “Membrane-Cleavable” system, where incorporation of a protease cleavage site (“C”) and a cell membrane tethering domain (“MT”) allow for regulated secretion of an effector molecule in a protease-dependent manner. Without wishing to be bound by theory, the components of the Membrane-Cleavable system present in the membrane-cleavable chimeric protein generally regulate secretion through the below cellular processes:

MT: The cell membrane tethering domain contains a transmembrane domain (or a transmembrane-intracellular domain) that directs cellular-trafficking of the chimeric protein such that the protein is inserted into, or otherwise associated with, a cell membrane (“tethered”)

C: Following expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released (“secreted”) into the extracellular space.

Generally, the protease cleavage site is protease-specific, including sites engineered to be protease- specific. The protease cleavage site can be selected or engineered to achieve optimal protein expression, cell-type specific cleavage, cell-state specific cleavage, and/or cleavage and release of the payload at desired kinetics (e.g., ratio of membrane-bound to secreted chimeric protein levels)

In some aspects, membrane-cleavable chimeric proteins (or engineered nucleic acids encoding the membrane-cleavable chimeric proteins) are provided for herein having an effector molecule e.g., an effector molecule that is or comprises IL15, or a functional fragment thereof), a protease cleavage site, and a cell membrane tethering domain.

In some embodiments, the subject has recovered from toxicities related to prior treatment to < Gr 2. In some embodiments, the subject has not been chronically administered an immunosuppressive agent or corticosteroids at >10mg/day prednisone or equivalent, optionally wherein the subject has not been chronically administered the immunosuppressive agent or corticosteroids at > 10 mg/day within 14 days prior to (a).

In some embodiments, the subject does not have a history of significant cardiac or pulmonary disease or dysfunction within 12 weeks of (a).

In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 500 million NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 1 billion NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 1.5 billion NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 2 billion NK cells. In some embodiments, first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 500 million to at least about 2 billion engineered NK cells, at least about 500 million to at least about 1.5 billion engineered NK cells, at least about 500 million to at least about 1 billion engineered NK cells, at least about 1 billion to at least about 2 billion engineered NK cells, at least about 1 billion to at least about 1.5 billion engineered NK cells, or at least about 1.5 billion to at least about 2 billion engineered NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise about 500 million NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise about 1 billion NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise about 1.5 billion NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise about 2 billion NK cells. In some embodiments, first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise about 500 million to about 2 billion engineered NK cells, about 500 million to about 1.5 billion engineered NK cells, about 500 million to about 1 billion engineered NK cells, about 1 billion to about 2 billion engineered NK cells, about 1 billion to about 1.5 billion engineered NK cells, or about 1.5 billion to about 2 billion engineered NK cells.

In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least 500 million NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least 1 billion NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least 1.5 billion NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least 2 billion NK cells. In some embodiments, first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least 500 million to at least 2 billion engineered NK cells, at least 500 million to at least 1.5 billion engineered NK cells, at least 500 million to at least 1 billion engineered NK cells, at least 1 billion to at least 2 billion engineered NK cells, at least 1 billion to at least 1.5 billion engineered NK cells, or at least 1.5 billion to at least 2 billion engineered NK cells.

In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 500 million NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 1 billion NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 1.5 billion NK cells. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 2 billion NK cells. In some embodiments, first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 500 million to 2 billion engineered NK cells, 500 million to 1.5 billion engineered NK cells, 500 million to 1 billion engineered NK cells, 1 billion to 2 billion engineered NK cells, 1 billion to 1.5 billion engineered NK cells, or 1.5 billion to 2 billion engineered NK cells.

In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 3xlO^A7 cells/kg body weight, 1.5xlO^A7 cells/kg body weight, or 4.5xlO^A7 cells/kg body weight. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 3xlO^A7 cells/kg body weight. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 1.5xlO^A7 cells/kg body weight. In some embodiments, the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 4.5xlO^A7 cells/kg body weight.

In some embodiments, the subject has, prior to (a), been administered one or more lymphodepletion agents. In some embodiments, the one or more lymphodepletion agents comprises fludarabine and cyclophosphamide. In some embodiments, the subject has been administered, in the five to three days prior to (a) fludarabine at about 30 mg/m²/day over 30 minutes daily, and cyclophosphamide at about 500 mg/m²/day over 30-60 minutes daily. In some embodiments, the method comprises, in the three to five days prior to (a), administering fludarabine at about 30 mg/m²/day over 30 minutes daily, and cyclophosphamide at about 500 mg/m²/day over 30-60 minutes daily. In some embodiments, the subject has been administered, in the five to three days prior to (a) fludarabine at 30 mg/m²/day over 30 minutes daily, and cyclophosphamide at 500 mg/m²/day over 30-60 minutes daily. In some embodiments, the method comprises, in the three to five days prior to (a), administering fludarabine at 30 mg/m²/day over 30 minutes daily, and cyclophosphamide at 500 mg/m²/day over 30-60 minutes daily. In some embodiments, the method comprises administering oral and IV hydration to the subject prior to the cyclophosphamide administration.

An “effector molecule,” refers to a molecule (e.g., a nucleic acid such as DNA or RNA, or a protein (polypeptide) or peptide) that binds to another molecule and modulates the biological activity of that molecule to which it binds. For example, an effector molecule may act as a ligand to increase or decrease enzymatic activity, gene expression, or cell signaling. Thus, in some embodiments, an effector molecule modulates (activates or inhibits) different immunomodulatory mechanisms. By directly binding to and modulating a molecule, an effector molecule may also indirectly modulate a second, downstream molecule.

The term “modulate” encompasses maintenance of a biological activity, inhibition (partial or complete) of a biological activity, and stimulation/activation (partial or complete) of a biological activity. The term also encompasses decreasing or increasing (e.g., enhancing) a biological activity.

Modulation by an effector molecule may be direct or indirect. Direct modulation occurs when an effector molecule binds to another molecule and modulates activity of that molecule. Indirect modulation occurs when an effector molecule binds to another molecule, modulates activity of that molecule, and as a result of that modulation, the activity of yet another molecule (to which the effector molecule is not bound) is modulated.

In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by the effector molecule results in an increase in an immunostimulatory and/or anti-tumor immune response (e.g., systemically or in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation of a tumor-mediated immunosuppressive mechanism may result in an increase in an immuno stimulatory and/or anti-tumor immune response by 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%. In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism results in an increase in an immuno stimulatory and/or anti-tumor immune response 10-20%, 10-30%, 10- 40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20- 50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50- 90%, 50-100%, or 50-200%. It should be understood that “an increase” in an immuno stimulatory and/or anti-tumor immune response, for example, systemically or in a tumor microenvironment, is relative to the immunostimulatory and/or anti-tumor immune response that would otherwise occur, in the absence of the effector molecule(s). In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by the effector molecule results in an increase in an immunostimulatory and/or anti-tumor immune response (e.g., systemically or in the tumor microenvironment) by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 fold). For example, modulation of a tumor-mediated immunosuppressive mechanism may result in an increase in an immuno stimulatory and/or antitumor immune response by at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, or at least 100 fold. In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism results in an increase in an immuno stimulatory and/or antitumor immune response by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold.

Non-limiting examples of immuno stimulatory and/or anti-tumor immune mechanisms include T cell signaling, activity and/or recruitment, antigen presentation and/or processing, natural killer cell-mediated cytotoxic signaling, activity and/or recruitment, dendritic cell differentiation and/or maturation, immune cell recruitment, pro-inflammatory macrophage signaling, activity and/or recruitment, stroma degradation, immuno stimulatory metabolite production, stimulator of interferon genes (STING) signaling (which increases the secretion of IFN and Thl polarization, promoting an anti-tumor immune response), and/or Type I interferon signaling. An effector molecule may stimulate at least one (one or more) of the foregoing immuno stimulatory mechanisms, thus resulting in an increase in an immunostimulatory response. Changes in the foregoing immuno stimulatory and/or anti-tumor immune mechanisms may be assessed, for example, using in vitro assays for T cell proliferation or cytotoxicity, in vitro antigen presentation assays, expression assays (e.g., of particular markers), and/or cell secretion assays (e.g., of cytokines).

In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by the effector molecule results in a decrease in an immunosuppressive response (e.g., systemically or in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation of a tumor-mediated immunosuppressive mechanism may result in a decrease in an immunosuppressive response by 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%. In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism results in a decrease in an immunosuppressive response 10- 20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20- 30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50- 70%, 50-80%, 50-90%, 50-100%, or 50-200%. It should be understood that “a decrease” in an immunosuppressive response, for example, systemically or in a tumor microenvironment, is relative to the immunosuppressive response that would otherwise occur, in the absence of the effector molecule(s).

In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism by the effector molecule results in a decrease in an immunosuppressive response (e.g., systemically or in the tumor microenvironment) by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 fold). For example, modulation of a tumor-mediated immunosuppressive mechanism may result in a decrease in an immunosuppressive response by at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, or at least 100 fold. In some embodiments, modulation of a tumor-mediated immunosuppressive mechanism results in a decrease in an immunosuppressive response by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold.

Non-limiting examples of immunosuppressive mechanisms include negative costimulatory signaling, pro-apoptotic signaling of cytotoxic cells (e.g., T cells and/or NK cells), T regulatory (Treg) cell signaling, tumor checkpoint molecule production/maintenance, myeloid-derived suppressor cell signaling, activity and/or recruitment, immunosuppressive factor/metabolite production, and/or vascular endothelial growth factor signaling. An effector molecule may inhibit at least one (one or more) of the foregoing immunosuppressive mechanisms, thus resulting in a decrease in an immunosuppressive response. Changes in the foregoing immunosuppressive mechanisms may be assessed, for example, by assaying for an increase in T cell proliferation and/or an increase in IFNy production (negative co- stimulatory signaling, T_reg cell signaling and/or MDSC); Annexin V/PI flow staining (pro-apoptotic signaling); flow staining for expression, e.g., PDL1 expression (tumor checkpoint molecule production/maintenance); ELISA, LUMINEX®, RNA via qPCR, enzymatic assays, e.g., IDO tryptophan catabolism (immunosuppressive factor/metabolite production); and phosphorylation of PI3K, Akt, p38 (VEGF signaling).

In some embodiments, the effector molecule stimulates an immunostimulatory mechanism in the tumor microenvironment and/or inhibits an immunosuppressive mechanism in the tumor microenvironment.

In some embodiments, the effector molecule (a) stimulates T cell signaling, activity and/or recruitment, (b) stimulates antigen presentation and/or processing, (c) stimulates natural killer cell-mediated cytotoxic signaling, activity and/or recruitment, (d) stimulates dendritic cell differentiation and/or maturation, (e) stimulates immune cell recruitment, (f) stimulates pro- inflammatory macrophage signaling, activity and/or recruitment or inhibits anti-inflammatory macrophage signaling, activity and/or recruitment, (g) stimulates stroma degradation, (h) stimulates immunostimulatory metabolite production, (i) stimulates Type I interferon signaling, (j) inhibits negative costimulatory signaling, (k) inhibits pro-apoptotic signaling of anti-tumor immune cells, (1) inhibits T regulatory (T_reg) cell signaling, activity and/or recruitment, (m) inhibits tumor checkpoint molecules, (n) stimulates stimulator of interferon genes (STING) signaling, (o) inhibits myeloid-derived suppressor cell signaling, activity and/or recruitment, (p) degrades immunosuppressive factors/metabolites, (q) inhibits vascular endothelial growth factor signaling, and/or (r) directly kills tumor cells.

Exemplary IL15 sequences are listed in Table 1. Effector molecules (that are or comprise IL15 or a functional fragment thereof) can be human, such as those listed in Table 1 or human equivalents of murine effector molecules listed in Table 1. Effector molecules can be human- derived, such as the endogenous human effector molecule or an effector molecule modified and/or optimized for function, e.g., codon optimized to improve expression, modified to improve stability, or modified at its signal sequence (see below). Various programs and algorithms for optimizing function are known to those skilled in the art and can be selected based on the improvement desired, such as codon optimization for a specific species (e.g., human, mouse, bacteria, etc.).

Table 1: Exemplary IL-15 sequences

The first engineered nucleic acid can include a nucleotide sequence 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% identical to SEQ ID NO: 309. The first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 309.

The first engineered nucleic acid can include a nucleotide sequence 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% identical to SEQ ID NO: 326. The first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 326. The first engineered nucleic acid can include a nucleotide sequence 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% identical to SEQ ID NO: 310. The first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 310.

The first engineered nucleic acid can include a nucleotide sequence 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% identical to SEQ ID NO: 327. The first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 327.

The first engineered nucleic acid can include a nucleotide sequence 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% identical to SEQ ID NO: 314. The first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 314.

The first engineered nucleic acid can include a nucleotide sequence 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% identical to SEQ ID NO: 315. The first engineered nucleic acid can include a nucleotide sequence having the sequence shown in SEQ ID NO: 315.

NK cells provided for herein can include any one of the engineered nucleic acids described herein. NK cells provided for herein can include combinations of any one of the engineered nucleic acids described herein. NK cells provided for herein can include two or more of any one of the engineered nucleic acids described herein.

NK cells provided for herein can include a nucleotide sequence 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% identical to SEQ ID NO: 309. NK cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 309.

NK cells provided for herein can include a nucleotide sequence 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% identical to SEQ ID NO: 326. NK cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 326.

NK cells provided for herein can include a nucleotide sequence 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% identical to SEQ ID NO: 310. NK cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 310.

NK cells provided for herein can include a nucleotide sequence 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% identical to SEQ ID NO: 327. NK cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 327.

NK cells provided for herein can include a nucleotide sequence 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% identical to SEQ ID NO: 314. NK cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 314. NK cells provided for herein can include a nucleotide sequence 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% identical to SEQ ID NO: 315. NK cells provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 315.

Expression vectors provided for herein can include any one of the engineered nucleic acids described herein. Expression vectors provided for herein can include combinations of any one of the engineered nucleic acids described herein. Expression vectors provided for herein can include two or more of any one of the engineered nucleic acids described herein.

Expression vectors provided for herein can include a nucleotide sequence 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% identical to SEQ ID NO: 309. Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 309.

Expression vectors provided for herein can include a nucleotide sequence 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% identical to SEQ ID NO: 326. Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 326.

Expression vectors provided for herein can include a nucleotide sequence 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% identical to SEQ ID NO: 310. Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 310.

Expression vectors provided for herein can include a nucleotide sequence 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% identical to SEQ ID NO: 327. Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 327.

Expression vectors provided for herein can include a nucleotide sequence 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% identical to SEQ ID NO: 314. Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 314.

Expression vectors provided for herein can include a nucleotide sequence 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% identical to SEQ ID NO: 315. Expression vectors provided for herein can include a nucleotide sequence having the sequence shown in SEQ ID NO: 315. Secretion Signals and Signal- Anchors

The one or more effector molecules (e.g., that is or comprises IL15 or a functional fragment thereof) of the membrane-cleavable chimeric proteins provided for herein are in general secretable effector molecules having a secretion signal peptide (also referred to as a signal peptide or signal sequence) at the chimeric protein’s N-terminus (e.g., an effector molecule’s N-terminus for S - C - MT) that direct newly synthesized proteins destined for secretion or membrane localization (also referred to as membrane insertion) to the proper protein processing pathways. For chimeric proteins having the formula MT - C - S, a membrane tethering domain generally has a signal-anchor sequence (e.g., signal-anchor sequences of a Type II transmembrane protein) that direct newly synthesized proteins destined for membrane localization to the proper protein processing pathways. For chimeric proteins having the formula S - C - MT, a membrane tethering domain having a reverse signal-anchor sequence (e.g., signal-anchor sequences of certain Type III transmembrane proteins) can be used, generally without a separate secretion signal peptide, that direct newly synthesized proteins destined for membrane localization to the proper protein processing pathways.

In general, for all membrane-cleavable chimeric proteins described herein, the one or more effector molecules are secretable effector molecules (referred to as “S” in the formula S - C - MT or MT - C - S). In embodiments with two or more chimeric proteins, each chimeric protein can comprise a secretion signal. In embodiments with two or more chimeric proteins, each chimeric protein can comprise a secretion signal such that each effector molecule is capable of secretion from an engineered cell following cleavage of the protease cleavage site.

A CAR disclosed herein may comprise a signal sequence. The signal sequence may direct the CAR to the cell membrane.

The secretion signal peptide operably associated with an effector molecule and/or CAR can be a native secretion signal peptide (e.g., the secretion signal peptide generally endogenously associated with the given effector molecule, such as a cytokine’s endogenous secretion signal peptide). The secretion signal peptide operably associated with an effector molecule and/or CAR can be a non-native secretion signal peptide native secretion signal peptide. Non-native secretion signal peptides can promote improved expression and function, such as maintained secretion, in particular environments, such as tumor microenvironments. Non-limiting examples of non-native secretion signal peptide are shown in Table 3. Table 3. Exemplary Signal Secretion Peptides

Protease Cleavage Site

In general, membrane-cleavable chimeric proteins described herein contain a protease cleavage site (comprised in “C” in the formula S - C - MT or MT - C - S). In general, the protease cleavage site can be any amino acid sequence motif capable of being cleaved by a protease. Examples of protease cleavage sites include, but are not limited to, an ADAM protease cleavage site (e.g., an ADAM8 protease cleavage site, an ADAM9 protease cleavage site, an ADAM10 protease cleavage site, an ADAM12 protease cleavage site, an ADAM15 protease cleavage site, an ADAM 17 protease cleavage site, an ADAM 19 protease cleavage site, an ADAM20 protease cleavage site, an ADAM21 protease cleavage site, an ADAM28 protease cleavage site, an ADAM30 protease cleavage site, an ADAM33 protease cleavage site).

An example of a protease cleavage site is an ADAM17-specific protease (also referred to as Tumor Necrosis Factor-a Converting Enzyme [TACE]) cleavage site. An ADAM 17 -specific protease cleavage site can be an endogenous sequence of a substrate naturally cleaved by ADAM 17. An ADAM 17 -specific protease cleavage site can be an engineered sequence capable of being cleaved by ADAM 17. An engineered ADAM17-specific protease cleavage site can be an engineered for specific desired properties including, but not limited to, optimal expression of the chimeric proteins, specificity for ADAM 17, rate-of-cleavage by ADAM 17, ratio of secreted and membrane-bound chimeric protein levels, and cleavage in different cell states. A protease cleavage site can be selected for specific cleavage by ADAM 17. For example, certain protease cleavage sites capable of being cleaved by ADAM 17 are also capable of cleavage by additional ADAM family proteases, such as ADAM10. Accordingly, an ADAM 17- specific protease cleavage site can be selected and/or engineered such that cleavage by other proteases, such as ADAM 10, is reduced or eliminated. A protease cleavage site can be selected for rate-of- cleavage by ADAM 17. For example, it can be desirable to select a protease cleavage site demonstrating a specific rate-of-cleavage by ADAM 17, such as reduced cleavage kinetics relative to an endogenous sequence of a substrate naturally cleaved by ADAM 17. In such cases, in general, a specific rate-of-cleavage can be selected to regulate the rate of processing of the chimeric protein, which in turn regulates the rate of release/secretion of the payload effector molecule. Accordingly, an ADAM 17- specific protease cleavage site can be selected and/or engineered such that the sequence demonstrates a desired rate-of-cleavage by ADAM 17. A protease cleavage site can be selected for both specific cleavage by ADAM 17 and rate-of- cleavage by ADAM 17. Exemplary ADAM 17 -specific protease cleavage sites, including those demonstrating particular specificity and rate-of-cleavage kinetics, are shown in Table 4A below with reference to the site of cleavage (P5-P1: N-terminal; Pl'-P5': C-terminal). Further details of ADAM 17 and ADAM10, including expression and protease cleavage sites, are described in Sharma, et al. (J Immunol October 15, 2017, 199 (8) 2865-2872), Pham et al. (Anticancer Res. 2017 Oct;37(10):5507-5513), Caescu et al. (Biochem J. 2009 Oct 23; 424(1): 79-88), and Tucher et al. (J. Proteome Res. 2014, 13, 4, 2205-2214), each herein incorporated by reference for purposes. Table 4A - Potential ADAM17 Protease Cleavage Site Sequences

In some embodiments, the protease cleavage site comprises a first region having the amino acid sequence of PRAE (SEQ ID NO: 176). In some embodiments, the protease cleavage site comprises a second region having the amino acid sequence of KGG (SEQ ID NO: 177). In some embodiments, the first region is located N-terminal to the second region. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEX1X2KGG (SEQ ID NO: 178), wherein Xi is A, Y, P, S, or F, and wherein X2is V, L, S, I, Y, T, or A. In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAVKGG (SEQ ID NO: 179). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEALKGG (SEQ ID NO: 180). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEYSKGG (SEQ ID NO: 181). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEPIKGG (SEQ ID NO: 182). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAYKGG (SEQ ID NO: 183). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAESSKGG (SEQ ID NO: 184). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEFTKGG (SEQ ID NO: 185). In some embodiments, the protease cleavage site comprises the amino acid sequence of PRAEAAKGG (SEQ ID NO: 186). In some embodiments, the protease cleavage site comprises the amino acid sequence of DEPHYSQRR (SEQ ID NO: 187). In some embodiments, the protease cleavage site comprises the amino acid sequence of PPLGPIFNPG (SEQ ID NO: 188). In some embodiments, the protease cleavage site comprises the amino acid sequence of PLAQAYRSS (SEQ ID NO: 189). In some embodiments, the protease cleavage site comprises the amino acid sequence of TPIDSSFNPD (SEQ ID NO: 190). In some embodiments, the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI (SEQ ID NO: 191).

