Engineered immune signaling constructs

Abstract

Disclosed herein are engineered immune signaling constructs that can comprise, for example, an extracellular region, transmembrane region, and intracellular region. The extracellular region can comprise a common gamma chain-binding domain, such as a tethered cytokine or gamma chain-binding domain thereof (e.g., "tetherkines") or an antigen-binding fragment of an antibody that binds gamma chain (e.g., "gripperkines"). The intracellular region can comprise an intracellular signaling domain of a common gamma chain family cytokine receptor subunit. The constructs can be configured such that the construct binds to common gamma chain and elicits constitutive gamma chain family cytokine receptor signaling in cells that express the engineered immune signaling construct and common gamma chain, for example, facilitating induction IL2R (e.g., IL2Rbg), IL4R, IL7R, IL9R, or IL21R signaling without requiring presence of a soluble ligand, such as a soluble cytokine.

Description

ENGINEERED IMMUNE SIGNALING CONSTRUCTSCROSS REFERENCE

[0001] This application claims priority to and the benefit of United States Provisional Patent Application No. 63 / 605,426, filed December 1, 2023, and United States Provisional Patent Application No. 63 / 605,932, filed December 4, 2023, each of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Immune-based therapies such as chimeric antigen receptor (CAR)-T-cell therapies have revolutionized the landscape of certain cancer treatments in recent years. Although this class of therapy has demonstrated impressive clinical efficacy against certain cancers that were once thought to be incurable, its success is many cases and contexts can be limited, for example, when engineered CAR-T cells exhibit insufficient proliferation, persistence, tumor cell killing, or cytokine production.SUMMARY

[0003] Disclosed herein, in some aspects, is a method of inducing signaling of a common gamma chain family cytokine receptor in a cell, the method comprising expressing an engineered immune signaling construct in the cell, wherein the engineered immune signaling construct comprises: an extracellular region that comprises a common gamma chain-binding domain; a transmembrane region; and an intracellular region comprising an intracellular signaling domain of a cytokine receptor subunit, wherein the cytokine receptor subunit is a common gamma chain family cytokine receptor subunit; wherein the common gamma chainbinding domain is heterologous to the cytokine receptor subunit, and the signaling is induced in a soluble ligand-independent manner.

[0004] In some embodiments, the signaling is constitutively induced if the cell expresses the common gamma chain. In some embodiments, the extracellular region or the gamma chain binding domain consists essentially of (i) the common gamma chain-binding domain, and (ii) optionally a hinge, linker, and / or spacer. In some embodiments, the extracellular region or the gamma chain binding domain consists of (i) the common gamma chain-binding domain, and (ii) optionally a hinge, linker, and / or spacer. In some embodiments, the extracellular region or the gamma chain-binding domain comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1-91. In some embodiments, the extracellular region or the gamma chain-binding domain comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 1-91. In some embodiments, the common gamma chain-binding domain comprises a cytokine or a gammachain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises a wild type cytokine or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises a mutein cytokine or a gamma chain-binding domain thereof. In some embodiments, the common gamma chainbinding domain comprises IL-7 or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-2 or a gamma chainbinding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-4 or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-9 or a gamma chain -binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-15 or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-21 or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises TSLP or a TSLP receptor (TSLPR) chainbinding domain thereof. In some embodiments, the common gamma chain-binding domain comprises an antibody or antigen-binding fragment thereof. In some embodiments, the common gamma chain-binding domain comprises a single chain variable fragment (scFv). In some embodiments, the common gamma chain-binding domain comprises a fragment antigen-binding (Fab). In some embodiments, the common gamma chain-binding domain comprises a single domain antibody. In some embodiments, the transmembrane region comprises a transmembrane domain of an immune receptor. In some embodiments, the transmembrane region comprises a transmembrane domain of a common gamma chain family cytokine receptor. In some embodiments, the transmembrane region comprises a transmembrane domain of IL-2Ra, IL- 2Rb, IL-4Ra, IL-7Ra, IL-9Ra, IL-15Ra, or IL-21R. In some embodiments, the transmembrane region comprises an IL-7Ra transmembrane domain. In some embodiments, the transmembrane region comprises a CD8a transmembrane domain. In some embodiments, the transmembrane region comprises a CD28 transmembrane domain. In some embodiments, the transmembrane region comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 92-104. In some embodiments, the transmembrane region comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 92-104. In some embodiments, the intracellular signaling domain comprises a JAK1 binding domain. In some embodiments, the intracellular signaling domain comprises a BOX1 motif. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of IL-2Rb, optionally wherein the signaling is IL-2R signaling. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of IL-7Ra, optionally wherein the signaling is IL-7R signaling. In some embodiments,the intracellular signaling domain comprises an intracellular signaling domain of IL-9Ra, optionally wherein the signaling is IL-9R signaling. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of IL-4Ra, optionally wherein the signaling is IL-4R signaling. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of IL-21R, optionally wherein the signaling is IL-21R signaling. In some embodiments, the intracellular region or the intracellular signaling domain comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 105-115 and 533-540. In some embodiments, the intracellular region or the intracellular signaling domain comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 105-115 and 533-540. In some embodiments, the engineered immune signaling construct consists essentially of the extracellular region, the transmembrane region, and the intracellular region. In some embodiments, the engineered immune signaling construct consists of the extracellular region, the transmembrane region, and the intracellular region. In some embodiments, the extracellular region lacks a ligand-binding domain of a cytokine receptor. In some embodiments, the extracellular region lacks a ligand-binding domain of an immune inhibitory receptor. In some embodiments, the method increases signaling of the common gamma chain family cytokine receptor in the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct. In some embodiments, the method increases signaling of the common gamma chain family cytokine receptor in a bystander cell that lacks the engineered immune signaling construct by at least 5%, as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signaling construct. In some embodiments, the increase in signaling is as determined by a STAT5 phosphorylation assay. In some embodiments, the method enhances survival or persistence of the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct. In some embodiments, the method enhances survival or persistence of a bystander cell that lacks the engineered immune signaling construct by at least 5% as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signaling construct. In some embodiments, the survival is as determined by an IL-2 starvation assay. In some embodiments, the method enhances proliferation of the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct. In some embodiments, the method enhances proliferation of a bystander cell that lacks the engineered immune signaling construct by at least 5% as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signalingconstruct. In some embodiments, the proliferation is as determined by a lymphoproliferation assay.