In certain embodiments of the membrane-cleavable chimeric protein having the formula S - C - MT or MT - C - S, C further comprises a linker sequence, e.g., a glycine- serine linker motif, e.g., comprising the sequence GGGS or GGGGS. A cleavage site may be flanked on the N terminal and/or C terminal sides by a linker sequence, e.g., a glycine-serine linker motif. For example and without limitation, the cleavage site may be flanked on both the N terminal and C terminal sides by a partial glycine-serine (GS) linker sequence.

In certain embodiments, the cleavage site and linker comprise the amino acid sequence of SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ (SEQ ID NO: 287). An exemplary nucleic acid sequence encoding SEQ ID NO: 287 is TCTGGCGGCGGAGGATCTGGCGGAGGTGGAAGCGGAGTTACACCCGAGCCTATCTT CAGCCTGATCGGAGGCGGTAGCGGAGGCGGAGGAAGTGGTGGCGGATCTCTGCAA (SEQ ID NO: 288). In some embodiments, nucleic acids encoding SEQ ID NO: 287 may comprise SEQ ID NO: 288, or a nucleic acid sequence that is 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% identical to SEQ ID NO: 288.

In certain embodiments, the protease cleavage site is N-terminal to a linker. In certain embodiments, the protease cleavage site and linker comprise the amino acid sequence of PRAEALKGGSGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 289). An exemplary nucleic acid sequence encoding SEQ ID NO: 289 is CCCAGAGCCGAGGCTCTGAAAGGCGGATCAGGCGGCGGTGGTAGTGGAGGCGGAG GCTCAGGCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAAT (SEQ ID NO: 292). In some embodiments, nucleic acids encoding SEQ ID NO: 289 may comprise SEQ ID NO: 292, or a nucleic acid sequence that is 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% identical to SEQ ID NO: 292.

In some embodiments, the protease cleavage site comprises the amino acid sequence of ITQGLAVSTISSFF (SEQ ID NO: 198), which is a cleavage site that is native to CD16 and is cleavable by ADAM 17. In certain embodiments, SEQ ID NO: 198 is comprised within a linker. In certain embodiments, the linker comprises the amino acid sequence of SGGGGSGGGGSGITQGLAVSTISSFFGGGSGGGGSGGGSLQ (SEQ ID NO: 290). An exemplary nucleic acid sequence encoding SEQ ID NO: 290 is AGCGGCGGAGGTGGTAGCGGAGGCGGAGGATCTGGAATTACACAGGGACTCGCCG TGTCTACAATCTCCAGCTTCTTTGGTGGCGGTAGTGGCGGCGGTGGCAGTGGCGGTG GATCTCTTCAA (SEQ ID NO: 291). In some embodiments, nucleic acids encoding SEQ ID NO: 290 may comprise SEQ ID NO: 291, or a nucleic acid sequence that is 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% identical to SEQ ID NO: 291.

The protease cleavage site can be C-terminal of the secretable effector molecule. The protease cleavage site can be N-terminal of the secretable effector molecule. In general, for all membrane-cleavable chimeric proteins described herein, the protease cleavage site is either: (1) C-terminal of the secretable effector molecule and N-terminal of the cell membrane tethering domain (in other words, the protease cleavage site is in between the secretable effector molecule and the cell membrane tethering domain); or (2) N-terminal of the secretable effector molecule and C-terminal of the cell membrane tethering domain (also between the secretable effector molecule and the cell membrane tethering domain with domain orientation inverted). The protease cleavage site can be connected to the secretable effector molecule by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the effector molecule or protease cleavage site. The protease cleavage site can be connected to the cell membrane tethering domain by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or protease cleavage site. A polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence. A polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g.. [GS]₄GG [SEQ ID NO: 182]), A(EAAAK)₃A (SEQ ID NO: 183), and Whitlow linkers (e.g., a “KEGS” linker such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), an eGK linker such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), an LR1 linker such as the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 215), and linkers described in more detail in Issued U.S. Pat. No. 5,990,275 herein incorporated by reference). Additional exemplary polypeptide linkers include SGGGGSGGGGSG (SEQ ID NO: 194), TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 196), and GGGSGGGGSGGGSLQ (SEQ ID NO: 197). Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition, etc.) and are known to those skilled in the art. An exemplary nucleic acid sequence encoding SEQ ID NO: 196 is ACCACCACACCAGCTCCTCGGCCACCAACTCCAGCTCCAACAATTGCCAGCCAGCC TCTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCAGGCGGAGCCGTGCATACAA GAGGACTGGATTTCGCCTGCGAC (SEQ ID NO: 337). In certain embodiments, a nucleic acid encoding SEQ ID NO: 196 comprises a sequence that is 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% identical to SEQ ID NO: 337.

In the Membrane-Cleavable system, following expression and localization of the chimeric protein into the cell membrane, the protease cleavage site directs cleavage of the chimeric protein such that the effector molecule is released (“secreted”) into the extracellular space of a cell.

In general, a protease that cleaves the protease cleavage site is a protease specific for that specific protease cleavage site. For example, in the case of a disintegrin and metalloproteinase (“ADAM”) family protease, the protease that cleaves a specific ADAM protease cleavage site is generally limited to the ADAM protease(s) that specifically recognize the specific ADAM protease cleavage site motif. A protease cleavage site can be selected and/or engineered such that cleavage by undesired proteases is reduced or eliminated. Proteases can be membranebound or membrane-associated. Proteases can be secreted, e.g., secreted in a specific cellular environment, such as a tumor microenvironment (“TME”).

A protease that cleaves the protease cleavage site of the chimeric protein can be expressed in the same cell that expresses the chimeric protein. A protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to a cell expressing the chimeric protein. In other words, a cell engineered to express the chimeric protein can endogenously express the protease specific for the protease cleavage site present in the chimeric protein. Endogenous expression of the protease refers to both expression under generally homeostatic conditions (e.g., a cell generally considered to be healthy), and also to differential expression under non-homeostatic conditions (e.g., upregulated expression in a tumor cell). The protease cleavage site can be selected based on the known proteases endogenously expressed by a desired cell population. In such cases, in general, the cleavage of the protease cleavage site (and thus release/secretion of a payload) can be restricted to only those cells of interest due to the cell-restricted protease needing to come in contact with the protease cleavage site of chimeric protein expressed in the same cell. For example, and without wishing to be bound by theory, ADAM 17 is believed to be restricted in its endogenous expression to NK cell and T cells. Thus, selection of an ADAM17-specific protease cleavage site may restrict the cleavage of the protease cleavage site to NK cell and T cells co-expressing the chimeric protein. In other examples, a protease cleavage site can be selected for a specific tumor- associated protease known to be expressed in a particular tumor population of interest (e.g., in a specific tumor cell engineered to express the chimeric protein). Protease and/or expression databases can be used to select an appropriate protease cleavage site, such as selecting a protease cleavage site cleaved by a tumor-associated proteases through consulting Oncomine (www.oncomine.org), the European Bioinformatic Institute (www.ebi.ac.uk) in particular (www.ebi.ac.uk/gxa), PMAP (www.proteolysis.org), ExPASy Peptide Cutter (ca.expasy.org/tools/peptide cutter) and PMAP. Cut DB (cutdb.burnham.org), each of which is incorporated by reference for all purposes.

A protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to a cell expressing the chimeric protein. For example, a cell engineered to express the chimeric protein can also be engineered to express a protease not generally expressed by the cell that is specific for the protease cleavage site present in the chimeric protein. A cell engineered to express both the chimeric protein and the protease can be engineered to express each from separate engineered nucleic acids or from a multicistronic systems (multicistronic and multi-promoter systems are described in greater detail in the Section herein titled “Multicistronic and Multiple Promoter Systems”). Heterologous proteases and their corresponding protease cleavage site can be selected as described above with reference to endogenous proteases.

A protease that cleaves the protease cleavage site of the chimeric protein can be expressed on a separate distinct cell than the cell that expresses the chimeric protein. For example, the protease can be generally expressed in a specific cellular environment, such as a tumor microenvironment. In such cases, in general, the cleavage of the protease cleavage site can be restricted to only those cellular environments of interest (e.g., a tumor microenvironment) due to the environment-restricted protease needing to come in contact with the protease cleavage site. In embodiments having membrane-cleavable chimeric proteins, in general, the secretion of the effector molecule can be restricted to only those cellular environments of interest (e.g., a tumor microenvironment) due to the environment-restricted protease needing to come in contact with the protease cleavage site. A protease that cleaves the protease cleavage site of the chimeric protein can be endogenous to the separate distinct cell. A protease that cleaves the protease cleavage site of the chimeric protein can be heterologous to the separate distinct cell. For example, the separate distinct cell can be engineered to express a protease not generally expressed by the separate distinct cell.

Proteases include, but are not limited to, an ADAM protease (e.g., ADAM8 protease, an ADAM9 protease, an ADAM 10 protease, an ADAM 12 protease, an ADAM 15 protease, an ADAM 17 protease, an ADAM 19 protease, an ADAM20 protease, an ADAM21 protease, an ADAM28 protease, an ADAM30 protease, an ADAM33 protease). A protease can be an ADAM17 protease. Proteases can also include, but are not limited to, proteases listed in Table 4B below.

Exemplary cognate protease cleavage sites for certain proteases are also listed in Table 4B.

Table 4B: Exemplary Proteases with Cognate Cleavage Sites and Inhibitors

A protease can be any of the following human proteases (MEROPS peptidase database number provided in parentheses; Rawlings N. D., Morton F. R., Kok, C. Y., Kong, J. & Barrett A. J. (2008) MEROPS: the peptidase database. Nucleic Acids Res. 36 Database issue, D320- 325; herein incorporated by reference for all purposes): ADAMTS9 peptidase (MER012092), AD AMTS 14 peptidase (MER016700), AD AMTS 15 peptidase (MER017029), AD AMTS 16 peptidase (MER015689), ADAMTS17 peptidase (MER016302), ADAMTS18 peptidase (MER016090), AD AMTS 19 peptidase (MER015663), ADAM8 peptidase (MER003902), ADAM9 peptidase (MER001140), ADAM10 peptidase (MER002382), ADAM12 peptidase (MER005107), ADAM19 peptidase (MER012241), ADAM15 peptidase (MER002386), ADAM 17 peptidase (MER003094), ADAM20 peptidase (MER004725), ADAMDEC1 peptidase (MER000743), ADAMTS3 peptidase (MER005100), ADAMTS4 peptidase (MER005101), AD AMTS 1 peptidase (MER005546), ADAM28 peptidase (Homo sapiens-type) (MER005495), ADAMTS5 peptidase (MER005548), ADAMTS8 peptidase (MER005545), ADAMTS6 peptidase (MER005893), ADAMTS7 peptidase (MER005894), ADAM30 peptidase (MER006268), ADAM21 peptidase (Homo sapiens-type) (MER004726), AD AMTS 10 peptidase (MER014331), AD AMTS 12 peptidase (MER014337), AD AMTS 13 peptidase (MER015450), ADAM33 peptidase (MER015143), ADAMTS20 peptidase (Homo sapiens- type) (MER026906), ADAM2 protein (MER003090), ADAM6 protein (MER047044), ADAM7 protein (MER005109), ADAM18 protein (MER012230), ADAM32 protein (MER026938), nonpeptidase homologue (Homo sapiens chromosome 4) (MER029973), ADAM3B protein (Homo sapiens-type) (MER005199), ADAMI 1 protein (MER001146), ADAM22 protein (MER005102), ADAM23 protein (MER005103), ADAM29 protein (MER006267), protein similar to ADAM21 peptidase preproprotein (Homo sapiens) (MER026944), putative ADAM pseudogene (chromosome 4, Homo sapiens) (MER029975), ADAM3A g.p. (Homo sapiens) (MER005200), ADAMI g.p. (Homo sapiens) (MER003912).

Protease enzymatic activity can be regulated through selection of a specific protease cleavage site. For example, a protease cleavage site can be selected and/or engineered such that the sequence demonstrates a desired rate-of-cleavage by a desired protease, such as reduced cleavage kinetics relative to an endogenous sequence of a substrate naturally cleaved by the desired protease. As another example, a protease cleavage site can be selected and/or engineered such that the sequence demonstrates a desired rate-of-cleavage in a cell-state specific manner. For example, various cell states (e.g., following cellular signaling, such as immune cell activation) can influence the expression and/or localization of certain proteases. As an illustrative example, ADAM 17 protein levels and localization is known to be influenced by signaling, such as through Protein kinase C (PKC) signaling pathways (e.g., activation by the PKC activator Phorbol-12-myristat-13-acetat [PMA]). Accordingly, a protease cleavage site can be selected and/or engineered such that cleavage of the protease cleavage site and subsequent release of an effector molecule is increased or decreased, as desired, depending on the protease properties (e.g., expression and/or localization) in a specific cell state. As another example, a protease cleavage site (particularly in combination with a specific membrane tethering domain) can be selected and/or engineered for optimal protein expression of the chimeric protein.

Cell Membrane Tethering Domain

The membrane-cleavable chimeric proteins provided for herein include a cell-membrane tethering domain (referred to as “MT” in the formula S - C - MT or MT - C - S). In general, the cell-membrane tethering domain can be any amino acid sequence motif capable of directing the chimeric protein to be localized to (e.g., inserted into), or otherwise associated with, the cell membrane of the cell expressing the chimeric protein. The cell-membrane tethering domain can be a transmembrane-intracellular domain. The cell-membrane tethering domain can be a transmembrane domain. The cell-membrane tethering domain can be an integral membrane protein domain (e.g., a transmembrane domain). The cell-membrane tethering domain can be derived from a Type I, Type II, or Type III transmembrane protein. The cell-membrane tethering domain can include post-translational modification tag, or motif capable of post-translational modification to modify the chimeric protein to include a post-translational modification tag, where the post-translational modification tag allows association with a cell membrane. Examples of post-translational modification tags include, but are not limited to, lipid-anchor domains (e.g., a GPI lipid- anchor, a myristoylation tag, or palmitoylation tag). Examples of cellmembrane tethering domains include, but are not limited to, a transmembrane-intracellular domain and/or transmembrane domain derived from PDGFR-beta, CD8, CD28, CD3zeta-chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA. The cell membrane tethering domain can be a cell surface receptor or a cell membrane-bound portion thereof. Sequences of exemplary cell membrane tethering domains are provided in Table 4C.

Table 4C.

In general, for all membrane-cleavable chimeric proteins described herein, the cell membrane tethering domain is either: (1) C-terminal of the protease cleavage site and N- terminal of any intracellular domain, if present (in other words, the cell membrane tethering domain is in between the protease cleavage site and, if present, an intracellular domain); or (2) N-terminal of the protease cleavage site and C-terminal of any intracellular domain, if present (also between the protease cleavage site and, if present, an intracellular domain with domain orientation inverted). In embodiments featuring a degron associated with the chimeric protein, the degron domain is the terminal cytoplasmic-oriented domain, specifically relative to the cell membrane tethering (in other words, the cell membrane tethering domain is in between the protease cleavage site and the degron). The cell membrane tethering domain can be connected to the protease cleavage site by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of cell membrane tethering domain or protease cleavage site. The cell membrane tethering domain can be connected to an intracellular domain, if present, by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or the intracellular domain. The cell membrane tethering domain can be connected to the degron, if present, by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or degron. A polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence. A polypeptide linker can be a flexible linker (e.g., a Gly- Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]₄GG [SEQ ID NO: 182]), A(EAAAK)₃A (SEQ ID NO: 183), and Whitlow linkers (e.g., a “KEGS” linker such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 184), an eGK linker such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 185), an LR1 linker such as the amino acid sequence SGGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 215), and linkers described in more detail in Issued U.S. Pat. No. 5,990,275 herein incorporated by reference). Additional polypeptide linkers include SEQ ID NO: 194, SEQ ID NO: 196, and SEQ ID NO: 197. Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition etc.) and are known to those skilled in the art.

In general, the cell-membrane tethering domain is oriented such that the secreted effector molecule and the protease cleavage site are extracellularly exposed following insertion into, or association with, the cell membrane, such that the protease cleavage site is capable of being cleaved by its respective protease and releasing (“secreting”) the effector molecule into the extracellular space.

Promoters

In general, in all embodiments described herein, the engineered nucleic acids encoding the proteins herein (e.g., a CAR and a membrane-cleavable chimeric protein described herein) encode an expression cassette containing a promoter and an exogenous polynucleotide sequence encoding the protein. In some embodiments, an engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a promoter operably linked to a nucleotide sequence (e.g. , an exogenous polynucleotide sequence) encoding at least 2 distinct proteins. For example, the engineered nucleic acid may comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 distinct proteins. In some embodiments, an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more distinct proteins. In some embodiments, an engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a promoter operably linked to a nucleotide sequence (e.g., an exogenous polynucleotide sequence) encoding at least 2 cytokines. For example, the engineered nucleic acid may comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 cytokines. In some embodiments, an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cytokines. In some embodiments, an engineered nucleic acid (e.g., an engineered nucleic acid comprising an expression cassette) comprises a promoter operably linked to a nucleotide sequence (e.g., an exogenous polynucleotide sequence) encoding at least 2 membrane-cleavable chimeric proteins. For example, the engineered nucleic acid may comprise a promoter operably linked to a nucleotide sequence encoding at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 8, at least 9, or at least 10 membrane-cleavable chimeric proteins. In some embodiments, an engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more membrane- cleavable chimeric proteins.

A “promoter” refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter may also contain sub-regions at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters may be constitutive, inducible, repressible, tissue-specific or any combination thereof. A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. Herein, a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and/or expression of that sequence.

A promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter can be referred to as “endogenous.” In some embodiments, a coding nucleic acid sequence may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded sequence in its natural environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring" such as, for example, those that contain different elements of different transcriptional regulatory regions and/or mutations that alter expression through methods of genetic engineering that are known in the art. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including polymerase chain reaction (PCR) (see, e.g., U.S. Pat. No. 4,683,202 and U.S. Pat. No. 5,928,906).

Promoters of an engineered nucleic acid may be “inducible promoters,” which refer to promoters that are characterized by regulating (e.g., initiating or activating) transcriptional activity when in the presence of, influenced by or contacted by a signal. The signal may be endogenous or a normally exogenous condition (e.g., light), compound (e.g., chemical or nonchemical compound) or protein (e.g., cytokine) that contacts an inducible promoter in such a way as to be active in regulating transcriptional activity from the inducible promoter. Activation of transcription may involve directly acting on a promoter to drive transcription or indirectly acting on a promoter by inactivation a repressor that is preventing the promoter from driving transcription. Conversely, deactivation of transcription may involve directly acting on a promoter to prevent transcription or indirectly acting on a promoter by activating a repressor that then acts on the promoter.

A promoter is “responsive to” or “modulated by” a local tumor state (e.g., inflammation or hypoxia) or signal if in the presence of that state or signal, transcription from the promoter is activated, deactivated, increased, or decreased. In some embodiments, the promoter comprises a response element. A “response element” is a short sequence of DNA within a promoter region that binds specific molecules (e.g., transcription factors) that modulate (regulate) gene expression from the promoter. Response elements that may be used in accordance with the present disclosure include, without limitation, a phloretin-adjustable control element (PEACE), a zinc-finger DNA-binding domain (DBD), an interferon-gamma-activated sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar; 17(3): 121-34, incorporated herein by reference), an interferon- stimulated response element (ISRE) (Han, K. J. et al. J Biol Chem. 2004 Apr 9;279(15): 15652-61, incorporated herein by reference), a NF-kappaB response element (Wang, V. et al. Cell Reports. 2012; 2(4): 824-839, incorporated herein by reference), and a STAT3 response element (Zhang, D. et al. J of Biol Chem. 1996; 271: 9503-9509, incorporated herein by reference). Other response elements are encompassed herein. Response elements can also contain tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element) to generally increase sensitivity of the response element to its cognate binding molecule. Tandem repeats can be labeled 2X, 3X, 4X, 5X, etc. to denote the number of repeats present.

Non-limiting examples of responsive promoters (also referred to as “inducible promoters”) (e.g., TGF-beta responsive promoters) are listed in Table 5A, which shows the design of the promoter and transcription factor, as well as the effect of the inducer molecule towards the transcription factor (TF) and transgene transcription (T) is shown (B, binding; D, dissociation; n.d., not determined) (A, activation; DA, deactivation; DR, derepression) (see Horner, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m, and references cited therein). Non-limiting examples of components of inducible promoters include those presented in Table 5B. Table 5A. Examples of Responsive Promoters

Table 5B. Exemplary Components of Inducible Promoters

Non-limiting examples of constitutive promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1-alpha (EFla) promoter, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK)

The promoter can be a tissue-specific promoter. In general, a tissue- specific promoter directs transcription of a nucleic acid, (e.g., the engineered nucleic acids encoding the proteins herein (e.g., a CAR, and a membrane-cleavable chimeric protein described herein) such that expression is limited to a specific cell type, organelle, or tissue. Tissue- specific promoters include, but are not limited to, albumin (liver specific, Pinkert et al., (1987)), lymphoid specific promoters (Calame and Eaton, 1988), particular promoters of T-cell receptors (Winoto and Baltimore, (1989)) and immunoglobulins; Banerji et al., (1983); Queen and Baltimore, 1983), neuron specific promoters (e.g. the neurofilament promoter; Byrne and Ruddle, 1989), pancreas specific promoters (Edlund et al., (1985)) or mammary gland specific promoters (milk whey promoter, U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166) as well as developmentally regulated promoters such as the murine hox promoters (Kessel and Gruss, Science 249:374-379 (1990)) or the a-fetoprotein promoter (Campes and Tilghman, Genes Dev. 3:537-546 (1989)), the contents of each of which are fully incorporated by reference herein. The promoter can be constitutive in the respective specific cell type, organelle, or tissue. Tissuespecific promoters and/or regulatory elements can also include promoters from the liver fatty acid binding (FAB) protein gene, specific for colon epithelial cells; the insulin gene, specific for pancreatic cells; the transphyretin, .alpha.1- antitrypsin, plasminogen activator inhibitor type 1 (PAI-I), apolipoprotein Al and LDL receptor genes, specific for liver cells; the myelin basic protein (MBP) gene, specific for oligodendrocytes; the glial fibrillary acidic protein (GFAP) gene, specific for glial cells; OPSIN, specific for targeting to the eye; and the neural-specific enolase (NSE) promoter that is specific for nerve cells. Examples of tissue-specific promoters include, but are not limited to, the promoter for creatine kinase, which has been used to direct expression in muscle and cardiac tissue and immunoglobulin heavy or light chain promoters for expression in B cells. Other tissue specific promoters include the human smooth muscle alphaactin promoter. Exemplary tissue- specific expression elements for the liver include but are not limited to HMG-COA reductase promoter, sterol regulatory element 1, phosphoenol pyruvate carboxy kinase (PEPCK) promoter, human C- reactive protein (CRP) promoter, human glucokinase promoter, cholesterol L 7-alpha hydroylase (CYP-7) promoter, beta- galactosidase alpha-2,6 sialylkansferase promoter, insulin-like growth factor binding protein (IGFBP-I) promoter, aldolase B promoter, human transferrin promoter, and collagen type I promoter. Exemplary tissue- specific expression elements for the prostate include but are not limited to the prostatic acid phosphatase (PAP) promoter, prostatic secretory protein of 94 (PSP 94) promoter, prostate specific antigen complex promoter, and human glandular kallikrein gene promoter (hgt- 1). Exemplary tissue- specific expression elements for gastric tissue include but are not limited to the human H+/K+-ATPase alpha subunit promoter. Exemplary tissue- specific expression elements for the pancreas include but are not limited to pancreatitis associated protein promoter (PAP), elastase 1 transcriptional enhancer, pancreas specific amylase and elastase enhancer promoter, and pancreatic cholesterol esterase gene promoter. Exemplary tissue-specific expression elements for the endometrium include, but are not limited to, the uteroglobin promoter. Exemplary tissue-specific expression elements for adrenal cells include, but are not limited to, cholesterol side-chain cleavage (SCC) promoter. Exemplary tissue-specific expression elements for the general nervous system include, but are not limited to, gammagamman enolase (neuron- specific enolase, NSE) promoter. Exemplary tissue-specific expression elements for the brain include, but are not limited to, the neurofilament heavy chain (NF-H) promoter. Exemplary tissue- specific expression elements for lymphocytes include, but are not limited to, the human CGL-l/granzyme B promoter, the terminal deoxy transferase (TdT), lambda 5, VpreB, and lek (lymphocyte specific tyrosine protein kinase p561ck) promoter, the humans CD2 promoter and its 3 ' transcriptional enhancer, and the human NK and T cell specific activation (NKG5) promoter. Exemplary tissue- specific expression elements for the colon include, but are not limited to, pp60c-src tyrosine kinase promoter, organ- specific neoantigens (OSNs) promoter, and colon specific antigen-P promoter. Tissue-specific expression elements for breast cells are for example, but are not limited to, the human alphalactalbumin promoter. Exemplary tissue- specific expression elements for the lung include, but are not limited to, the cystic fibrosis transmembrane conductance regulator (CFTR) gene promoter.

In some embodiments, a promoter of the present disclosure is modulated by signals within a tumor microenvironment. A tumor microenvironment is considered to modulate a promoter if, in the presence of the tumor microenvironment, the activity of the promoter is increased or decreased by at least 10%, relative to activity of the promoter in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased by 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%, relative to activity of the promoter in the absence of the tumor microenvironment. For example, the activity of the promoter is increased or decreased by 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, or 50-200%, relative to activity of the promoter in the absence of the tumor microenvironment.

In some embodiments, the activity of the promoter is increased or decreased by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or 100 fold), relative to activity of the promoter in the absence of the tumor microenvironment. For example, the activity of the promoter is increased or decreased by at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, or at least 100 fold, relative to activity of the promoter in the absence of the tumor microenvironment. In some embodiments, the activity of the promoter is increased or decreased by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold, relative to activity of the promoter in the absence of the tumor microenvironment.

In some embodiments, a promoter of the present disclosure is activated under a hypoxic condition. A “hypoxic condition” is a condition where the body or a region of the body is deprived of adequate oxygen supply at the tissue level. Hypoxic conditions can cause inflammation (e.g., the level of inflammatory cytokines increase under hypoxic conditions). In some embodiments, the promoter that is activated under hypoxic condition is operably linked to a nucleotide encoding a protein that decreases the expression of activity of inflammatory cytokines, thus reducing the inflammation caused by the hypoxic condition. In some embodiments, the promoter that is activated under hypoxic conditions comprises a hypoxia responsive element (HRE). A “hypoxia responsive element (HRE)” is a response element that responds to hypoxia-inducible factor (HIF). The HRE, in some embodiments, comprises a consensus motif NCGTG (where N is either A or G).

Multicistronic and Multiple Promoter Systems

In some embodiments, engineered nucleic acids (e.g., an engineered nucleic acid comprising an expression cassette) are configured to produce multiple proteins (e.g., a CAR, membrane-cleavable chimeric protein, and/or combinations thereof). For example, nucleic acids may be configured to produce 2-20 different proteins. In some embodiments, nucleic acids are configured to produce 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8,

2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8,

3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20,

6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17,

7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13,

8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10- 19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13- 19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20 proteins. In some embodiments, nucleic acids are configured to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 proteins.

In some embodiments, engineered nucleic acids can be multicistronic, i.e., more than one separate polypeptide (e.g., multiple proteins, such as a CAR, and a membrane-cleavable chimeric protein described herein) can be produced from a single mRNA transcript. Engineered nucleic acids can be multicistronic through the use of various linkers, e.g., a polynucleotide sequence encoding a first protein can be linked to a nucleotide sequence encoding a second protein, such as in a first gene: I inker: second gene 5’ to 3’ orientation. A linker can encode a 2A ribosome skipping element, such as T2A. Other 2A ribosome skipping elements include, but are not limited to, E2A, P2A, and F2A. 2A ribosome skipping elements allow production of separate polypeptides encoded by the first and second genes are produced during translation. A linker can encode a cleavable linker polypeptide sequence, such as a Furin cleavage site or a TEV cleavage site, wherein following expression the cleavable linker polypeptide is cleaved such that separate polypeptides encoded by the first and second genes are produced. A cleavable linker can include a polypeptide sequence, such as such a flexible linker e.g., a Gly-Ser-Gly sequence), that further promotes cleavage. In some embodiments, an engineered nucleic acid disclosed herein comprises a sequence encoding an E2A/T2A ribosome skipping element. In certain embodiments, the E2A/T2A ribosome skipping element comprises the amino acid sequence of GSGQCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 281). An exemplary nucleic acid encoding SEQ ID NO: 281 is GGTAGCGGCCAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATC TAATCCTGGACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACG TGGAGGAAAACCCTGGACCT (SEQ ID NO: 282). In certain embodiments, a nucleic acid encoding SEQ ID NO: 281 comprises a sequence that is 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% identical to SEQ ID NO: 282. In some embodiments, an engineered nucleic acid disclosed herein encodes an E2A/T2A ribosome skipping element. In certain embodiments, the E2A/T2A ribosome skipping element comprises the amino acid sequence of QCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 283). An exemplary nucleic acid encoding SEQ ID NO: 283 is CAGTGTACCAACTACGCCCTGCTGAAACTGGCCGGCGACGTGGAATCTAATCCTGG ACCTGGATCTGGCGAGGGACGCGGGAGTCTACTGACGTGTGGAGACGTGGAGGAA AACCCTGGACCT (SEQ ID NO: 284). In certain embodiments, a nucleic acid encoding SEQ ID NO: 283 comprises a sequence that is 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% identical to SEQ ID NO: 284.

A linker can encode an Internal Ribosome Entry Site (IRES), such that separate polypeptides encoded by the first and second genes are produced during translation. A linker can encode a splice acceptor, such as a viral splice acceptor.

A linker can be a combination of linkers, such as a Furin-2A linker that can produce separate polypeptides through 2A ribosome skipping followed by further cleavage of the Furin site to allow for complete removal of 2A residues. In some embodiments, a combination of linkers can include a Furin sequence, a flexible linker, and 2A linker. Accordingly, in some embodiments, the linker is a Furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, a linker of the present disclosure is a Furin-Gly-Ser-Gly-T2A fusion polypeptide.

In general, a multicistronic system can use any number or combination of linkers, to express any number of genes or portions thereof (e.g., an engineered nucleic acid can encode a first, a second, and a third protein, each separated by linkers such that separate polypeptides encoded by the first, second, and third proteins are produced).

Engineered nucleic acids can use multiple promoters to express genes from multiple ORFs, i.e., more than one separate mRNA transcript can be produced from a single engineered nucleic acid. For example, a first promoter can be operably linked to a polynucleotide sequence encoding a first protein, and a second promoter can be operably linked to a polynucleotide sequence encoding a second protein. In general, any number of promoters can be used to express any number of proteins. In some embodiments, at least one of the ORFs expressed from the multiple promoters can be multicistronic.

Expression cassettes encoded on the same engineered nucleic acid can be oriented in any manner suitable for expression of the encoded exogenous polynucleotide sequences. Expression cassettes encoded on the same engineered nucleic acid can be oriented in the same direction, i.e., transcription of separate cassettes proceeds in the same direction. Constructs oriented in the same direction can be organized in a head-to-tail format referring to the 5' end (head) of the first gene being adjacent to the 3' end (tail) of the upstream gene. Expression cassettes encoded on the same engineered nucleic acid can be oriented in an opposite direction, i.e., transcription of separate cassettes proceeds in the opposite direction (also referred to herein as “bidirectional”). Expression cassettes encoded on the same engineered nucleic acid oriented in opposite directions can be oriented in a “head-to-head” directionality. As used herein, head-to-head refers to the 5' end (head) of a first gene of a bidirectional construct being adjacent to the 5' end (head) of an upstream gene of the bidirectional construct. Expression cassettes encoded on the same engineered nucleic acid oriented in opposite directions can be oriented in a “tail-to-tail” directionality. As used herein, tail-to-tail refers to the 3' end (tail) of a first gene of a bidirectional construct being adjacent to the 3' end (tail) of an upstream gene of the bidirectional construct. For example, and without limitation, FIG. 1 schematically depicts a cytokine-CAR bidirectional construct in head-to-head directionality (FIG. 1A), head-to-tail directionality (FIG. IB), and tail-to-tail directionality (FIG. 1C).

“Linkers,” as used herein can refer to polypeptides that link a first polypeptide sequence and a second polypeptide sequence, the multicistronic linkers described above, or the additional promoters that are operably linked to additional ORFs described above.

Exogenous polynucleotide sequences encoded by the expression cassette can include a 3 ’untranslated region (UTR) comprising an mRNA-destabilizing element that is operably linked to the exogenous polynucleotide sequence, such as exogenous polynucleotide sequences encoding a cytokine (e.g., IL12 or IL12p70). In some embodiments, the mRNA-destabilizing element comprises an AU-rich element and/or a stem-loop destabilizing element (SLDE). In some embodiments, the mRNA-destabilizing element comprises an AU-rich element. In some embodiments, the AU-rich element includes at least two overlapping motifs of the sequence ATTTA (SEQ ID NO: 209). In some embodiments, the AU-rich element comprises ATTTATTTATTTATTTATTTA (SEQ ID NO: 210). In some embodiments, the mRNA- destabilizing element comprises a stem-loop destabilizing element (SLDE). In some embodiments, the SLDE comprises CTGTTTAATATTTAAACAG (SEQ ID NO: 211). In some embodiments, the mRNA-destabilizing element comprises at least one AU-rich element and at least one SLDE. “AuSLDE” as used herein refers to an AU-rich element operably linked to a stem- loop destabilizing element (SLDE). An exemplary AuSLDE sequence comprises ATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG (SEQ ID NO: 212). In some embodiments, the mRNA-destabilizing element comprises a 2X AuSLDE. An exemplary AuSLDE sequence is provided as ATTTATTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAGtgcggtaagcATTTA TTTATTTATTTATTTAacatcggttccCTGTTTAATATTTAAACAG (SEQ ID NO: 213). In certain embodiments, an engineered nucleic acid described herein comprises an insulator sequence. Such insulator sequences function to prevent inappropriate interactions between adjacent regions of a construct. In certain embodiments, an insulator sequence comprises the nucleic acid sequence of ACAATGGCTGGCCCATAGTAAATGCCGTGTTAGTGTGTTAGTTGCTGTTCTTCCACG TCAGAAGAGGCACAGACAAATTACCACCAGGTGGCGCTCAGAGTCTGCGGAGGCAT CACAACAGCCCTGAATTTGAATCCTGCTCTGCCACTGCCTAGTTGAGACCTTTTACT ACCTGACTAGCTGAGACATTTACGACATTTACTGGCTCTAGGACTCATTTTATTCAT TTCATTACTTTTTTTTTCTTTGAGACGGAATCTCGCTCT (SEQ ID NO: 300). In certain embodiments, an insulator sequence comprises a sequence that is 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% identical to SEQ ID NO: 300.

Engineered Cells

Provided herein are engineered immunoresponsive cells, and methods of producing the engineered immunoresponsive cells, that produce a protein described herein (e.g., a CAR, and a membrane-cleavable chimeric protein described herein). In general, engineered NK cells of the present disclosure may be engineered to express the proteins provided for herein, such as a CAR, and/or the membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein. These cells are referred to herein as “engineered cells.” These cells, which typically contain engineered nucleic acid, do not occur in nature. In some embodiments, the cells are engineered to include a nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding a protein, for example, a CAR, and/or a membrane-cleavable chimeric protein. An engineered cell can comprise an engineered nucleic acid integrated into the cell’s genome. An engineered cell can comprise an engineered nucleic acid capable of expression without integrating into the cell’s genome, for example, engineered with a transient expression system such as a plasmid or mRNA.

The present disclosure also encompasses additivity and synergy between a protein(s) and the engineered cell from which they are produced. In some embodiments, cells are engineered to produce at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) proteins, for example at least each of a CAR, and membrane-cleavable chimeric protein. In general, NK cells provide herein are engineered to produce at least one membrane-cleavable chimeric protein having an effector molecule that is or comprises IL 15 or a functional fragment thereof, and a CAR. Such an effector molecule may, for example, complement the function of effector molecules natively produced by the cells. In some embodiments, engineered cells comprise one or more engineered nucleic acids encoding a promoter operably linked to a nucleotide sequence encoding a protein (e.g., an expression cassette). Engineered cells can comprise an engineered nucleic acid encoding at least one of the linkers described above, such as polypeptides that link a first polypeptide sequence and a second polypeptide sequence, one or more multicistronic linker described above, one or more additional promoters operably linked to additional ORFs, or a combination thereof.

In some embodiments, a cell (e.g., an immune cell) is engineered to express a protease. In some embodiments, a cell is engineered to express a protease heterologous to a cell. In some embodiments, a cell is engineered to express a protease heterologous to a cell expressing a protein, such as a heterologous protease that cleaves the protease cleavage site of a membrane- cleavable chimeric protein. In some embodiments, engineered cells comprise one or more engineered nucleic acids encoding a promoter operably linked to a nucleotide sequence encoding a protease, such as a heterologous protease. Protease and protease cleavage sites are described in greater detail in the Section herein titled “Protease Cleavage site.”

Also provided herein are engineered cells that are engineered to produce multiple proteins, at least two of which include effector molecules that modulate different tumor- mediated immunosuppressive mechanisms. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) protein includes an effector molecule that stimulates at least one immuno stimulatory mechanism in the tumor microenvironment, or inhibits at least one immunosuppressive mechanism in the tumor microenvironment. In some embodiments, at least one (e.g., 1, 2, 3, 4, 5, or more) protein includes an effector molecule that inhibits at least one immunosuppressive mechanism in the tumor microenvironment, and at least one protein (e.g., 1, 2, 3, 4, 5, or more) inhibits at least one immunosuppressive mechanism in the tumor microenvironment. In yet other embodiments, at least two (e.g., 2, 3, 4, 5, or more) of the proteins are effector molecules that each stimulate at least one immuno stimulatory mechanism in the tumor microenvironment. In still other embodiments, at least two (e.g., 1, 2, 3, 4, 5, or more) of the proteins are effector molecules that each inhibit at least one immunosuppressive mechanism in the tumor microenvironment.

In some embodiments, a cell (e.g., an immune cell) is engineered to produce at least one protein including an effector molecule that stimulates T cell or NK cell signaling, activity and/or recruitment. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates antigen presentation and/or processing. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates natural killer cell-mediated cytotoxic signaling, activity and/or recruitment. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates dendritic cell differentiation and/or maturation. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates immune cell recruitment. In some embodiments, a cell is engineered to produce at least one protein includes an effector molecule that that stimulates Ml macrophage signaling, activity and/or recruitment. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates Thl polarization. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates stroma degradation. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates immuno stimulatory metabolite production. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that stimulates Type I interferon signaling. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that inhibits negative costimulatory signaling. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that inhibits pro-apoptotic signaling (e.g., via TRAIL) of anti-tumor immune cells. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that inhibits T regulatory (T_reg) cell signaling, activity and/or recruitment. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that inhibits tumor checkpoint molecules. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that activates stimulator of interferon genes (STING) signaling. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that inhibits myeloid-derived suppressor cell signaling, activity and/or recruitment. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that degrades immunosuppressive factors/metabolites. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that inhibits vascular endothelial growth factor signaling. In some embodiments, a cell is engineered to produce at least one protein that includes an effector molecule that directly kills tumor cells (e.g., granzyme, perforin, oncolytic viruses, cytolytic peptides and enzymes, anti-tumor antibodies, e.g., that trigger ADCC).

In some embodiments, at least one protein including an effector molecule that: stimulates T cell signaling, activity and/or recruitment, stimulates antigen presentation and/or processing, stimulates natural killer cell-mediated cytotoxic signaling , activity and/or recruitment, stimulates dendritic cell differentiation and/or maturation, stimulates immune cell recruitment, stimulates macrophage signaling, stimulates stroma degradation, stimulates immunostimulatory metabolite production, or stimulates Type I interferon signaling; and at least one protein including an effector molecule that inhibits negative costimulatory signaling, inhibits pro- apoptotic signaling of anti-tumor immune cells, inhibits T regulatory (Treg) cell signaling, activity and/or recruitment, inhibits tumor checkpoint molecules, activates stimulator of interferon genes (STING) signaling, inhibits myeloid-derived suppressor cell signaling, activity and/or recruitment, degrades immunosuppressive factors/metabolites, inhibits vascular endothelial growth factor signaling, or directly kills tumor cells.

In some embodiments, an immunoresponsive cell is engineered to produce at least one membrane-cleavable chimeric protein including an effector molecule cytokine that is or comprises IL- 15.

In certain embodiments, the IL 15 comprises the amino acid sequence of NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 285). An exemplary nucleic acid sequence encoding SEQ ID NO: 285 is AATTGGGTCAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTGATCCAGAGCAT GCACATCGACGCCACACTGTACACCGAGAGCGACGTGCACCCTAGCTGTAAAGTGA CCGCCATGAAGTGCTTTCTGCTGGAACTGCAAGTGATCAGCCTGGAAAGCGGCGAC GCCAGCATCCACGACACCGTGGAAAACCTGATCATCCTGGCCAACAACAGCCTGAG CAGCAACGGCAATGTGACCGAGTCCGGCTGCAAAGAGTGCGAGGAACTGGAAGAG AAGAATATCAAAGAGTTCCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAA CACAAGC (SEQ ID NO: 286). In certain embodiments, a nucleic acid encoding SEQ ID NO: 285 comprises a sequence that is 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% identical to SEQ ID NO: 286.

In some embodiments, a cell is engineered to produce at least one membrane-cleavable chimeric protein where the secretable effector molecule (e.g., “S” in the formula S - C - MT or MT - C - S) is or comprises IL15.

A cell can also be further engineered to express additional proteins in addition to the cytokines and/or the membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein. In some embodiments, an immunoresponsive cell is engineered to express a chimeric antigen receptor (CAR) that binds to GPC3 and the membrane-cleavable chimeric protein.

A CAR can include an antigen-binding domain, such as an antibody, an antigen-binding fragment of an antibody, a F(ab) fragment, a F(ab') fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb). An antigen recognizing receptors can include an scFv. An scFv can include a heavy chain variable domain (VH) and a light chain variable domain (VL), which can be separated by a peptide linker. For example, an scFv can include the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. In certain embodiments, the peptide linker is a gly-ser linker. In certain embodiments, the peptide linker is a (GGGGS)3 linker comprising the sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 223). An exemplary nucleic acid sequence encoding SEQ ID NO: 223 is GGCGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGATCT (SEQ ID NO: 224) or GGCGGCGGAGGAAGCGGAGGCGGAGGATCCGGTGGTGGTGGATCT (SEQ ID NO: 332). In certain embodiments, a nucleic acid encoding SEQ ID NO: 223 comprises a sequence that is 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% identical to SEQ ID NO: 224 or SEQ ID NO: 332.