[0005] Disclosed herein, in some aspects, is an engineered immune signaling construct comprising: an extracellular region comprising a common gamma chain-binding domain, wherein the extracellular region lacks a soluble ligand-binding domain of a receptor; a transmembrane region; and an intracellular region comprising an intracellular signaling domain of a cytokine receptor subunit, wherein the cytokine receptor subunit is a common gamma chain family cytokine receptor subunit and is heterologous to the common gamma chain-binding domain.

[0006] In some embodiments, the extracellular region consists essentially of (i) the common gamma chain-binding domain, and (ii) optionally a hinge, linker, and / or spacer.

[0007] In some embodiments, the extracellular region consists of (i) the common gamma chain-binding domain, and (ii) optionally a hinge, linker, and / or spacer. In some embodiments, the extracellular region or the gamma chain-binding domain comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1-91. In some embodiments, the extracellular region or the gamma chain-binding domain comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 1-91. In some embodiments, the common gamma chain-binding domain comprises a cytokine or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises a wild type cytokine or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises a mutein cytokine or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-7 or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-2 or a gamma chainbinding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-4 or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-9 or a gamma chain -binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-15 or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-21 or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises TSLP or a TSLP receptor (TSLPR) chainbinding domain thereof. In some embodiments, the common gamma chain-binding domain comprises an antibody or antigen-binding fragment thereof. In some embodiments, the common gamma chain-binding domain comprises a single chain variable fragment (scFv). In some embodiments, the common gamma chain-binding domain comprises a fragment antigen-binding(Fab). In some embodiments, the common gamma chain-binding domain comprises a single domain antibody. In some embodiments, the transmembrane region comprises a transmembrane domain of an immune receptor. In some embodiments, the transmembrane region comprises a transmembrane domain of a cytokine receptor. In some embodiments, the transmembrane region comprises a transmembrane domain of a common gamma chain family cytokine receptor. In some embodiments, the transmembrane region comprises a transmembrane domain of IL-2Ra, IL-2Rb, IL-4Ra, IL-7Ra, IL-9Ra, IL-15Ra, or IL-21R. In some embodiments, the transmembrane region comprises an IL-7Ra transmembrane domain. In some embodiments, the transmembrane region comprises a CD8a transmembrane domain. In some embodiments, the transmembrane region comprises a CD28 transmembrane domain. In some embodiments, the transmembrane region comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 92-104. In some embodiments, the transmembrane region comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 92-104. In some embodiments, the intracellular signaling domain comprises a JAK1 binding domain. In some embodiments, the intracellular signaling domain comprises a BOXl motif. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of IL-2Rb, optionally wherein the signaling is IL-2R signaling. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of IL-7Ra, optionally wherein the signaling is IL-7R signaling. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of IL-9Ra, optionally wherein the signaling is IL-9R signaling. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of IL-4Ra, optionally wherein the signaling is IL-4R signaling. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of IL-21R, optionally wherein the signaling is IL-21R signaling. In some embodiments, the intracellular region or the intracellular signaling domain comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 105-115 and 533-540. In some embodiments, the intracellular region or the intracellular signaling domain comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 105-115 and 533-540. In some embodiments, the engineered immune signaling construct consists essentially of the extracellular region, the transmembrane region, and the intracellular region. In some embodiments, the engineered immune signaling construct consists of the extracellular region, the transmembrane region, and the intracellular region. In some embodiments, the extracellular region lacks a soluble ligand-binding domain.