A CAR can have one or more intracellular signaling domains, such as a CD3zeta-chain intracellular signaling domain, a CD97 intracellular signaling domain, a CDl la-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD 154 intracellular signaling domain, a CD8 intracellular signaling domain, an 0X40 intracellular signaling domain, a 4- IBB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP 10 intracellular signaling domain, a DAP 12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD 16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS 1 intracellular signaling domain, a KIR3DS 1 intracellular signaling domain, a NKp44 intracellular signaling domain, a NKp46 intracellular signaling domain, a FceRlg intracellular signaling domain, a NKG2D intracellular signaling domain, an EAT-2 intracellular signaling domain, fragments thereof, combinations thereof, or combinations of fragments thereof. In some embodiments, the intracellular signaling domain comprises a sequence from Table 6A.

Table 6A.

In some embodiments, a CAR can also comprise a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region may be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In some embodiments, the spacer region may be a hinge from a human protein. For example, the hinge may be a human Ig (immunoglobulin) hinge, including without limitation an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge. In some embodiments, the spacer region may comprise an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-beta extracellular linker. In some embodiments, the spacer region comprises a sequence from Table 6B.

Table 6B.

A CAR can have a transmembrane domain, such as a CD8 transmembrane domain, a CD28 transmembrane domain a CD3zeta-chain transmembrane domain, a CD4 transmembrane domain, a 4- IBB transmembrane domain, an 0X40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, a BTLA transmembrane domain, an 0X40 transmembrane domain, a DAP 10 transmembrane domain, a DAP 12 transmembrane domain, a CD 16a transmembrane domain, a DNAM-1 transmembrane domain, a KIR2DS1 transmembrane domain, a KIR3DS 1 transmembrane domain, an NKp44 transmembrane domain, an NKp46 transmembrane domain, an FceRlg transmembrane domain, an NKG2D transmembrane domain, fragments thereof, combinations thereof, or combinations of fragments thereof. A CAR can have a spacer region between the antigen-binding domain and the transmembrane domain. Exemplary transmembrane domain sequences are provided in Table 6C. Table 6C.

In some embodiments, the CAR antigen-binding domain that binds to GPC3 includes a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH includes: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 199), a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 200), and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 201), and wherein the VL includes: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 202), a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO: 203), and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 204). In some embodiments, the antigen-binding domain that binds to GPC3 includes a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 199). In some embodiments, the antigenbinding domain that binds to GPC3 includes a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 200). In some embodiments, the antigen-binding domain that binds to GPC3 includes a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 201). In some embodiments, the antigen-binding domain that binds to GPC3 includes a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 202). In some embodiments, the antigen-binding domain that binds to GPC3 includes a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO: 203). In some embodiments, the antigen-binding domain that binds to GPC3 includes a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 204).

In some embodiments, the antigen-binding domain that binds to GPC3 includes a VH region having an amino acid sequence with 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% identity to the amino acid sequence of EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIRNKTN NYATYYADSVKARFTISRDDSQSMLYLQMNNLKIEDTAMYYCVAGNSFA YWGQGTLVTVSA (SEQ ID NO: 205) or EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIRNKTNN YATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFAYWGQGTLVT VSA (SEQ ID NO: 206). An exemplary nucleic acid sequence encoding SEQ ID NO: 206 is GAAGTGCAGCTGGTGGAATCTGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAG ACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAACAAGAACGCCATGAACTGGGTCCG ACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGACGGATCCGGAACAAGACCAAC AACTACGCCACCTACTACGCCGACAGCGTGAAGGCCAGGTTCACCATCTCCAGAGA TGACAGCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAAAACCGAGGACACCG CCGTGTACTATTGCGTGGCCGGCAATAGCTTTGCCTACTGGGGACAGGGCACCCTG GTTACAGTTTCTGCT (SEQ ID NO: 222) or GAAGTGCAGCTGGTTGAATCAGGTGGCGGCCTGGTTCAACCTGGCGGATCTCTGAG ACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAACAAGAACGCCATGAACTGGGTCC GACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGACGGATCCGGAACAAGACCAA CAACTACGCCACCTACTACGCCGACAGCGTGAAGGCCAGATTCACCATCAGCCGGG ACGACAGCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAAAACCGAGGACACC GCCGTGTATTATTGCGTGGCCGGCAACAGCTTTGCCTACTGGGGACAGGGAACCCT GGTCACCGTGTCTGCC (SEQ ID NO: 330). In certain embodiments, a nucleic acid encoding SEQ ID NO: 206 comprises a sequence that is 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% identical to SEQ ID NO: 222 or SEQ ID NO: 330.

In some embodiments, the antigen-binding domain that binds to GPC3 includes a VL region having an amino acid sequence with 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% identity to the amino acid sequence of DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASS RESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLELK (SEQ ID NO: 207), or DIVMTQSPDSEAVSEGERATINCKSSQSEEYSSNQKNYEAWYQQKPGQPPKEEIYWASS RESGVPDRFSGSGSGTDFTETISSEQAEDVAVYYCQQYYNYPETFGQGTKEEIK (SEQ ID NO: 208). An exemplary nucleic acid sequence encoding SEQ ID NO: 208 is GACATCGTGATGACACAGAGCCCCGATAGCCTGGCCGTGTCTCTGGGAGAAAGAGC CACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGTACTCCAGCAACCAGAAGAACT ACCTGGCCTGGTATCAGCAAAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTATTGG GCCAGCTCCAGAGAAAGCGGCGTGCCCGATAGATTTTCTGGCTCTGGCAGCGGCAC CGACTTCACCCTGACAATTTCTAGCCTGCAAGCCGAGGACGTGGCCGTGTACTACTG CCAGCAGTACTACAACTACCCTCTGACCTTCGGCCAGGGCACCAAGCTGGAAATCA AA (SEQ ID NO: 221) or GACATCGTGATGACACAGAGCCCCGATAGCCTGGCCGTGTCTCTGGGAGAAAGAGC CACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGTACTCCAGCAACCAGAAGAACT ACCTGGCCTGGTATCAGCAAAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTATTGG GCCAGCTCCAGAGAAAGCGGCGTGCCCGATAGATTTTCTGGCTCTGGCAGCGGCAC CGACTTCACCCTGACAATTTCTAGCCTGCAAGCCGAGGACGTGGCCGTGTATTACTG CCAGCAGTACTACAACTACCCTCTGACCTTCGGCCAGGGCACCAAGCTGGAAATCA AA (SEQ ID NO: 333) or GACATCGTGATGACACAGAGCCCCGATAGCCTGGCCGTGTCTCTGGGAGAAAGAGC CACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGTACTCCAGCAACCAGAAGAACT ACCTGGCCTGGTATCAGCAAAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTATTGG GCCAGCTCCAGAGAAAGCGGCGTGCCCGATAGATTTTCTGGCTCTGGCAGCGGCAC CGACTTCACCCTGACAATTTCTAGCCTGCAAGCCGAGGACGTGGCCGTGTATTACTG CCAGCAGTACTACAACTACCCTCTGACCTTCGGCCAGGGCACCAAGCTGGAAATCA AG (SEQ ID NO: 336). In certain embodiments, a nucleic acid encoding SEQ ID NO: 208 comprises a sequence that is 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% identical to SEQ ID NO: 221 or SEQ ID NO: 336.

Engineered Cell Types

Also provided herein are engineered NK cells. Engineered NK cells can be engineered to comprise any of the engineered nucleic acids described herein (e.g., any of the engineered nucleic acids encoding a membrane-cleavable chimeric protein described herein, and a CAR described herein). Cells can be engineered to possess any of the features of any of the engineered cells described herein. In a particular aspect, provided herein are cells engineered to produce a CAR that binds to GPC3 and a membrane-cleavable chimeric protein having the formula S - C - MT or MT - C - S described herein.

The engineered NK cells include, but are not limited to, a Natural Killer (NK) cell, such as an ESC-derived NK cell, and an iPSC-derived NK cell.

A cell can be engineered to produce the proteins described herein using methods known to those skilled in the art. For example, cells can be transduced, e.g., using a virus.

In a particular embodiment, the cell is transduced using an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof.

The virus, including any of the oncolytic viruses described herein, can be a recombinant virus that encodes one more transgenes encoding one or more proteins, such as any of the engineered nucleic acids described herein. The virus, including any of the oncolytic viruses described herein, can be a recombinant virus that encodes one more transgenes encoding one or more of the two or more proteins, such as any of the engineered nucleic acids described herein.

An engineered cell can be a human cell. An engineered cell can be a human primary cell. An engineered primary cell can be a tumor infiltrating primary cell. An engineered primary cell can be a natural killer (NK) cell. Human cells (e.g., NK cells) can be engineered to comprise any of the engineered nucleic acids described herein. Human cells (e.g., NK cells) can be engineered to possess any of the features of any of the engineered cells described herein. In a particular aspect, provided herein are human cells (e.g., NK cells) engineered to produce one or more of the proteins described herein. In a particular aspect, provided herein are human cells (e.g., NK cells) engineered to produce two or more of the proteins described herein.

An engineered cell can be isolated from a subject (autologous), such as a subject known or suspected to have cancer. Cell isolation methods are known to those skilled in the art and include, but are not limited to, sorting techniques based on cell-surface marker expression, such as FACS sorting, positive isolation techniques, and negative isolation, magnetic isolation, and combinations thereof.

An engineered cell can be allogenic with reference to the subject being administered a treatment. Allogenic modified cells can be HLA-matched to the subject being administered a treatment. Allogeneic engineered cells can include engineered NK cells. An engineered cell can be a cultured cell, such as an ex vivo cultured cell. An engineered cell can be an ex vivo cultured cell, such as a primary cell isolated from a subject. Cultured cell can be cultured with one or more cytokines.

Also provided herein are methods that include culturing the engineered cells of the present disclosure. Methods of culturing the engineered cells described herein are known. One skilled in the art will recognize that culturing conditions will depend on the particular engineered cell of interest. One skilled in the art will recognize that culturing conditions will depend on the specific downstream use of the engineered cell, for example, specific culturing conditions for subsequent administration of the engineered cell to a subject.

Methods of Engineering Cells

Also provided herein are compositions and methods for engineering NK cells to produce a CAR and a membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein).

In general, cells are engineered to produce proteins of interest through introduction (z.e., delivery) of polynucleotides encoding the one or more proteins of interest or effector molecules, e.g., the chimeric proteins described herein including the protein of interest or effector molecule, into the cell’s cytosol and/or nucleus. For example, the polynucleotides encoding the one or more chimeric proteins can be any of the engineered nucleic acids encoding a CAR, and a membrane-cleavable chimeric protein having the formula S - C - MT or MT - C - S described herein. Delivery methods include, but are not limited to, viral-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means. One skilled in the art will appreciate the choice of delivery method can depend on the specific cell type to be engineered.

Viral-Mediated Delivery

Viral vector-based delivery platforms can be used to engineer cells. In general, a viral vector-based delivery platform engineers a cell through introducing (i.e., delivering) into a host cell. For example, a viral vector-based delivery platform can engineer a cell through introducing any of the engineered nucleic acids described herein (e.g., any of the exogenous polynucleotide sequences encoding a CAR disclosed herein, and a the membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein, and/or any of the expression cassettes described herein containing a promoter and an exogenous polynucleotide sequence encoding the proteins, oriented from N-terminal to C-terminal). A viral vector-based delivery platform can be a nucleic acid, and as such, an engineered nucleic acid can also encompass an engineered virally-derived nucleic acid. Such engineered virally-derived nucleic acids can also be referred to as recombinant viruses or engineered viruses.

A viral vector-based delivery platform can encode more than one engineered nucleic acid, gene, or transgene within the same nucleic acid. For example, an engineered virally- derived nucleic acid, e.g., a recombinant virus or an engineered virus, can encode one or more transgenes, including, but not limited to, any of the engineered nucleic acids described herein that encode one or more of the proteins described herein. The one or more transgenes encoding the one or more proteins can be configured to express the one or more proteins and/or other protein of interest. A viral vector-based delivery platform can encode one or more genes in addition to the one or more transgenes (e.g., transgenes encoding the one or more proteins and/or other protein of interest), such as viral genes needed for viral infectivity and/or viral production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.), referred to as cis-acting elements or genes.

A viral vector-based delivery platform can comprise more than one viral vector, such as separate viral vectors encoding the engineered nucleic acids, genes, or transgenes described herein, and referred to as trans-acting elements or genes. For example, a helper-dependent viral vector-based delivery platform can provide additional genes needed for viral infectivity and/or viral production on one or more additional separate vectors in addition to the vector encoding the one or more proteins and/or other protein of interest. One viral vector can deliver more than one engineered nucleic acids, such as one vector that delivers engineered nucleic acids that are configured to produce two or more proteins and/or other protein of interest. More than one viral vector can deliver more than one engineered nucleic acids, such as more than one vector that delivers one or more engineered nucleic acid configured to produce one or more proteins and/or other protein of interest. The number of viral vectors used can depend on the packaging capacity of the above mentioned viral vector-based vaccine platforms, and one skilled in the art can select the appropriate number of viral vectors.

In general, any of the viral vector-based systems can be used for the in vitro production of molecules, such as the proteins, effector molecules, and/or other protein of interest described herein, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids encoding one or more proteins and/or other protein of interest. The selection of an appropriate viral vector-based system will depend on a variety of factors, such as cargo/payload size, immunogenicity of the viral system, target cell of interest, gene expression strength and timing, and other factors appreciated by one skilled in the art.

Viral vector-based delivery platforms can be RNA-based viruses or DNA-based viruses. Exemplary viral vector-based delivery platforms include, but are not limited to, a herpes simplex virus, a adenovirus, a measles virus, an influenza virus, a Indiana vesiculovirus, a Newcastle disease virus, a vaccinia virus, a poliovirus, a myxoma virus, a reovirus, a mumps virus, a Maraba virus, a rabies virus, a rotavirus, a hepatitis virus, a rubella virus, a dengue virus, a chikungunya virus, a respiratory syncytial virus, a lymphocytic choriomeningitis virus, a morbillivirus, a lentivirus, a replicating retrovirus, a rhabdovirus, a Seneca Valley virus, a sindbis virus, and any variant or derivative thereof. Other exemplary viral vector-based delivery platforms are described in the art, such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616 — 629), or lentivirus, including but not limited to second, third or hybrid second/third generation lentivirus and recombinant lentivirus of any generation designed to target specific cell types or receptors (See, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1): 45-61, Sakuman et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873- 9880).

The sequences may be preceded with one or more sequences targeting a subcellular compartment. Upon introduction (i.e. delivery) into a host cell, infected cells (i.e., an engineered cell) can express the proteins and/or other protein of interest. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456- 460 (1991)). A wide variety of other vectors useful for the introduction (z.e., delivery) of engineered nucleic acids, e.g., Salmonella typhi vectors, and the like will be apparent to those skilled in the art from the description herein.

The viral vector-based delivery platforms can be a virus that targets a cell, herein referred to as an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof. Any of the oncolytic viruses described herein can be a recombinant oncolytic virus comprising one more transgenes (e.g., an engineered nucleic acid) encoding one or more proteins and/or other protein of interest. The transgenes encoding the one or more proteins and/or other protein of interest can be configured to express the proteins and/or other protein of interest.

The viral vector-based delivery platform can be retrovirus-based. In general, retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the one or more engineered nucleic acids (e.g., transgenes encoding the one or more proteins and/or other protein of interest) into the target cell to provide permanent transgene expression. Retroviral-based delivery systems include, but are not limited to, those based upon murine leukemia, virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency vims (SIV), human immuno deficiency vims (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et ah, J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et ah, J. Virol. 63:2374-2378 (1989); Miller et al, J, Virol. 65:2220-2224 (1991); PCT/US94/05700). Other retroviral systems include the Phoenix retrovirus system.

The viral vector-based delivery platform can be lentivirus-based. In general, lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Lentiviral-based delivery platforms can be HIV -based, such as ViraPower systems (ThermoFisher) or pLenti systems (Cell Biolabs). Lentiviral-based delivery platforms can be SIV, or FIV-based. Other exemplary lentivirus-based delivery platforms are described in more detail in U.S. Pat. Nos. 7,311,907; 7,262,049; 7,250,299; 7,226,780; 7,220,578; 7,211,247; 7,160,721; 7,078,031; 7,070,993; 7,056,699; 6,955,919, each herein incorporated by reference for all purposes.

The viral vector-based delivery platform can be adenovirus-based. In general, adenoviral based vectors are capable of very high transduction efficiency in many cell types, do not require cell division, achieve high titer and levels of expression, and can be produced in large quantities in a relatively simple system. In general, adenoviruses can be used for transient expression of a transgene within an infected cell since adenoviruses do not typically integrate into a host’s genome. Adenovirus-based delivery platforms are described in more detail in Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655, each herein incorporated by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Pat. Nos. 5585362; 6,083,716, 7,371,570; 7,348,178; 7,323,177; 7,319,033; 7,318,919; and 7,306,793 and International Patent Application WO96/13597, each herein incorporated by reference for all purposes.

The viral vector-based delivery platform can be adeno-associated virus (AAV)-based. Adeno-associated virus (“AAV”) vectors may be used to transduce cells with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). AAV systems can be used for the in vitro production of proteins of interest, such as the proteins described herein and/or effector molecules, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids encoding one or more proteins and/or other protein of interest (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. Nos. 4,797,368; 5,436,146; 6,632,670; 6,642,051; 7,078,387; 7,314,912; 6,498,244; 7,906,111; US patent publications US 2003-0138772, US 2007/0036760, and US 2009/0197338; Gao, et al., J. Virol, 78( 12):6381- 6388 (June 2004); Gao, et al, Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); and International Patent applications WO 2010/138263 and WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994), each herein incorporated by reference for all purposes). Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Pat. No, 5,173,414; Tratschin et ah, Mol. Cell. Biol. 5:3251- 3260 (1985); Tratschin, et ah, Mol. Cell, Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:64666470 (1984); and Samuiski et ah, J. Virol. 63:03822-3828 (1989), each herein incorporated by reference for all purposes. In general, an AAV-based vector comprises a capsid protein having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.RhlO, AAV11 and variants thereof. In particular examples, an AAV-based vector has a capsid protein having an amino acid sequence corresponding to AAV2. In particular examples, an AAV-based vector has a capsid protein having an amino acid sequence corresponding to AAV8.

AAV vectors can be engineered to have any of the exogenous polynucleotide sequences encoding the proteins described herein, such as a CAR described herein, and membrane- cleavable chimeric proteins described herein having the formula: S - C - MT or MT - C - S.

The viral vector-based delivery platform can be a virus-like particle (VLP) platform. In general, VLPs are constructed by producing viral structural proteins and purifying resulting viral particles. Then, following purification, a cargo/payload (e.g., any of the engineered nucleic acids described herein) is encapsulated within the purified particle ex vivo. Accordingly, production of VLPs maintains separation of the nucleic acids encoding viral structural proteins and the nucleic acids encoding the cargo/payload. The viral structural proteins used in VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translation expression systems. The purified viral particles can be denatured and reformed in the presence of the desired cargo to produce VLPs using methods known to those skilled in the art. Production of VLPs are described in more detail in Seow et al. (Mol Ther. 2009 May; 17(5): 767-777), herein incorporated by reference for all purposes.

The viral vector-based delivery platform can be engineered to target (z.e., infect) a range of cells, target a narrow subset of cells, or target a specific cell. In general, the envelope protein chosen for the viral vector-based delivery platform will determine the viral tropism. The virus used in the viral vector-based delivery platform can be pseudotyped to target a specific cell of interest. The viral vector-based delivery platform can be pantropic and infect a range of cells. For example, pantropic viral vector-based delivery platforms can include the VSV-G envelope. The viral vector-based delivery platform can be amphotropic and infect mammalian cells. Accordingly, one skilled in the art can select the appropriate tropism, pseudotype, and/or envelope protein for targeting a desired cell type.

Lipid Structure Delivery Systems

Engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) can be introduced into a cell using a lipid-mediated delivery system. In general, a lipid-mediated delivery system uses a structure composed of an outer lipid membrane enveloping an internal compartment. Examples of lipid-based structures include, but are not limited to, a lipid-based nanoparticle, a liposome, a micelle, an exosome, a vesicle, an extracellular vesicle, a cell, or a tissue. Lipid structure delivery systems can deliver a cargo/payload (e.g., any of the engineered nucleic acids described herein) in vitro, in vivo, or ex vivo. A lipid-based nanoparticle can include, but is not limited to, a unilamellar liposome, a multilamellar liposome, and a lipid preparation. As used herein, a “liposome” is a generic term encompassing in vitro preparations of lipid vehicles formed by enclosing a desired cargo, e.g., an engineered nucleic acid, such as any of the engineered nucleic acids described herein, within a lipid shell or a lipid aggregate. Liposomes may be characterized as having vesicular structures with a bilayer membrane, generally comprising a phospholipid, and an inner medium that generally comprises an aqueous composition. Liposomes include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes can be unilamellar liposomes. Liposomes can be multilamellar liposomes. Liposomes can be multivesicular liposomes. Liposomes can be positively charged, negatively charged, or neutrally charged. In certain embodiments, the liposomes are neutral in charge. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of a desired purpose, e.g., criteria for in vivo delivery, such as liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szokan et al., Ann. Rev. Biophys. Bioeng. 9; 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369, each herein incorporated by reference for all purposes.

A multilamellar liposome is generated spontaneously when lipids comprising phospholipids are suspended in an excess of aqueous solution such that multiple lipid layers are separated by an aqueous medium. Water and dissolved solutes are entrapped in closed structures between the lipid bilayers following the lipid components undergoing self-rearrangement. A desired cargo e.g., a polypeptide, a nucleic acid, a small molecule drug, an engineered nucleic acid, such as any of the engineered nucleic acids described herein, a viral vector, a viral-based delivery system, etc.) can be encapsulated in the aqueous interior of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the polypeptide/nucleic acid, interspersed within the lipid bilayer of a liposome, entrapped in a liposome, complexed with a liposome, or otherwise associated with the liposome such that it can be delivered to a target entity. Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with the lipid bilayer.

A liposome used according to the present embodiments can be made by different methods, as would be known to one of ordinary skill in the art. Preparations of liposomes are described in further detail in WO 2016/201323, International Applications PCT/US85/01161 and PCT/US89/05040, and U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; each herein incorporated by reference for all purposes.

Liposomes can be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patent No. 5,962,016; 5,030,453; 6,680,068, U.S. Application 2004/0208921, and International Patent Applications W003/015757A1, WO04029213A2, and W002/100435A1, each hereby incorporated by reference in their entirety.

Lipid-mediated gene delivery methods are described, for instance, in WO 96/18372; WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833 Rose U.S. Pat. No. 5,279,833; W091/06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987), each herein incorporated by reference for all purposes.

Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. The size of exosomes ranges between 30 and 100 nm in diameter. Their surface consists of a lipid bilayer from the donor cell's cell membrane, and they contain cytosol from the cell that produced the exosome, and exhibit membrane proteins from the parental cell on the surface. Exosomes useful for the delivery of nucleic acids are known to those skilled in the art, e.g., the exosomes described in more detail in U.S. Pat. No. 9,889,210, herein incorporated by reference for all purposes.

As used herein, the term “extracellular vesicle” or “EV” refers to a cell-derived vesicle comprising a membrane that encloses an internal space. In general, extracellular vesicles comprise all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived. Generally extracellular vesicles range in diameter from 20 nm to 1000 nm, and can comprise various macromolecular cargo either within the internal space, displayed on the external surface of the extracellular vesicle, and/or spanning the membrane. The cargo can comprise nucleic acids (e.g., any of the engineered nucleic acids described herein), proteins, carbohydrates, lipids, small molecules, and/or combinations thereof. By way of example and without limitation, extracellular vesicles include apoptotic bodies, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane). Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, and/or cultured cells.

As used herein the term “exosome” refers to a cell-derived small (between 20-300 nm in diameter, more preferably 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. The exosome comprises lipid or fatty acid and polypeptide and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The exosome can be derived from a producer cell, and isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof. An exosome is a species of extracellular vesicle. Generally, exosome production/biogenesis does not result in the destruction of the producer cell. Exosomes and preparation of exosomes are described in further detail in WO 2016/201323, which is hereby incorporated by reference in its entirety.

As used herein, the term “nanovesicle” (also referred to as a “microvesicle”) refers to a cell-derived small (between 20-250 nm in diameter, more preferably 30-150 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct or indirect manipulation such that said nanovesicle would not be produced by said producer cell without said manipulation. In general, a nanovesicle is a sub-species of an extracellular vesicle. Appropriate manipulations of the producer cell include but are not limited to serial extrusion, treatment with alkaline solutions, sonication, or combinations thereof. The production of nanovesicles may, in some instances, result in the destruction of said producer cell. Preferably, populations of nanovesicles are substantially free of vesicles that are derived from producer cells by way of direct budding from the plasma membrane or fusion of the late endosome with the plasma membrane. The nanovesicle comprises lipid or fatty acid and polypeptide, and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The nanovesicle, once it is derived from a producer cell according to said manipulation, may be isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof.

Lipid nanoparticles (LNPs), in general, are synthetic lipid structures that rely on the amphiphilic nature of lipids to form membranes and vesicle like structures (Riley 2017). In general, these vesicles deliver cargo/pay loads, such as any of the engineered nucleic acids or viral systems described herein, by absorbing into the membrane of target cells and releasing the cargo into the cytosol. Lipids used in LNP formation can be cationic, anionic, or neutral. The lipids can be synthetic or naturally derived, and in some instances biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethyleneglycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat soluble vitamins. Lipid compositions generally include defined mixtures of materials, such as the cationic, neutral, anionic, and amphipathic lipids. In some instances, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups that facilitate attachment of additional moieties. Lipid composition can influence overall LNP size and stability. In an example, the lipid composition comprises dilinoleylmethyl- 4- dimethylaminobutyrate (MC3) or MC3-like molecules. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as a PEG or PEG-conjugated lipid, a sterol, or neutral lipids. In addition, LNPs can be further engineered or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity.

Micelles, in general, are spherical synthetic lipid structures that are formed using singlechain lipids, where the single-chain lipid’s hydrophilic head forms an outer layer or membrane and the single-chain lipid’s hydrophobic tails form the micelle center. Micelles typically refer to lipid structures only containing a lipid mono-layer. Micelles are described in more detail in Quader et al. (Mol Ther. 2017 Jul 5; 25(7): 1501-1513), herein incorporated by reference for all purposes.

Nucleic-acid vectors, such as expression vectors, exposed directly to serum can have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by the free nucleic acids. Similarly, viral delivery systems exposed directly to serum can trigger an undesired immune response and/or neutralization of the viral delivery system. Therefore, encapsulation of an engineered nucleic acid and/or viral delivery system can be used to avoid degradation, while also avoiding potential off-target affects. In certain examples, an engineered nucleic acid and/or viral delivery system is fully encapsulated within the delivery vehicle, such as within the aqueous interior of an LNP. Encapsulation of an engineered nucleic acid and/or viral delivery system within an LNP can be carried out by techniques well-known to those skilled in the art, such as microfluidic mixing and droplet generation carried out on a microfluidic droplet generating device. Such devices include, but are not limited to, standard T-junction devices or flow-focusing devices. In an example, the desired lipid formulation, such as MC3 or MC3-like containing compositions, is provided to the droplet generating device in parallel with an engineered nucleic acid or viral delivery system and any other desired agents, such that the delivery vector and desired agents are fully encapsulated within the interior of the MC3 or MC3-like based LNP. In an example, the droplet generating device can control the size range and size distribution of the LNPs produced. For example, the LNP can have a size ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. Following droplet generation, the delivery vehicles encapsulating the cargo/payload (e.g., an engineered nucleic acid and/or viral delivery system) can be further treated or engineered to prepare them for administration.

Nanoparticle Delivery

Nanomaterials can be used to deliver engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). Nanomaterial vehicles, importantly, can be made of non-immunogenic materials and generally avoid eliciting immunity to the delivery vector itself. These materials can include, but are not limited to, lipids (as previously described), inorganic nanomaterials, and other polymeric materials. Nanomaterial particles are described in more detail in Riley et al. (Recent Advances in Nanomaterials for Gene Delivery — A Review. Nanomaterials 2017, 7(5), 94), herein incorporated by reference for all purposes.

Genomic Editing Systems

A genomic editing systems can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid encoding a CAR disclosed herein, and a membrane-cleavable chimeric protein having the formula S - C - MT or MT - C - S described herein. In general, a “genomic editing system” refers to any system for integrating an exogenous gene into a host cell’s genome. Genomic editing systems include, but are not limited to, a transposon system, a nuclease genomic editing system, and a viral vector-based delivery platform.

A transposon system can be used to integrate an engineered nucleic acid, such as an engineered nucleic acid encoding a CAR, and a membrane-cleavable chimeric protein having the formula S - C - MT or MT - C - S described herein, into a host genome. Transposons generally comprise terminal inverted repeats (TIR) that flank a cargo/payload nucleic acid and a transposase. The transposon system can provide the transposon in cis or in trans with the TIR- flanked cargo. A transposon system can be a retrotransposon system or a DNA transposon system. In general, transposon systems integrate a cargo/payload (e.g., an engineered nucleic acid) randomly into a host genome. Examples of transposon systems include systems using a transposon of the Tcl/mariner transposon superfamily, such as a Sleeping Beauty transposon system, described in more detail in Hudecek et al. (Crit Rev Biochem Mol Biol. 2017 Aug;52(4):355-380), and U.S. Patent Nos. 6,489,458, 6,613,752 and 7,985,739, each of which is herein incorporated by reference for all purposes. Another example of a transposon system includes a PiggyBac transposon system, described in more detail in U.S. Patent Nos. 6,218,185 and 6,962,810, each of which is herein incorporated by reference for all purposes. A nuclease genomic editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid encoding a CAR, and a membrane- cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein. Without wishing to be bound by theory, in general, the nuclease-mediated gene editing systems used to introduce an exogenous gene take advantage of a cell’s natural DNA repair mechanisms, particularly homologous recombination (HR) repair pathways. Briefly, following an insult to genomic DNA (typically a double-stranded break), a cell can resolve the insult by using another DNA source that has identical, or substantially identical, sequences at both its 5’ and 3’ ends as a template during DNA synthesis to repair the lesion. In a natural context, HDR can use the other chromosome present in a cell as a template. In gene editing systems, exogenous polynucleotides are introduced into the cell to be used as a homologous recombination template (HRT or HR template). In general, any additional exogenous sequence not originally found in the chromosome with the lesion that is included between the 5’ and 3’ complimentary ends within the HRT (e.g., a gene or a portion of a gene) can be incorporated (z.e., “integrated”) into the given genomic locus during templated HDR. Thus, a typical HR template for a given genomic locus has a nucleotide sequence identical to a first region of an endogenous genomic target locus, a nucleotide sequence identical to a second region of the endogenous genomic target locus, and a nucleotide sequence encoding a cargo/payload nucleic acid (e.g., any of the engineered nucleic acids described herein, such as any of the engineered nucleic acids encoding a CAR, and a membrane-cleavable chimeric protein having the formula S - C - MT or MT - C - S described herein).

In some examples, a HR template can be linear. Examples of linear HR templates include, but are not limited to, a linearized plasmid vector, a ssDNA, a synthesized DNA, and a PCR amplified DNA. In particular examples, a HR template can be circular, such as a plasmid. A circular template can include a supercoiled template.

The identical, or substantially identical, sequences found at the 5’ and 3’ ends of the HR template, with respect to the exogenous sequence to be introduced, are generally referred to as arms (HR arms). HR arms can be identical to regions of the endogenous genomic target locus (z.e., 100% identical). HR arms in some examples can be substantially identical to regions of the endogenous genomic target locus. While substantially identical HR arms can be used, it can be advantageous for HR arms to be identical as the efficiency of the HDR pathway may be impacted by HR arms having less than 100% identity.

Each HR arm, i.e., the 5’ and 3’ HR arms, can be the same size or different sizes. Each HR arm can each be greater than or equal to 50, 100, 200, 300, 400, or 500 bases in length. Although HR arms can, in general, be of any length, practical considerations, such as the impact of HR arm length and overall template size on overall editing efficiency, can also be taken into account. An HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site. Each HR arms can be identical to, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site. Each HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus within a certain distance of a cleavage site, such as 1 base-pair, less than or equal to 10 base-pairs, less than or equal to 50 base-pairs, or less than or equal to 100 base-pairs of each other.

A nuclease genomic editing system can use a variety of nucleases to cut a target genomic locus, including, but not limited to, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease or derivative thereof, a Transcription activator-like effector nuclease (TALEN) or derivative thereof, a zinc-finger nuclease (ZFN) or derivative thereof, and a homing endonuclease (HE) or derivative thereof.

A CRISPR-mediated gene editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid encoding a CAR and a membrane-cleavable chimeric protein having the formula S - C - MT or MT - C - S described herein. CRISPR systems are described in more detail in M. Adli (“The CRISPR tool kit for genome editing and beyond” Nature Communications; volume 9 (2018), Article number: 1911), herein incorporated by reference for all that it teaches. In general, a CRISPR-mediated gene editing system comprises a CRIS PR-associated (Cas) nuclease and a RNA(s) that directs cleavage to a particular target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR/Cas9 systems comprised of a Cas9 nuclease and a RNA(s) that has a CRISPR RNA (crRNA) domain and a trans-activating CRISPR (tracrRNA) domain. The crRNA typically has two RNA domains: a guide RNA sequence (gRNA) that directs specificity through base-pair hybridization to a target sequence (“a defined nucleotide sequence”), e.g., a genomic sequence; and an RNA domain that hybridizes to a tracrRNA. A tracrRNA can interact with and thereby promote recruitment of a nuclease e.g., Cas9) to a genomic locus. The crRNA and tracrRNA polynucleotides can be separate polynucleotides. The crRNA and tracrRNA polynucleotides can be a single polynucleotide, also referred to as a single guide RNA (sgRNA). While the Cas9 system is illustrated here, other CRISPR systems can be used, such as the Cpfl/Casl2 or Casl3 systems. Nucleases can include derivatives thereof, such as Cas9 functional mutants, e.g., a Cas9 “nickase” mutant that in general mediates cleavage of only a single strand of a defined nucleotide sequence as opposed to a complete double- stranded break typically produced by Cas9 enzymes. In general, the components of a CRISPR system interact with each other to form a Ribonucleoprotein (RNP) complex to mediate sequence specific cleavage. In some CRISPR systems, each component can be separately produced and used to form the RNP complex. In some CRISPR systems, each component can be separately produced in vitro and contacted (z.e., “complexed”) with each other in vitro to form the RNP complex. The in vitro produced RNP can then be introduced (z.e., “delivered”) into a cell’s cytosol and/or nucleus, e.g., a T cell’s cytosol and/or nucleus. The in vitro produced RNP complexes can be delivered to a cell by a variety of means including, but not limited to, electroporation, lipid-mediated transfection, cell membrane deformation by physical means, lipid nanoparticles (LNP), virus like particles (VLP), and sonication. In a particular example, in vitro produced RNP complexes can be delivered to a cell using a Nucleofactor/Nucleofection® electroporation-based delivery system (Lonza®). Other electroporation systems include, but are not limited to, MaxCyte electroporation systems, Miltenyi CliniMACS electroporation systems, Neon electroporation systems, and BTX electroporation systems. CRISPR nucleases, e.g., Cas9, can be produced in vitro (i.e., synthesized and purified) using a variety of protein production techniques known to those skilled in the art. CRISPR system RNAs, e.g., an sgRNA, can be produced in vitro (i.e., synthesized and purified) using a variety of RNA production techniques known to those skilled in the art, such as in vitro transcription or chemical synthesis.

An in vitro produced RNP complex can be complexed at different ratios of nuclease to gRNA. An in vitro produced RNP complex can also be used at different amounts in a CRISPR- mediated editing system. For example, depending on the number of cells desired to be edited, the total RNP amount added can be adjusted, such as a reduction in the amount of RNP complex added when editing a large number of cells in a reaction.

In some CRISPR systems, each component (e.g., Cas9 and an sgRNA) can be separately encoded by a polynucleotide with each polynucleotide introduced into a cell together or separately. In some CRISPR systems, each component can be encoded by a single polynucleotide (i.e., a multi-promoter or multicistronic vector, see description of exemplary multicistronic systems below) and introduced into a cell. Following expression of each polynucleotide encoded CRISPR component within a cell (e.g., translation of a nuclease and transcription of CRISPR RNAs), an RNP complex can form within the cell and can then direct site-specific cleavage.

Some RNPs can be engineered to have moieties that promote delivery of the RNP into the nucleus. For example, a Cas9 nuclease can have a nuclear localization signal (NLS) domain such that if a Cas9 RNP complex is delivered into a cell’s cytosol or following translation of Cas9 and subsequent RNP formation, the NLS can promote further trafficking of a Cas9 RNP into the nucleus.

The engineered cells described herein can be engineered using non-viral methods, e.g., the nuclease and/or CRISPR mediated gene editing systems described herein can be delivered to a cell using non-viral methods. The engineered cells described herein can be engineered using viral methods, e.g., the nuclease and/or CRISPR mediated gene editing systems described herein can be delivered to a cell using viral methods such as adenoviral, retroviral, lentiviral, or any of the other viral-based delivery methods described herein.

In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target the same gene or general genomic locus at more than target nucleotide sequence. For example, two separate CRISPR compositions can be provided to direct cleavage at two different target nucleotide sequences within a certain distance of each other. In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target opposite strands of the same gene or general genomic locus. For example, two separate CRISPR “nickase” compositions can be provided to direct cleavage at the same gene or general genomic locus at opposite strands.

In general, the features of a CRISPR-mediated editing system described herein can apply to other nuclease-based genomic editing systems. TALEN is an engineered site-specific nuclease, which is composed of the DNA- binding domain of TALE (transcription activator-like effectors) and the catalytic domain of restriction endonuclease Fokl. By changing the amino acids present in the highly variable residue region of the monomers of the DNA binding domain, different artificial TALENs can be created to target various nucleotides sequences. The DNA binding domain subsequently directs the nuclease to the target sequences and creates a doublestranded break. TALEN-based systems are described in more detail in U.S. Ser. No. 12/965,590; U.S. Pat. No. 8,450,471; U.S. Pat. No. 8,440,431; U.S. Pat. No. 8,440,432; U.S. Pat. No. 10,172,880; and U.S. Ser. No. 13/738,381, all of which are incorporated by reference herein in their entirety. ZFN-based editing systems are described in more detail in U.S. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated herein by reference in their entireties for all purposes. Other Engineering Delivery Systems

Various additional means to introduce engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into a cell or other target recipient entity, such as any of the lipid structures described herein.

Electroporation can used to deliver polynucleotides to recipient entities. Electroporation is a method of internalizing a cargo/payload into a target cell or entity’s interior compartment through applying an electrical field to transiently permeabilize the outer membrane or shell of the target cell or entity. In general, the method involves placing cells or target entities between two electrodes in a solution containing a cargo of interest (e.g., any of the engineered nucleic acids described herein). The lipid membrane of the cells is then disrupted, i.e., permeabilized, by applying a transient set voltage that allows the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the example of cells, at least some, if not a majority, of the cells remain viable. Cells and other entities can be electroporated in vitro, in vivo, or ex vivo. Electroporation conditions (e.g., number of cells, concentration of cargo, recovery conditions, voltage, time, capacitance, pulse type, pulse length, volume, cuvette length, electroporation solution composition, etc.) vary depending on several factors including, but not limited to, the type of cell or other recipient entity, the cargo to be delivered, the efficiency of internalization desired, and the viability desired. Optimization of such criteria are within the scope of those skilled in the art. A variety devices and protocols can be used for electroporation. Examples include, but are not limited to, Neon® Transfection System, MaxCyte® Flow Electroporation™, Lonza® Nucleofector™ systems, and Bio-Rad® electroporation systems.

Other means for introducing engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into a cell or other target recipient entity include, but are not limited to, sonication, gene gun, hydrodynamic injection, and cell membrane deformation by physical means.

Compositions and methods for delivering engineered mRNAs in vivo, such as naked plasmids or mRNA, are described in detail in Kowalski et al. (Mol Ther. 2019 Apr 10; 27(4): 710-728) and Kaczmarek et al. (Genome Med. 2017; 9: 60.), each herein incorporated by reference for all purposes.

Delivery Vehicles

Also provided herein are compositions for delivering a cargo/payload (a “delivery vehicle”).

The cargo can comprise nucleic acids (e.g., any of the engineered nucleic acids described herein, such as any of the engineered nucleic acids described herein encoding a CAR, and a membrane-cleavable chimeric protein having the formula S - C - MT or MT - C - S described herein), as described above. The cargo can comprise proteins, carbohydrates, lipids, small molecules, and/or combinations thereof.

The delivery vehicle can comprise any composition suitable for delivering a cargo. The delivery vehicle can comprise any composition suitable for delivering a protein (e.g., any of the proteins described herein). The delivery vehicle can be any of the lipid structure delivery systems described herein. For example, a delivery vehicle can be a lipid-based structure including, but not limited to, a lipid-based nanoparticle, a liposome, a micelle, an exosome, a vesicle, an extracellular vesicle, a cell, or a tissue. The delivery vehicle can be any of the nanoparticles described herein, such as nanoparticles comprising lipids (as previously described), inorganic nanomaterials, and other polymeric materials.

The delivery vehicle can be capable of delivering the cargo to a cell, such as delivering any of the proteins described herein to a cell. The delivery vehicle can be capable of delivering the cargo to a cell, such as delivering any of the proteins described herein to a cell. The delivery vehicle can be configured to target a specific cell, such as configured with a re-directing antibody to target a specific cell. The delivery vehicle can be capable of delivering the cargo to a cell in vivo.

The delivery vehicle can be capable of delivering the cargo to a tissue or tissue environment (e.g., a tumor microenvironment), such as delivering any of the proteins described herein to a tissue or tissue environment in vivo. Delivering a cargo can include secreting the cargo, such as secreting any of the proteins described herein. Accordingly, the delivery vehicle can be capable of secreting the cargo, such as secreting any of the proteins described herein. The delivery vehicle can be capable of secreting the cargo to a tissue or tissue environment (e.g., a tumor microenvironment), such as secreting any of the proteins described herein into a tissue or tissue environment. The delivery vehicle can be configured to target a specific tissue or tissue environment (e.g., a tumor microenvironment), such as configured with a re-directing antibody to target a specific tissue or tissue environment.

Methods of Treatment

Further provided herein are methods that include delivering, or administering, to a subject (e.g., a human subject) engineered cells as provided herein to produce in vivo at least two proteins of interest, e.g., a CAR, and a membrane-cleavable chimeric protein having the formula S - C - MT or MT - C - S described herein, produced by the engineered cells.

Further provided herein are methods that include delivering, or administering, to a subject (e.g., a human subject) any of the delivery vehicles described herein, such as any of the delivery vehicles described herein comprising any of the proteins of interest described herein, e.g., a CAR and a membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein.

In some embodiments, the engineered cells or delivery vehicles are administered via intravenous, intraperitoneal, intratracheal, subcutaneous, intratumoral, oral, anal, intranasal (e.g., packed in a delivery particle), or arterial (e.g., internal carotid artery) routes. Thus, the engineered cells or delivery vehicles may be administered systemically or locally (e.g., to a TME or via intratumoral administration). An engineered cell can be isolated from a subject, such as a subject known or suspected to have cancer. An engineered cell can be allogenic with reference to the subject being administered a treatment. Allogenic modified cells can be HLA- matched to the subject being administered a treatment. Delivery vehicles can be any of the lipid structure delivery systems described herein. Delivery vehicles can be any of the nanoparticles described herein.

Engineered cells or delivery vehicles can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.. For example, engineered cells or delivery vehicles can be administered in combination with a checkpoint inhibitor therapy. Exemplary checkpoint inhibitors include, but are not limited to, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, anti-TIM-3 antibodies, anti-TIGIT antibodies, anti- VISTA antibodies, anti-KIR antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-HVEM antibodies, anti- BTLA antibodies, anti-GAL9 antibodies, anti-A2AR antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies, anti-TNFa antibodies, anti-TREMl antibodies, and anti- TREM2 antibodies. Illustrative immune checkpoint inhibitors include pembrolizumab (anti-PD- 1; MK-3475/Keytruda® - Merck), nivolumamb (anti-PD-1; Opdivo® - BMS), pidilizumab (anti-PD-1 antibody; CT-011 - Teva/CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti- PD-Ll; Bavencio® - Pfizer), durvalumab (anti-PD-Ll; MEDI4736/Imfinzi® - Medimmune/AstraZeneca), atezolizumab (anti-PD-Ll; Tecentriq® - Roche/Genentech), BMS- 936559 (anti-PD-Ll - BMS), tremelimumab (anti-CTLA-4; Medimmune/AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy ® - BMS), lirilumab (anti-KIR; BMS), monalizumab (anti- NKG2A; Innate Pharma/AstraZeneca). In other examples, engineered cells or delivery vehicles can be administered in combination with TGFbeta inhibitors, VEGF inhibitors, or HPGE2. In another example, engineered cells or delivery vehicles can be administered in combination with an anti-CD40 antibody. Some methods comprise selecting a subject (or patient population) having a tumor (or cancer) and treating that subject with engineered cells or delivery vehicles that modulate tumor- mediated immunosuppressive mechanisms.

The engineered cells or delivery vehicles of the present disclosure may be used, in some instances, to treat cancer, such as ovarian cancer. Other cancers are described herein. For example, the engineered cells may be used to treat bladder tumors, brain tumors, breast tumors, cervical tumors, colorectal tumors, esophageal tumors, gliomas, kidney tumors, liver tumors, lung tumors, melanomas, ovarian tumors, pancreatic tumors, prostate tumors, skin tumors, thyroid tumors, and/or uterine tumors. The engineered cells or delivery vehicles of the present disclosure can be used to treat cancers with tumors located in the peritoneal space of a subject.

The methods provided herein also include delivering a preparation of engineered cells or delivery vehicles. A preparation, in some embodiments, is a substantially pure preparation, containing, for example, less than 5% (e.g., less than 4%, 3%, 2%, or 1%) of cells other than engineered cells. A preparation may comprise IxlO⁵ cells/kg to IxlO⁷ cells/kg cells. Preparation of engineered cells or delivery vehicles can include pharmaceutical compositions having one or more pharmaceutically acceptable carriers. For example, preparations of engineered cells or delivery vehicles can include any of the engineered viruses, such as an engineered AAV virus, or any of the engineered viral vectors, such as AAV vector, described herein.

In vivo Expression

The methods provided herein also include delivering a composition in vivo capable of producing the engineered cells described herein, e.g., capable of delivering any of the engineered nucleic acids described herein to a cell in vivo. Such compositions include any of the viral-mediated delivery platforms, any of the lipid structure delivery systems, any of the nanoparticle delivery systems, any of the genomic editing systems, or any of the other engineering delivery systems described herein capable of engineering a cell in vivo.

The methods provided herein also include delivering a composition in vivo capable of producing proteins of interest described herein, e.g., a CAR, and membrane-cleavable chimeric proteins having the formula S - C - MT or MT - C - S described herein. The methods provided herein also include delivering a composition in vivo capable of producing two or more of the proteins of interest described herein. Compositions capable of in vivo production of proteins of interest include, but are not limited to, any of the engineered nucleic acids described herein. Compositions capable of in vivo production proteins of interest can be a naked mRNA or a naked plasmid. Enumerated Embodiments:

Embodiment 1 A method of stimulating a cell-mediated immune response to a tumor, reducing tumor volume, or providing an anti-tumor immunity in a human subject in need thereof, the method comprising a. administering to the subject a first dose of engineered NK cells; b. about seven days following (a), administering to the subject a second dose of engineered NK cells; and c. about seven days following (b), administering to the subject a third dose of engineered NK cells, wherein the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 500 million engineered NK cells, at least about 1 billion engineered NK cells, at least about 1.5 billion engineered NK cells, or at least about 2 billion engineered NK cells, wherein the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells comprise an engineered nucleic acid comprising: a first promoter operably linked to a first exogenous polynucleotide sequence and a second exogenous polynucleotide sequence, wherein the first exogenous polynucleotide sequence encodes a chimeric antigen receptor (CAR) that binds to GPC3, and wherein the second exogenous polynucleotide sequence encodes a membrane- cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:

S - C - MT or MT - C - S, wherein S comprises a secretable effector molecule that is or comprises IL 15, C comprises a protease cleavage site, and MT comprises a cell membrane tethering domain, and wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide.

Embodiment 2 The method of embodiment Embodiment 1, wherein the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise engineered about 500 million engineered NK cells. Embodiment 3 The method of embodiment Embodiment 1, wherein the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 1 billion engineered NK cells.

Embodiment 4 The method of embodiment Embodiment 1, wherein the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 1.5 billion engineered NK cells.

Embodiment 5 The method of embodiment 1, wherein the first dose, wherein the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 2 billion engineered NK cells.

Embodiment 6 The method of any one of the preceding embodiments, wherein steps (a), (b), and (c) are comprised in a first dosing cycle, and wherein the method further comprises repeating the dosing cycle for a total of 2, 3, 4, 5, or 6 dosing cycles.

Embodiment 7 The method of embodiment Embodiment 5, wherein each dosing cycle has a duration of 28 days, with (a) occurring on day 0, (b) occurring on day 7, and (c) occurring on day 14 of the 28 day duration.

Embodiment 8 The method of any one of the preceding embodiments, wherein the method further comprises administering to the subject an anti-histamine and an antipyretic agent 30-60 minutes prior to the 1^st dose, the second dose, and/or the third dose, and then every 8 hours for 24 hours.

Embodiment 9 The method of any one of the preceding embodiments, wherein the subject has, prior to (a), been administered one or more lymphodepletion agents.

Embodiment 10 The method of embodiment Embodiment 8, wherein the one or more lymphodepletion agents comprises fludarabine and cyclophosphamide.

Embodiment 11 The method of embodiment 10, wherein the subject has been administered, in the five to three days prior to (a), a. fludarabine at about 25-30 mg/m2/day over 30 minutes daily, and b. cyclophosphamide at about 250-500 mg/m2/day over 30-60 minutes daily.

Embodiment 12 The method of embodiment 10, wherein the subject has been administered, in the five to three days prior to (a), a. fludarabine at about 30 mg/m²/day over 30 minutes daily, and b. cyclophosphamide at about 500 mg/m²/day over 30-60 minutes daily.

Embodiment 13 The method of embodiment 10, wherein the method comprises, in the three to five days prior to (a), administering a. fludarabine at about 30 mg/m²/day over 30 minutes daily, and b. cyclophosphamide at about 500 mg/m²/day over 30-60 minutes daily.

Embodiment 14 The method of embodiment 10, wherein the method comprises administering oral and IV hydration to the subject prior to the cyclophosphamide administration.

Embodiment 15 The method of any one of the preceding embodiments, wherein the CAR comprises an antigen-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of KNAMN, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY , and wherein the VL comprises: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of WASSRES, and a light chain complementarity determining region 3 (CDR- L3) having the amino acid sequence of QQYYNYPLT.

Embodiment 16 The method of embodiment 15, wherein the VH region comprises an amino acid sequence with 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% identity to the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIR NKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFA YWGQGTLVTVSA.

Embodiment 17 The method of Embodiment 15, wherein the VH region comprises the amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIR NKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFA YWGQGTLVTVSA, optionally wherein the VH region comprises the amino acid sequence MEVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRI RNKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSF AYWGQGTLVTVSA. Embodiment 18 The method of any one of embodiments Embodiment 14-Embodiment 16, wherein the VL region comprises an amino acid sequence with 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% identity to the amino acid sequence of: DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIY WASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTK EEIK.

Embodiment 19 The method of embodiment Embodiment 17, wherein the VE region comprises the amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIY WASSRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEIK, optionally wherein the VL region comprises the amino acid sequence DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIY WASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTK LEIKS.

Embodiment 20 The method of any one of embodiments Embodiment 14-Embodiment 18, wherein the antigen-binding domain comprises a single chain variable fragment (scFv).

Embodiment 21 The method of any one of embodiments Embodiment 14-Embodiment 19, wherein the VH region and the VL region are separated by a peptide linker (L).

Embodiment 22 The method of embodiment Embodiment 20, wherein the scFv comprises the structure VH-L-VL or VL-L-VH.

Embodiment 23 The method of embodiment Embodiment 20, wherein the peptide linker (L) comprises a glycine- serine (GS) linker.

Embodiment 24 The method of embodiment Embodiment 22, wherein the GS linker comprises the amino acid sequence of (GGGGS)3.

Embodiment 25 The method of any one of the preceding embodiments, wherein the CAR comprises a hinge domain, optionally wherein the hinge domain is derived from CD8, optionally wherein the hinge domain comprises the sequence GALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACD.

Embodiment 26 The method of any one of the preceding embodiments, wherein the CAR comprises a transmembrane domain.

Embodiment 27 The method of embodiment Embodiment 25, wherein the transmembrane domain is derived from CD28 or CD8. Embodiment 28 The method of embodiment Embodiment 26, wherein the transmembrane domain is derived from CD8.

Embodiment 29 The method of embodiment Embodiment 27, wherein the transmembrane domain comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYCNHR.

Embodiment 30 The method of any one of the preceding embodiments, wherein the CAR comprises one or more intracellular signaling domains.

Embodiment 31 The method of embodiment Embodiment 29, wherein at least one of the one or more intracellular signaling domains is derived from CD28.

Embodiment 32 The method of embodiment Embodiment 30, wherein the intracellular signaling domain derived from CD28 comprises the sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.

Embodiment 33 The method of embodiment Embodiment 29, wherein the CAR comprises an intracellular signaling domain derived from CD28 and an intracellular signaling domain derived from CD3zeta.

Embodiment 34 The method of embodiment Embodiment 32, wherein the ICD derived from CD3zeta comprises the amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR.

Embodiment 35 The method of any one of the preceding embodiments, wherein the CAR further comprises a signal sequence.

Embodiment 36 The method of embodiment Embodiment 34, wherein the signal sequence comprised in the CAR is derived from GM-CSF-Ra, optionally wherein the signal sequence comprises the amino acid sequence MLLLVTSLLLCELPHPAFLLIP, optionally wherein the signal sequence operably linked to the CAR comprises the amino acid sequence MLLLVTSLLLCELPHPAFLLIPH.

Embodiment 37 The method of any one of the preceding embodiments, wherein the CAR comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence: MLLLVTSLLLCELPHPAFLLIPHMEVQLVESGGGLVQPGGSLRLSCAASGFTFNK NAMNWVRQAPGKGLEWVGRIRNKTNNYATYYADSVKARFTISRDDSKNSLYL QMNSLKTEDTAVYYCVAGNSFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIV MTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWA SSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEI KSGALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRRSKRSRLLHSDYMNM TPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLG RREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

Embodiment 38 The method of any one of the preceding embodiments, wherein the IL 15 comprises an amino acid sequence at least 80% identical to NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS.

Embodiment 39 The method of embodiment Embodiment 37, wherein the IL 15 comprises an amino acid sequence at least 85% identical to NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS.

Embodiment 40 The method of embodiment Embodiment 39, wherein the IL 15 comprises an amino acid sequence at least 90% identical to NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS.

Embodiment 41 The method of embodiment Embodiment 39, wherein the IL 15 comprises an amino acid sequence at least 95% identical to NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS.

Embodiment 42 The method of embodiment Embodiment 40, wherein the IL 15 comprises an amino acid sequence at least 99% identical to NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS.

Embodiment 43 The method of embodiment Embodiment 41, wherein the IL 15 comprises the amino acid sequence NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESG DASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS. Embodiment 44 The method of any one of the preceding embodiments, wherein the secretable effector molecule further comprises a signal sequence.

Embodiment 45 The method of embodiment Embodiment 43, wherein the signal sequence is derived from IgE.

Embodiment 46 The method of embodiment Embodiment 44, wherein the signal sequence comprises the amino acid sequence MDWTWILFLVAAATRVHS

Embodiment 47 The method of any one of the preceding embodiments, wherein the protease cleavage site comprises the amino acid sequence of VTPEPIFSLI.

Embodiment 48 The method of any one of the preceding embodiments, wherein the protease cleavage site is cleavable by a protease, optionally wherein the protease cleavage site is cleavable by an ADAM 17 protease.

Embodiment 49 The method of embodiment Embodiment 47, wherein the protease is endogenously expressed by at least a portion of the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells.

Embodiment 50 The method of any one of the preceding embodiments, wherein C further comprises one or more glycine-serine linker motifs, optionally wherein the glycineserine linker motif comprises (GGGS) or (GGGGS).

Embodiment 51 The method of embodiment Embodiment 49, wherein C comprises the amino acid sequence SGGGGSGGGGSGVTPEPIFSLIGGGSGGGGSGGGSLQ.

Embodiment 52 The method of any one of the preceding embodiments, wherein the cell membrane tethering domain is derived from B7-1.

Embodiment 53 The method of embodiment Embodiment 51, wherein the cell membrane tethering domain comprises the amino acid sequence LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV.

Embodiment 54 The method of any one of the preceding embodiments, wherein the first exogenous polynucleotide sequence and the second exogenous polynucleotide sequence are separated by a linker polynucleotide sequence encoding an E2A/T2A ribosome skipping element.

Embodiment 55 The method of embodiment Embodiment 53, wherein the E2A/T2a ribosome skipping element comprises the amino acid sequence GSGQCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP.

Embodiment 56 The method of any one of the preceding embodiments, wherein the tumor is a GPC3 expressing tumor.

Embodiment 57 The method of embodiment Embodiment 55, wherein the GPC3 expressing tumor is selected from the group consisting of: hepatocellular carcinoma (HCC), lung squamous cell cancer, esophageal squamous cell cancer, pancreatic cancer, papillary thyroid cancer, lung large cell cancer, AFP producing gastric cancer, follicular thyroid cancer, medullary thyroid cancer, ovarian clear cell carcinoma, melanoma, hepatoblastoma, nephroblastoma (Wilms tumor), hepatoblastoma, and yolk sac tumor.

Embodiment 58 The method of embodiment Embodiment 56, wherein the tumor is HCC, optionally wherein the tumor is unresectable, recurrent, and/or metastatic HCC.

Embodiment 59 The method of embodiment Embodiment 57, wherein over 50% of the subject’s liver is not occupied by the HCC tumor.

Embodiment 60 The method of any one of the preceding embodiments, wherein the subject had received at least one prior treatment and have been exposed to a checkpoint inhibitor and a tyrosine kinase inhibitor.

Embodiment 61 The method of any one of the preceding embodiments, wherein the subject had received a prior systemic treatment for the tumor.

Embodiment 62 The method of embodiment Embodiment 60, wherein the prior systemic treatment comprises a chemotherapy per respective NCCN guidelines for the indication, an immune checkpoint inhibitor approved for commercial use for the indication, and/or an agent that targets a tumor-associated mutation.

Embodiment 63 The method of any one of the preceding embodiments, wherein the subject does not have a history of organ transplantation and is not on a waiting list for organ transplantation, including liver transplantation.

Embodiment 64 The method of any one of the preceding embodiments, wherein tumor thrombus is not present in the portal vein, mesenteric vein, or inferior vena of the subject based on imaging.

Embodiment 65 The method of any one of the preceding embodiments, wherein the subject is not or has not been diagnosed with brain or leptomeningeal metastases.

Embodiment 66 The method of any one of the preceding embodiments, wherein the subject has not been previously administered a prior adoptive cell therapy, a prior GPC3 targeted anti-cancer therapy.

Embodiment 67 The method of any one of the preceding embodiments, wherein the subject has not been previously administered an investigational therapy within 14 days prior to (a).

Embodiment 68 The method of any one of the preceding embodiments, wherein the subject has not been administered an anti-cancer chemotherapeutic or targeted small molecule drug within 14 days or 5 half-lives (whichever is shorter), or an anti-cancer biologic within 28 days prior to (a). Embodiment 69 The method of any one of the preceding embodiments, wherein the subject has recovered from toxicities related to prior treatment to < Gr 2.

Embodiment 70 The method of any one of the preceding embodiments, wherein the subject has not been chronically administered an immunosuppressive agent or corticosteroids at >10mg/day prednisone or equivalent, optionally wherein the subject has not been chronically administered the immunosuppressive agent or corticosteroids at > 10 mg/day within 14 days prio to (a).

Embodiment 71 The method of any one of the preceding embodiments, wherein the subject does not have a history of significant cardiac or pulmonary disease or dysfunction within 12 weeks of (a).

Embodiment 72 The method of any one of the preceding embodiments, wherein the subject does not have a history of infection selected from: (i) known active HIV infection, (ii) active or latent hepatitis B or C infection in cases wherein the tumor is not an HCC, and (iii) ongoing active infection requiring systemic anti-infectives within 7 days prior to the first dose, in cases wherein the tumor is not an HCC and the systemic anti-infectives within 7 days prior to the first dose is for use in treatment of hepatitis B or C infection.

Embodiment 73 The method of any one of the preceding embodiments, wherein the subject does not have a history of prior malignancy, unless the prior malignancy comprises adequately treated basal cell or squamous cell skin cancer, in-situ cervical cancer, prostate cancer with stable PSA, or other prior malignancy wherein the subject has been malignancy free for 2 years prior to selection for administration of the first dose, the second dose, and the third dose.

Embodiment 74 The method of any one of the preceding embodiments, wherein the subject is not or has not been chronically administered an immunosuppression agent.

Embodiment 75 A kit, comprising: a first dose of engineered NK cells, comprising at least about 500 million engineered NK cells, at least about 1 billion engineered NK cells, at least about 1.5 billion engineered NK cells, or at least about 2 billion engineered NK cells, wherein the first dose of engineered NK cells comprises an engineered nucleic acid comprising: a. a first promoter operably linked to a first exogenous polynucleotide sequence and a second exogenous polynucleotide sequence, b. wherein the first exogenous polynucleotide sequence encodes a chimeric antigen receptor (CAR) that binds to GPC3, and c. wherein the second exogenous polynucleotide sequence encodes a membrane- cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:

S - C - MT or MT - C - S wherein

S comprises a secretable effector molecule that is or comprises IL15,

C comprises a protease cleavage site, and

MT comprises a cell membrane tethering domain, and (a) wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide; and

(b) (b) instructions for use in performing the method of any one of the preceding embodiments.

EXAMPLES

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. For example, the experiments described and performed below demonstrate the general utility of engineering cells to secrete payloads (e.g., effector molecules) and delivering those cells to induce an immunogenic response against tumors.

Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

Example 1: Expression and Function of an anti-GPC3 CAR + IL15 Bidirectional Construct

Protein expression, cellular activation, and killing activity of cells transduced with anti- GPC3 CAR + IL 15 bidirectional constructs were assessed. A cartoon diagram of the bidirectional orientation of the constructs is shown in FIG. 1.

Materials and Methods

Primary, donor-derived NK cells were transduced (50,000 to 100,000 cells/transduction) in a non-TC treated retronectin coated plate with lentivirus (at a multiplicity of infection, MOI, of 40) or retrovirus (SinVec, approximately 400pl each) encoding constructs having a first expression cassette encoding an anti-GPC3 CAR and a second expression cassette encoding IL15, with the two expression cassettes in a head-to-head bidirectional orientation. Constructs varied in the intracellular domains of the CAR, having 4- IBB and CD3-zeta signaling domains (41BBz), CD28 and CD3-zeta signaling domains (CD28z), 0X40 and CD3-zeta signaling domains (OX40z) or a KIR3DS1 signaling domain (KIR3DS1), and transductions using either a lentivirus or a retrovirus system were compared for each construct. As a control, transductions were also performed with retroviruses and lentiviruses encoding each of the same CARs, but without the IL15 expression cassette (“CAR-only). After transduction, NK cells were rested in the same plate for 3 days before transfer to a 24- well non-adherent cell-optimized plate. NK cells were expanded to a total of 5 ml with a first cytokine spike-in on day 7 following transduction and a second cytokine spike-in on day 15 (each spike-in included 500 lU/ml IL12 for the CAR+IL15 transductions and the CAR-only transductions, and lOng/ml IL 15 for the CAR only constructs).

On days five and seven following transduction, CAR expression was assessed by flow cytometry for each construct. Day seven CAR expression from cells transduced with lentivirus encoding a bidirectional CAR + IL 15 bidirectional construct and cells transduced with a lentivirus encoding the CAR-only is shown in FIG. 2. Day seven CAR expression from cells transduced with retrovirus encoding a bidirectional CAR + IL 15 bidirectional construct and cells transduced with a retrovirus encoding the CAR-only is shown in FIG. 3. Day fifteen CAR expression from cells transduced with lentivirus encoding a bidirectional CAR + IL 15 bidirectional construct and cells transduced with a lentivirus encoding the CAR-only is shown in FIG. 4. Day fifteen CAR expression from cells transduced with retrovirus encoding a bidirectional CAR + IL 15 bidirectional construct and cells transduced with a retrovirus encoding the CAR-only is shown in FIG. 5.

On day seven following transduction, a payload assay was conducted to assess IL15 levels for each construct. 200,000 cells per well were plated in 200pl media (NK MACs complete media with IL2) in a 96-well plate. NK cells were incubated for 48 hours, and then IL15 levels were assessed by immunoassay. IL15 expression is shown in FIG. 6.

Co-culture killing assays were then performed. 25,000 target cells (a Huh7 mKate cell line or a HepG2 mKate cell line) per well were plated in a 96-well plate. Effector cells (the NK cells expressing each construct) were added to the plate at effector to target (E to T) cell ratios of 1:1 or 0.5:1, and the cells were cultured with NK MACs complete media without cytokines in a total volume of 200pl. Two to three days following co-culture, real-time, fluorescence-based assays to measure mKate levels were performed to assess target cell killing. Killing by lentivirus-transduced NK cells expressing each construct is shown in FIG. 7, and killing by retrovirus-transduced NK cells expressing each construct is shown in FIG. 8.

Results

CAR expression from NK cells transduced with each construct was assessed. As shown in FIG. 2, at day seven transduced NK cells had measurable CAR expression for each construct, with at least 10% of cells in each transduced population positive for CAR expression. As shown in FIG. 3, at day fifteen lentivirus-transduced NK cells had measurable CAR expression for each construct (top panel), with at least 20% of cells in each transduced population positive for CAR expression. Additionally, as shown in FIG. 3, retrovirus-transduced NK cells expressing the 28z CAR + IL 15 bidirectional construct had measurable CAR expression, with at least 42% of cells in the transduced population positive for CAR expression.

IL15 expression by NK cells transduced with each construct was also assessed. Assay of IL15 expression by non-transduced cells and Ox40z CAR-only cells was performed as a negative control. As shown in FIG. 6, retrovirus-transduced NK cells expressing bidirectional CAR + IL15 had statistically significant increase in IL15 production over reciprocal lentivirus- transduced NK cells. Killing by NK cells transduced with each construct was then assessed. As shown in FIG. 7, lentivirus-transduced NK cells expressing the CAR + IL 15 bidirectional construct had statistically significant increased killing over lentivirus-transduced NK cells expressing the CAR alone (without the IL15 expression cassette). As shown in FIG. 8, retrovirus-transduced NK cells expressing the CAR + IL 15 bidirectional construct had statistically significant increased killing over retrovirus-transduced NK cells expressing the CAR alone (without the IL 15 expression cassette).

Example 2: Expression and Function of anti-GPC3 CAR + IL15 Bidirectional Constructs

Protein expression, cellular activation, and killing activity of cells transduced with anti- GPC3 CAR + cleavable release IL 15 bidirectional constructs were assessed. The expression cassette encoding the cleavable release IL15 includes a chimeric polypeptide including the IL15 and a transmembrane domain. Between the IL15 and the transmembrane domain is a protease cleavage domain that is cleavable by a protease endogenous to NK cells. A cartoon diagram of the bidirectional construct encoding a cleavable release IL 15 is shown in FIG. 9.

Briefly, primary, donor-derived NK cells were transduced with viral vectors encoding constructs having a first expression cassette encoding an anti-GPC3 CAR and a second expression cassette encoding a cleavable release IL15 expression cassette, with the two expression cassettes in a head-to-head bidirectional orientation.

Culture Supernatant'. Spinoculation of NK cells was performed (day 0). A partial culture media exchange was performed on days 1, 2, and 6. Cell culture supernatant was harvested on day 8.

Flow cytometry: On day 10 following transduction, CAR and mbIL15 expression was assessed by flow cytometry for each construct. NK cells were stained with an IL-15 primary antibody and PE-secondary, and rhGPC3-FITC and Sytox blue (viability stain). Cells were run on Cytoflex and analyzed using Flowjo for CAR/mbIE15 expression.

Payload assay: On day 7 or 8 following transduction, a payload assay was conducted to assess IE15 levels for each construct. 200,000 cells per well were plated in 200 pl media (NK MACs complete media with IE2 only) in a 96-well plate, run in duplicates. Cells were incubated for 48 hours, and then cleaved IE15 levels were assessed by Euminex immunoassay.

Serial killing assay: Co-culture killing assays were performed. About 25,000 target cells (a Huh7 mKate cell line or a HepG2 mKate cell line) per well were plated in a 96-well plate. Effector cells (the NK cells expressing each construct) were added to the plate at effector to target (E to T) cell ratios of 1:1 in triplicates, and the cells were cultured with NK MAC complete media (no cytokines) in a total volume of 200 pl. Real-time, fluorescence-based assays were used to measure mKate to assess target cell killing in a serial-killing assay performed at 37° C; initial killing was at day 9 post-transduction, serial one was at day 11 posttransduction, and serial 2 was at day 14 post transduction.

Over 150 IL15 cleavable release (crIL15) constructs were designed, and 33 constructs were selected for experimental testing, (see Table 7A). Each construct was tested in two viral backbones (e.g., SB06250 and SB06256, as shown in Table 7A). A summary of expression and killing activity of cells expressing a subset of bicistronic constructs is shown in Table 7B. Full- length sequences of a subset of constructs are shown in Table 7C. A summary of bicistronic constructs tested and their functional activities is provided in FIG. 10.

Table 7A.

Table 7B. a Normalized to Target cells alone

* crIL-15 control did not function as expected

* crIL-15 control did not killed as expected

Table 7C.

NK cells comprising CARs comprising 0X40 transmembrane (TM) and co-stimulatory (co-stim) domains, SB06251, SB06257, and SB06254, were assessed for expression of constructs as described above. Results as determined by flow cytometry are shown in FIG. 11A and FIG. 11B. Secreted IL- 15 was measured as described above; results are summarized in FIG. 12A and FIG. 12B. To assess killing of the target cell population, cell growth was determined as described above (FIG. 13A and FIG. 13B).

Serial killing by the NK cells comprising SB06257 was also assessed. Target cells were added at Days 0, 2, and 5, and Huh7 target cell count was calculated using an Incucyte. Results are shown in FIG. 14.

NK cells comprising CARs comprising CD28 co-stimulatory (co-stim) domains, SB06252, SB06258, and SB06255, were assessed for expression of constructs as described above. Results as determined by flow cytometry FACS are shown in FIG. 15A and FIG. 15B. Secreted IL- 15 was measured as described above; results are summarized in FIG. 16A and FIG. 16B. To assess killing of the target cell population, cell growth was determined as described above (FIG. 17A and FIG. 17B).

Serial killing by the NK cells comprising SB06252 and SB06258 was also assessed. Target cells were added at Days 0, 2, and 5, and Huh7 target cell count was calculated using an Incucyte. Results are shown in FIG. 18.

Screening for bicistronic constructs

0.5e6 NK donor 7B cells were expanded in the presence of fresh irradiated mbIL21/IL15 K562 feeder cells on retronectin coated non-TC 24-well plates. Spinoculation was performed at 800g at 32°C for 2 hr. For viral transduction, 300 pl of virus added, for a total transduction volume of 500 pl.

Cells were cultured in the same plate for the entire expansion period, in 2 ml final volume. Three partial media exchanges were performed as described above before assessing expression and using the cells in functional assays. Results of expression and cytotoxicity against target cells are shown in Table 8. As shown, SB06261, SB6294, and SB6298 showed good CAR and IL- 15 expression levels as determined by flow and good cytotoxicity in serial killing assay (n=2). Flow cytometry expression data is shown in FIG. 19A and FIG. 19B, IL- 15 levels are shown in FIG. 20A and FIG. 20B, and cell growth of the target cell population (as a measure of cell killing by the NK cells) is shown in FIG. 21 A and FIG. 21B.

Due to its high CAR and IL- 15 expression and performance in functional assays, SB06294, a retroviral vector with crIL15 2A 0X40 CAR design, was selected for further study.

Table 8.

Analysis of TACE-OPT constructs

Bicistronic TACE-OPT constructs comprising a TACE10 cleavage site, were analyzed for CAR and IL- 15 expression, CNA assay, and payload assay for secreted cytokines, as described above. A TACE10 cleavage site was modified to increase cleavage kinetics, resulting in “TACE-OPT,” which results in higher cytokine secretion levels as compared to the parent TACE10. Tricistronic constructs were analyzed for CAR and IL- 15 expression, and IL- 12 induction.

Briefly, 0.5e6 NK donor 7B cells were expanded in the presence of fresh irradiated mbIL21/IL15 K562 feeder cells on retronectin coated non-TC 24-well plates. Spinoculation was performed at 800g at 32°C for 2 hr. For viral transduction, 300 pl of virus was added, for a total transduction volume of 500 pl.

Bicistronic constructs SB6691 (comprising 41BB co- stimulatory domain), SB6692 (comprising 0X40 co- stimulatory domain), and SB6693 (comprising CD28 co-stimulatory domain) were assessed by flow cytometry for expression of CAR and IL- 15 (FIG. 22A). Copy number of each construct per cell is shown in Table 9. IL-15 secretion was quantified as described above at 48 hours and 24 weeks post-transduction (FIG. 22B). While the TACE-OPT constructs tested have similar expression levels and cytokine secretion, SB06692 (comprising an 0X40 co-stimulatory domain) has the highest CAR expression.

Table 9.

SB06258, SB06257, SB06294 and SB06692 demonstrated high CAR expression, high crIL-15 expression (both membrane-bound and secreted), and high serial killing function in vitro. Example 3: Screening of GPC3 CAR / IL15 Expression Constructs

Assessment of the expression and function of the GPC3 CAR/IL15 expression constructs in NK cells was performed. 2e6 NK cells were plated into a 6-well non-TC treated, retronectin coated plate. A single viral transduction via spinoculation (MOI = 15) was performed on plated NK cells. The NK cells were transduced using lentivirus or retrovirus containing the expression construct. Expression of the CAR and membrane IL15 were assessed as seen in FIG. 23A. NK cells transduced with constructs SB06257, SB06258, SB06294, and SB06692 exhibited expression of greater than 65% of cells in the gated population. In addition, FIG. 23A shows the measured copy numbers of YP7 and IL15 of each transduced NK cell population.

In addition to CAR expression being assessed, secreted IL- 15 was also measured using the same expression constructs. To measure the levels of secreted IL- 15, 200,000 transduced NK cells were suspended in 200 f L of MACS media in the presence of IL2. Secreted IL- 15 was measured 48 hours after transduction. The concentrations of secreted IL- 15 were measured for each construct and the results are shown in FIG. 23B.

Serial killing by NK cells transduced with the constructs was also assessed. Target cells were added at Days 0, 2, and 5, and target cell killing was measured over the course of the study. Results for serial NK cell killing of HepG2 target cells are shown in FIG. 23C and FIG. 24A. FIG. 24B shows results of serial NK cell killing of HuH-7 target cells.

Table 14 shows the exemplary constructs and their components used in this study.

Table 14

Example 4: Measuring GPC3 CAR / IL15 Expression and Function in Expanded NK cells

In this study, the expression and function of GPC3 CAR/IL15 were measured for NK cells that were expanded using the G-Rex (Gas rapid expansion) system.

7-day-old donor-derived 7B NK cells (mbIL21/IL15 K562 feeders) were transduced and expanded in two different G-Rex experimental methods. Experiment 1 transduced 7-day donor 7B NK cells (mbIL21/IL15 K562 feeders) in G-Rex 6M culture containers for 11 days and harvested 11 days after transduction. Experiment 2 transduced 7-day donor 7B NK cells (mbIL21/IL15 K562 feeders) in G-Rex IL culture containers for 7 days and harvested 10 days after transduction. FIG. 25A demonstrated the effects of the different expansion conditions have on the expression of different proteins of interest in the engineered NK cells. FIG. 25B shows the serial killing assay measurements from the NK Cells derived from the different experiments. Table 15 shows a summary of the study performed in Example 6. The top number corresponds to results obtained from NK cells expanded using the method of Experiment 1. The bottom number corresponds to results obtained from NK cells expended using the method of Experiment 2.

Table 15

Example 5: Assessment of GPC3 CAR / IL15 Bicistronic Constructs in a Xenograft Tumor Model

The in vivo function of selected engineered NK cells was assessed using a HepG2 xenotransplantation tumor model. Two studies were conducted: a double NK dose and a triple NK dose.

Double NK Dose In vivo Xenograft Tumor Model

The tumor was implanted in NSG mice at day 0. Mice were randomized at day 9. NK cells were injected twice over the course of the study on days 10 and 17. Table 16 summarizes the study set-up.

Table 16: Summary of double NK dosing in vivo xenograft tumor model

For this survival study, Jackson Labs NSG mice were also injected with 50,000 IU rhIL2 per mouse twice per week. Bioluminescence imaging (BLI), body weight, and overall health measurements were conducted twice a week. Upon euthanizing mice, tumor were collected, weighed, and formalin fixed paraffin embedded (FFPE) for histology. IP fluid and cells were collected from the IP space and the % NK cells were assessed by flow cytometry. FIG. 26 summarizes the results the fold change in normalized mean BLI measurement in the HepG2 xenotransplantation tumor model. SB06258 showed the lowest normalized mean BLI compared to other treatment groups and was found to be statistically significant compared to the no virus (NV) group. FIG. 27A shows a survival curve of animals and FIG. 27B shows a summary of the median survival of each of the treatment groups. Each of the different CAR constructs tested were found to be statistically significant compared to un-engineered NK cells. FIG. 27C shows an survival curve with extended time points and median survival from the extended time points, of animals dosed with PBS, unengineered NK cells, and NK cells engineered with SB06258.

FIG. 28 shows a time course of the mice treated with different CAR-NK cells as measured and observed through bioluminescence imaging (BLI). The animals shown here were imaged 3 days, 10 days, 34 days, 48 days, and 69 days after treatment. In FIG. 29, BLI measurements were normalized to day 10 (first dose).

Triple Dosing - In Vivo HepG2 Xenograft Tumor Model

The in vivo function of selected engineered NK cells was assessed using a HepG2 xenotransplantation tumor model. The tumor was implanted in NSG mice at day 0 in another in vivo experiments. Mice were randomized at day 9 and day 20. 30e6 NK cells were injected (IP) three times over the course of the study on days 10, 15, and 22. Table 17 summarizes the study set-up. On day 21, half of the mice were euthanized. The other half were euthanized on day 50 of the study. Upon euthanizing mice, tumor were collected, weighed, and formalin fixed paraffin embedded (FFPE) for histology.

Table 17: Study Design of HepG2 xenograft model

For this survival study, Jackson Labs NSG mice were also injected with 50,000 IU rhIL2 per mouse twice per week. Bioluminescence imaging (BLI), body weight, and overall health measurements were conducted twice a week. IP fluid and cells were collected from the IP space and the % NK cells were assessed by flow cytometry. FIG. 30A shows a representative BLI image at day 23 of the study. FIG. 30B summarizes the results the fold change in normalized mean BLI measurement in the HepG2 xenograft tumor model.

The fold change of BLI measurements were assessed at different stages of the experiments to assess the effect of a single or double dose of the engineered NK cells had an effect. FIG. 31A shows the fold change of BLI measurements on day 13, in which the mice had undergone one dose of the engineered NK cells. FIG. 31B shows the fold change of BLI measurements on day 20, in which the mice had undergone two doses of the engineered NK cells.

Comparison of the results from the two in vivo experiments are presented in FIG. 32A and FIG. 32B. In FIG. 32A, the different CAR constructs were tested in a xenograft model, plotting fold change of BLI over the course of the study. As seen in FIG. 32A and FIG. 32B, the two in vivo experiments exhibit differences in antitumor function of SB06257 and SB06258. GPC3 CAR- crIL-15 NK cell therapy shows statically significant in vivo anti-tumor efficacy compared to unengineered NK cells in an IP HCC (HepG2+luciferase) xenotransplantation model. All 3 groups treated with GPC3 CAR-crIL-15 engineered NK cells show significant increased survival over untreated (PBS) and unengineered NK cell-treated groups.

In vivo Xenograft model - Intratumoral Injection ofNK cells

Another experimental approach was used to demonstrate NK-mediated anti-tumor killing for an HepG2 (HCC) subcutaneous xenograft tumor model. In this survival study, mice were injected three times with 3e6 NK cells on days 20, 25, and 32. FIG. 33A demonstrates tumor growth in mice in the absence or presence of injected engineered NK cells. GPC3 CAR- crIL-15 NK cell therapy shows significant in vivo anti-tumor efficacy compared to unengineered NK cells injected intratumorally (IT) within a subcutaneous HCC (HepG2+luciferase) xenotransplantation model. NK cells transduced with SB05605 show significantly increased survival over untreated (PBS) and unengineered NK cell-treated groups. Table 18 provides the constructs used for intratumoral injection of NK cells. SB05009 and SB06205 contain IL15 and the GPC3 CAR that are separate, and their expression is driven by separate promoters (SV40 promoter = GPC3 CAR, hPGK promoter = IL15). In addition, these constructs are oriented such that the reading frames are oriented in opposing directions.

Table 18

Example 6: Selection of GPC3 CAR / IL15 clones

Selection of clones were performed by transducing NK cells that have stably integrated the expression construct. A lower MOI was used (MOI=3) was used for clonal selection of SB06258. A control transient transduction (MOI = 15) was also performed used in SB06258 and SB07273 (identical to SB06258 but contains a kanamycin resistance marker instead of an ampicillin resistance marker). 8 days after transduction, the cells were assessed. The copies per cell was lower in the PCB clones as compared to the transient transduction using SB06258 (FIG. 45 A). CAR expression was relatively constant across the different PCB clones (FIG. 45B), as well as the IL15+ population (FIG. 45C). Secreted IL15 of PCB clones was measured to be greater than 30 pg/mL (FIG. 45D). Flow cytometry was also used to assess the expression of the GPC3 CAR and IL 15 in the

PCM clones. As a control, SB07473 was used to transduced NK cells at an MOI=15. PCB clones were transduced at an MOI of 3.0. For all PCR clones, GPC3 CAR expression was greater than 20% (FIG. 46 A).

For select clones, SB05042 was also co-transduced to assess the expression of the GPC3 CAR, membrane bound IL15 and membrane bound IL12 9 days after transduction. Clone 3 (MOI=3.0) and clone 4 (MOI=3.0) was co-transduced with SB05042 (MOI = 0.05). During cotransduction, there was similar expression of the GPC3 CAR and membrane bound IL12 (FIG. 46B). Table 19 shows a summary of the expression levels of the PCB clones transduced with SB06258.

Table 19 Table 20

Table 21:

Example 7: assessment of antitumor function in the presence of full-length soluble GPC3

GPC3 shedding has been shown to neutralized GPC3-specific CAR-T cells and compete with binding to target cells. 5-10 ug/ml of soluble recombinant GPC3 is sufficient to inhibit GPC3 CAR-T cell mediated cytotoxicity (Sun L. et al, J Imm Therapy of Cancer, 2021). The effect of engineered NK cells modified with SB06258 vs. unengineered NK cells on Huh7 target cell cytotoxicity in the presence of various concentrations of soluble GPC3 was assessed using an LDH assay. As shown in FIG. 34, SB06258 maintained its anti-tumor function in the presence of soluble GPC3.

Example 8: Clinical Assessment of GPC3 CAR / IL15 NK Therapies

Appendix A is a document 16 pages in length (including title slip sheet) describing embodiments of the invention. Appendix A is hereby incorporated by reference, in its entirety, for all purposes.

It should be noted that the language used in Appendix A has been principally selected for readability and instructional purposes, and it may not have been selected to delineate or circumscribe the inventive subject matter. Accordingly, the disclosure of Appendix A is intended to be illustrative, but not limiting, of the scope of the invention.

Appendix A describes a clinical protocol for Phase 1, open-label study assessing efficacy and safety of off-the-shelf CAR NK cell therapies in subjects with GPC3-expressing solid tumors. Among the aspects shown in Appendix A, and described below, include (1) subject eligibility criteria; (2) study design; (3) study treatment/dosing regimens; and (4) endpoint/evaluation criteria.

Assessed are CAR NK cell therapies that include membrane-cleavable IL- 15 (also referred to as cleavable release IL- 15 [crIL-15]) and GPC3-targeting CARs, which are described herein. At least assessed is an NK therapy including NK cells engineered to express the following construct: (GM-CSF-Ra (SS) - aGPC3 hPY7 vH - (GGGGS)3 - aGPC3 hPY7 vL - CD8FA (Hinge) - CD8 (TM) - CD28 (ICD) - CD3z (ICD) - E2A T2A - IgE (SS) - IL-15 - TacelO (cleavage site) - B7-1 (TM)).

Subject inclusion criteria include:

■ Having advanced GPC3 expressing solid tumors, including: o Unresectable, recurrent, and/or metastatic hepatocellular carcinoma (HCC)

- Have received at least one prior treatment and have been exposed to a checkpoint inhibitor (CPI) and a tyrosine kinase inhibitor (TKI) o Other advanced GPC3 expressing solid tumors (E.g. squamous cell lung (SCC) and large cell lung (LCLC), ovarian clear cell, pancreatic, and thyroid (follicular, medullary, papillary) carcinomas)

- Have received at least 1 prior systemic therapy that includes:

• A regimen containing chemotherapy per respective NCCN guidelines for that indication (e.g., see www.nccn.org/guidelines/category_l)

• An immune checkpoint inhibitor if approved for commercial use for that indication, and

• A mutationally selected agent that is approved for commercial use for that indication in patients with tumors with driver mutations o Measurable disease per RECIST vl.l and at least one biopsy-accessible lesion o Confirmation of GPC3+ by immunochemistry (IHC) during pre-screening

■ ECOG 0-1 (e.g., see ecog-acrin.org/resources/ecog-performance-status/)

■ Child-Pugh < 6 and BCLC Stage A, B, or C (HCC subjects only)

■ Adequate organ function: o LVEF > 40% o Estimated creatinine clearance > 60 mL/min/1.73m² o Total bilirubin < 1.5 x ULN or < 3x ULN for subjects with liver tumors o AST and ALT < 2.5 x ULN or < 5 x ULN for subjects with liver tumors o Platelets > 50 x 10⁹/L (platelet transfusions acceptable) o ANC > 1.5 x 10⁹/L o Hgb > 8.0 g/dL o INR < 1.7 (HCC subjects only)

■ Permitted concomitant medications include: o Anti-infective prophylaxis (strongly recommended) o Growth factors, blood transfusion products o Management of AEs

- Steroids permitted at doses > 10 mg/d of prednisone or equivalent to manage AEs

- Tocilizumab or similar o Anti-epileptic prophylaxis depending on history o Hydration o Supportive therapies per flu/cy labels

Subject exclusion criteria include:

■ No history of organ transplantation or patients currently on the waiting list for organ transplantation, including but not limited to liver transplantation

■ Over 50% of the liver occupied by HCC tumor or the presence of tumor thrombus in the portal vein, mesenteric vein, or inferior vena based on imaging

■ Known brain or leptomeningeal metastases

■ No prior adoptive cell therapy including NK cell or CAR T therapy

■ No prior GPC3 targeted anti-cancer therapy

■ No investigational therapy within 14 days prior to first dose

■ No anti-cancer chemotherapeutic or targeted small molecule drug within 14 days or 5 half-lives (whichever is shorter) or use of anti-cancer biologies within 28 days prior to the first dose

■ Recovered from toxicities related to prior treatment to < Gr 2 and not on chronic immunosuppressives or corticosteroids >10mg/day prednisone or equivalent

■ History of significant cardiac or pulmonary disease or dysfunction within 12 weeks of dosing: o > class III NYHA congestive heart failure o Unstable angina pectoris or life-threatening cardiac arrhythmia o Persistent and prolonged QTcF >480msec or Long QT Syndrome

■ History of infection including: o Known active HIV infection o Active or latent hepatitis B or C infection (non-HCC subjects only) o Ongoing active infection requiring systemic anti-infectives within 7 days prior to the first dose (with the exception of hepatitis B or C for HCC subjects)

■ Prior malignancy except adequately treated basal cell or squamous cell skin cancer, in-situ cervical cancer, prostate cancer with stable PSA. Other prior malignancies acceptable if disease free for at least 2 year prior to study entry

■ Treatment with systemic corticosteroids >10 mg/day of prednisone or equivalent or other chronic immunosuppressive medications within 14 days prior to first dose, or anticipated requirement for systemic immunosuppressive medications during the trial

■ Active autoimmune disease requiring chronic immunosuppression ■ Non-cancer related clinically significant illness or major surgery within 4 weeks before the first dose. Subjects must have recovered from surgery without current complications

■ Prohibited concomitant medications include: o Other anti-cancer therapies o Other investigational treatments o Chronic use of steroids > 10 mg/day prednisone or equivalent and immunosuppressives except as needed acutely to treat CRS or other adverse events o COVID vaccines should be administered at least 2 weeks prior to or after Cycle 1 o Live vaccines administration per fludarabine/cyclophosphamide restrictions

Appendix A describes the study design and dosing regimens. NK cells are engineered as described herein to express GPC3 targeting CARs and membrane-cleavable IL- 15. The Phase 1 trial includes a dose finding modified “3+3” design.

Engineered NK therapy dose finding includes:

■ Starting Dosing Level/Cohort 1: IxlO⁹ CAR+ NK cells for days 0, 7, 14

■ Depending on adverse events (AEs) and dose-limiting toxicities (DLTs) of Cohort 1 per study design, either: o Dosing Level/Cohort -1: 5xl0⁸ CAR+ NK cells o Dosing Level/Cohort 2: 1.5xl0⁹ CAR+ NK cells o Dosing Level/Cohort 3: 2xl0⁹ CAR+ NK cells

■ Dosing above is based on subjects that are above 50 kg. For subjects less than 50 kg, the following doses are administered: o Dosing Level/Cohort 1: 3xl0⁷ CAR+ NK cells/kg body weight o Dosing Level/Cohort -1: 1.5xl0⁷ CAR+ NK cells/kg body weight o Dosing Level/Cohort 2: 4.5xl0⁷ CAR+ NK cells/kg body weight

Dosing regimens include:

■ Conditioning chemotherapy prior to each treatment cycle: o Fludarabine (Flu) 30 mg/m²/day over 30 minutes daily and cyclophosphamide (Cy) 500 mg/m²/day over 30-60 minutes daily on Days -5 to -3 o Should there be conditions such as infection during the screening period for the subject, the investigator may adjust the lymphodepletion dosage at their discretion. ■ Engineered NK cells are dosed in 3 weekly doses per 28-day treatment cycle, spaced one week apart (Dose on Days 0, 7, 14) o Medication with anti-histamines and anti-pyretics are administered 30-60’ predose and every 8 hours (Q8h) for 24 hours to limit and/or prevent infusion reactions o If it is assessed that the lymphocyte depletion before infusion is not sufficiently thorough, which may lead to a shorter immune rejection window and affect clinical benefits, the investigator may decide whether to advance the infusion of CAR+NK cells based on the actual situation, and the originally scheduled infusion times on D7 and D14 can be adjusted by up to ±3 days.

■ Adequate oral and IV hydration is administered prior to cyclophosphamide dosing to limit and/or prevent urinary tract toxicity

■ Dosing in cycles continues for up to 5 cycles based on safety criteria until complete response (CR)/optimal response: o Resolution of engineered NK therapy attributable G3 non-hematologic adverse events (AEs) o ANC > 500/ mm3 , PLT > 25,000/ mm3 (transfusions allowed) to receive additional conditioning chemotherapy plus engineered NK therapy o Hold of up to 28 days allowed for count recovery and for resolution of AEs o Absence of PD per RECIST 1.1 criteria, may be allowed one other cycle if meets criteria for iPD without clinical deterioration

Appendix A describes an assessment schedule. Safety, efficacy, immunogenicity, pharmacokinetics (PK), and Pharmacodynamics (PDn) are assessed. A recommended Phase 2 dose (RP2D) is determined based on the assessment, in particular determining an efficacious dose that limits and/or avoids toxicity.

Monitoring and assessment includes:

■ Post-Dose Safety Observation o Cycle 1: Patients are hospitalized during each infusion & for minimum of 24 hours thereafter o Cycles 2+: Can be administered outpatient with minimum of 8 hours post-dose observation, if Cycle 1 doses are well tolerated without the occurrence of Gr3 hemodynamic instability. Sponsor approval required. o Patients reside within a 2-3 hour drive of the clinic for at least a week following dosing for all cycles

■ Dose-limiting toxicities monitored include: o Events attributable to the dosing of the engineered NK cells that occur during Cycle 1 (28 days)

- Gr 4 hematological toxicity that does not recover to baseline or < Gr 2 by Day 28 (as expected with LD chemo)

- Non-hematological toxicity of Gr > 3, except:

• Asymptomatic lab abnormalities that resolve to baseline or < Gr 2 within 3 days

• The following Gr 3 events that resolve within 3 days:

• Nausea and vomiting controlled with antiemetic therapy

• Cytokine release syndrome (CRS) that is responsive to treatment

• Infusion reaction that is responsive to treatment and resolves within 1 day

- The inability to receive the second and/or third dose of Cycle 1 due to toxicity related to the dosing of the engineered NK cells

- Any medically important AE of any Grade as assessed by the SRC

■ Study stopping criteria include paused or stopping enrollment to allow for safety review when either (1) 2 non-hematologic Grade 4 toxicity in 2 subjects; or (2) 1 Grade 5 toxicity is observed

■ Efficacy o Assessed at Day 28 of each cycle (end of cycle 1, 3, and 5) o Assessed by methods such as:

- RECIST (e.g., see ctep.cancer.gov/protocoldevelopment/docs/recist_guideline.pdf)

- iRECIST (e.g., see Seymour et al. Lancet Oncol. 2017 Mar; 18(3): el43- el52.)

- mRECIST (e.g., see Llovet et al. J Hepatol. 2020 Feb;72(2):288-306.)

- Alpha Fetoprotein (AFP) assay (e.g., see Hu et al. Int J Biol Sci. 2022; 18(2): 536-551.) Interpretations

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Certain numerical values are presented herein as preceded by the term “about”. The term “about” is used herein to provide literal support for the exact value that it precedes, as well as a values within a range of normal tolerance in the art, for example within 2 standard deviations of the mean.. A value may be within a range of normal tolerance for a specifically recited number, if it provides the substantial equivalent of the specifically recited number. If the range of normal tolerance is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. About can be understood as within +/- 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

Claims

CLAIMS What is claimed is:

1. A method of stimulating a cell-mediated immune response to a tumor, reducing tumor volume, or providing an anti-tumor immunity in a human subject in need thereof, the method comprising a) administering to the subject a first dose of engineered NK cells; b) about seven days following (a), administering to the subject a second dose of engineered NK cells; and c) about seven days following (b), administering to the subject a third dose of engineered NK cells, wherein the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise at least about 500 million engineered NK cells, at least about 1 billion engineered NK cells, at least about 1.5 billion engineered NK cells, at least about 2 billion engineered NK cells, from 500 million to 2 billion engineered NK cells, from 500 million to 1.5 billion engineered NK cells, from 500 million to 1 billion engineered NK cells, from 1 billion to 2 billion engineered NK cells, from 1 billion to 1.5 billion engineered NK cells, from 1.5 billion to 2 billion engineered NK cells, wherein the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells comprise an engineered nucleic acid comprising: a first promoter operably linked to a first exogenous polynucleotide sequence and a second exogenous polynucleotide sequence, wherein the first exogenous polynucleotide sequence encodes a chimeric antigen receptor (CAR) that binds to GPC3, and wherein the second exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:

S - C - MT or MT - C - S wherein

2. The method of any one of the preceding claims, wherein the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 500 million engineered NK cells, 1 billion engineered NK cells, 1.5 billion engineered NK cells, or 2 billion engineered NK cells.

3. The method of any one of the preceding claims, wherein the second dose of engineered NK cells is administered seven days following (a) and the third dose of engineered NK cells is administered seven days following (b).

4. The method of any one of the preceding claims, wherein steps (a), (b), and (c) are comprised in a first dosing cycle, and wherein the method further comprises repeating the dosing cycle for a total of 2, 3, 4, 5, or 6 dosing cycles, optionally wherein each dosing cycle has a duration of 28 days, with (a) occurring on day 0, (b) occurring on day 7, and (c) occurring on day 14 of the 28 day duration, optionally wherein the method further comprises administering to the subject an anti-histamine and an anti-pyretic agent 30-60 minutes prior to the 1^st dose, the second dose, and/or the third dose, and then every 8 hours for 24 hours.

5. The method of any one of the preceding claims, wherein the subject has, prior to (a), been administered one or more lymphodepletion agents, optionally wherein the one or more lymphodepletion agents comprises fludarabine and cyclophosphamide, optionally wherein the subject has been administered, in the five to three days prior to (a), i. fludarabine at about 25- 30 mg/m2/day over 30 minutes daily, and ii. cyclophosphamide at about 250 - 500 mg/m2/day over 30-60 minutes daily; optionally wherein the method comprises administering oral and IV hydration to the subject prior to the cyclophosphamide administration.

6. The method of any one of the preceding claims, wherein the CAR comprises an antigenbinding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein i. the VH comprises: a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of KNAMN, a heavy chain complementarity determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA, and a heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY , and wherein the VL comprises: a light chain complementarity determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA, a light chain complementarity determining region 2 (CDR-L2) having the amino acid sequence of WASSRES, and a light chain complementarity determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT; and/or ii. the VH region comprises an amino acid sequence with 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% identity to the amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPG KGLEWVGRIRNKTNNYATYYADSVKARFTISRDDSKNSLYLQM NSLKTEDTAVYYCVAGNSFAYWGQGTLVTVSA; and/or the VL region comprises an amino acid sequence with 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% identity to the amino acid sequence of: DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQ KPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDV AVYYCQQYYNYPLTFGQGTKLEIK; and/or iii. wherein the antigen-binding domain comprises a single chain variable fragment (scFv), optionally wherein the VH region and the VL region are separated by a peptide linker (L), optionally wherein the scFv comprises the structure VH-L-VL or VL-L-VH, optionally wherein the peptide linker (L) comprises a glycine-serine (GS) linker, optionally wherein the GS linker comprises the amino acid sequence of (GGGGS)3.

7. The method of any one of the preceding claims, wherein the CAR comprises i. a hinge domain, optionally wherein the hinge domain is derived from CD8, optionally wherein the hinge domain comprises the sequence GALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAA GGAVHTRGLDFACD; and/or ii. a transmembrane domain, optionally wherein the transmembrane domain is derived from CD28 or CD8, optionally wherein the transmembrane domain is derived from CD8 comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYCNHR; and/or iii. at least one or more intracellular signaling domains, optionally wherein at least one of the one or more intracellular signaling domains is derived from CD28, optionally wherein the intracellular signaling domain derived from CD28 comprises the sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRS, optionally wherein the at least one or more intracellular signaling domains further comprises an intracellular signaling domain derived from CD3zeta comprising the amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR; and/or iv. a signal sequence, wherein the signal sequence comprised in the CAR is derived from GM-CSF-Ra, optionally wherein the signal sequence comprises the amino acid sequence MLLLVTSLLLCELPHPAFLLIP, optionally wherein the signal sequence operably linked to the CAR comprises the amino acid sequence MLLLVTSLLLCELPHPAFLLIPH, optionally wherein the CAR comprises an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence:

MLLLVTSLLLCELPHPAFLLIPHMEVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMN WVRQAPGKGLEWVGRIRNKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTED TAVYYCVAGNSFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGE RATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTD FTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEIKSGALSNSIMYFSHFVPVFLPAKP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL LSLVITLYCNHRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

8. The method of any one of the preceding claims, wherein the membrane-cleavable chimeric protein comprises: i. an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to NWVNVISDEKKIEDEIQSMHIDATEYTESDVHPSCKVTAMKCFE LELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEE LEEKNIKEFLQSFVHIVQMFINTS, optionally wherein the IE15 further comprises a signal sequence, optionally wherein the signal sequence is derived from IgE, optionally wherein the signal sequence comprises the amino acid sequence MDWTWIEFEVAAATRVHS, and/or ii. wherein the protease cleavage site comprises the amino acid sequence of VTPEPIFSEI, optionally wherein the protease cleavage site is cleavable by a protease, optionally wherein the protease cleavage site is cleavable by an ADAM 17 protease, optionally wherein C further comprises one or more glycine- serine linker motifs, optionally wherein the glycine-serine linker motif comprises (GGGS) or (GGGGS), optionally wherein C comprises the amino acid sequence SGGGGSGGGGSGVTPEPIFSEIGGGSGGGGSGGGSEQ, optionally wherein the cell membrane tethering domain is derived from B7-1, optionally wherein the cell membrane tethering domain comprises the amino acid sequence EEPSWAITEISVNGIFVICCETYCFAPRCRERRRNERERRESVRPV, optionally wherein the protease is endogenously expressed by at least a portion of the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells.

9. The method of any one of the preceding claims, wherein the first exogenous polynucleotide sequence and the second exogenous polynucleotide sequence are separated by a linker polynucleotide sequence encoding an E2A/T2A ribosome skipping element, optionally wherein the E2A/T2a ribosome skipping element comprises the amino acid sequence GSGQCTNYAEEKEAGDVESNPGPGSGEGRGSEETCGDVEENPGP.

10. The method of any one of the preceding claims, wherein the tumor is a GPC3 expressing tumor, optionally wherein the GPC3 expressing tumor is selected from the group consisting of: hepatocellular carcinoma (HCC), lung squamous cell cancer, esophageal squamous cell cancer, pancreatic cancer, papillary thyroid cancer, lung large cell cancer, AFP producing gastric cancer, follicular thyroid cancer, medullary thyroid cancer, ovarian clear cell carcinoma, melanoma, hepatoblastoma, nephroblastoma (Wilms tumor), hepatoblastoma, and yolk sac tumor, optionally wherein the tumor is HCC, optionally wherein the tumor is unresectable, recurrent, and/or metastatic HCC, optionally wherein over 50% of the subject’s liver is not occupied by the HCC tumor.

11. The method of any one of the preceding claims, wherein the subject had received: i. at least one prior treatment and have been exposed to a checkpoint inhibitor and a tyrosine kinase inhibitor, and/or ii. a prior systemic treatment for the tumor, optionally wherein the prior systemic treatment comprises a chemotherapy per respective NCCN guidelines for the indication, an immune checkpoint inhibitor approved for commercial use for the indication, and/or an agent that targets a tumor-associated mutation.

12. The method of any one of the preceding claims, wherein the subject does not have a history of organ transplantation and is not on a waiting list for organ transplantation, including liver transplantation.

13. The method of any one of the preceding claims, wherein tumor thrombus is not present in the portal vein, mesenteric vein, or inferior vena of the subject based on imaging.

14. The method of any one of the preceding claims, wherein the subject is not or has not been diagnosed with brain or leptomeningeal metastases.

15. The method of any one of the preceding claims, wherein the subject has not been previously administered a prior adoptive cell therapy, a prior GPC3 targeted anti-cancer therapy

16. The method of any one of the preceding claims, wherein the subject has not been previously administered an investigational therapy within 14 days prior to (a).

17. The method of any one of the preceding claims, wherein the subject has not been administered an anti-cancer chemotherapeutic or targeted small molecule drug within 14 days or 5 half-lives (whichever is shorter), or an anti-cancer biologic within 28 days prior to (a).

18. The method of any one of the preceding claims, wherein the subject has recovered from toxicities related to prior treatment to < Gr 2.

19. The method of any one of the preceding claims, wherein the subject has not been chronically administered an immunosuppressive agent or corticosteroids at >10mg/day prednisone or equivalent, optionally wherein the subject has not been chronically administered the immunosuppressive agent or corticosteroids at > 10 mg/day within 14 days prior to (a).

20. The method of any one of the preceding claims, wherein the subject does not have a history of significant cardiac or pulmonary disease or dysfunction within 12 weeks of (a).

21. The method of any one of the preceding claims, wherein the subject does not have a history of infection selected from: (i) known active HIV infection, (ii) active or latent hepatitis B or C infection in cases wherein the tumor is not an HCC, and (iii) ongoing active infection requiring systemic anti-infectives within 7 days prior to the first dose, in cases wherein the tumor is not an HCC and the systemic anti-infectives within 7 days prior to the first dose is for use in treatment of hepatitis B or C infection.

22. The method of any one of the preceding claims, wherein the subject does not have a history of prior malignancy, unless the prior malignancy comprises adequately treated basal cell or squamous cell skin cancer, in-situ cervical cancer, prostate cancer with stable PSA, or other prior malignancy wherein the subject has been malignancy free for 2 years prior to selection for administration of the first dose, the second dose, and the third dose.

23. The method of any one of the preceding claims, wherein the subject is not or has not been chronically administered an immunosuppression agent.

24. A kit, comprising: a) A first dose of engineered NK cells, comprising at least about 500 million engineered NK cells, at least about 1 billion engineered NK cells, at least about 1.5 billion engineered NK cells, at least about 2 billion engineered NK cells, from 500 million to 2 billion engineered NK cells, from 500 million to 1.5 billion engineered NK cells, from 500 million to 1 billion engineered NK cells, from 1 billion to 2 billion engineered NK cells, from 1 billion to 1.5 billion engineered NK cells, or from 1.5 billion to 2 billion engineered NK cells, wherein the first dose of engineered NK cells comprises an engineered nucleic acid comprising: a first promoter operably linked to a first exogenous polynucleotide sequence and a second exogenous polynucleotide sequence, wherein the first exogenous polynucleotide sequence encodes a chimeric antigen receptor (CAR) that binds to GPC3, and wherein the second exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:

S - C - MT or MT - C - S wherein

MT comprises a cell membrane tethering domain, and wherein S - C - MT or MT - C - S is configured to be expressed as a single polypeptide; and (b) instructions for use in performing the method of any one of the preceding claims.

25. A method of stimulating a cell-mediated immune response to a tumor, reducing tumor volume, or providing an anti-tumor immunity in a human subject in need thereof, the method comprising

(a) administering to the subject a first dose of engineered NK cells;

(b) about seven days following (a), administering to the subject a second dose of engineered NK cells; and

(c) about seven days following (b), administering to the subject a third dose of engineered NK cells, wherein the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells each comprise 3xlO^A7 cells/kg body weight, 1.5xlO^A7 cells/kg body weight, or 4.5xlO^A7 cells/kg body weight, wherein the first dose of engineered NK cells, the second dose of engineered NK cells, and the third dose of engineered NK cells comprise an engineered nucleic acid comprising: a first promoter operably linked to a first exogenous polynucleotide sequence and a second exogenous polynucleotide sequence, wherein the first exogenous polynucleotide sequence encodes a chimeric antigen receptor (CAR) that binds to GPC3, and wherein the second exogenous polynucleotide sequence encodes a membrane-cleavable chimeric protein, oriented from N-terminal to C-terminal, having the formula:

S - C - MT or MT - C - S wherein

S comprises a secretable effector molecule that is or comprises IL15,

C comprises a protease cleavage site, and