[0008] Disclosed herein, in some aspects, is a polynucleotide encoding the engineered immune signaling construct of any one of the previous embodiments.

[0009] Disclosed herein, in some aspects, is a vector comprising the polynucleotide of any one of the previous embodiments.

[0010] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector.

[0011] Disclosed herein, in some aspects, is a method of making an engineered cell, the method comprising contacting a cell with the vector of any one of the previous embodiments.

[0012] In some embodiments, the contacting is in vitro or ex vivo. In some embodiments, the contacting is in vivo.

[0013] Disclosed herein, in some aspects, is a cell expressing the engineered immune signaling construct of any one of the previous embodiments.

[0014] In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T cell. In some embodiments, the cell is an NK cell. In some embodiments, the cell expresses an exogenous antigen-recognition receptor. In some embodiments, the exogenous antigen-recognition receptor is a chimeric antigen receptor. In some embodiments, the exogenous antigen-recognition receptor is a T cell receptor. In some embodiments, if the cell expresses common gamma chain, the engineered immune signaling construct constitutively induces signaling of the intracellular signaling domain in the cell. In some embodiments, the cell expresses common gamma chain. In some embodiments, the engineered immune signaling construct constitutively binds to the common gamma chain. In some embodiments, the cell expresses common gamma chain, and the engineered immune signaling construct constitutively induces signaling of the intracellular signaling domain in the cell. In some embodiments, the engineered immune signaling construct increases signaling of a common gamma chain family cytokine receptor comprising the cytokine receptor subunit and common gamma chain in the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct. In some embodiments, the engineered immune signaling construct increases signaling of a gamma chain family cytokine receptor in a bystander cell that lacks the engineered immune signaling construct by at least 5%, as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signaling construct. In some embodiments, the increase in signaling is as determined by a STAT5 phosphorylation assay. In some embodiments, the engineered immune signaling construct enhances survival or persistence of the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct. In some embodiments, the engineered immune signaling construct enhances survival or persistence of abystander cell that lacks the engineered immune signaling construct by at least 5% as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signaling construct. In some embodiments, the survival is as determined by an IL-2 starvation assay. In some embodiments, the engineered immune signaling construct enhances proliferation of the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct. In some embodiments, the engineered immune signaling construct enhances proliferation of a bystander cell that lacks the engineered immune signaling construct by at least 5% as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signaling construct. In some embodiments, the proliferation is as determined by a lymphoproliferation assay.

[0015] Disclosed herein, in some aspects, is a pharmaceutical composition comprising the polynucleotide of any one of the previous embodiments and a pharmaceutically-acceptable excipient, vehicle, carrier, or diluent

[0016] Disclosed herein, in some aspects, is a pharmaceutical composition comprising the cell of any one of the previous embodiments and a pharmaceutically-acceptable excipient, vehicle, carrier, or diluent.

[0017] Disclosed herein, in some aspects, is a method of treating a condition in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of any one of the previous embodiments.

[0018] In some embodiments, the condition comprises or is cancer. In some embodiments, the condition comprises or is an infectious disease. In some embodiments, the condition comprises or is an autoimmune disease.

[0019] Disclosed herein, in some aspects, is a method of enhancing the efficacy of a therapeutic regimen that comprises an engineered cell, the method comprising expressing the engineered immune signaling construct of any one of the previous embodiments in the engineered cell, thereby enhancing the efficacy of the therapeutic regimen.

[0020] Disclosed herein, in some aspects, is a method of enhancing survival, proliferation, differentiation, persistence, or effector function of an engineered cell, the method comprising expressing the engineered immune signaling construct of any one of the previous embodiments in the engineered cell, thereby enhancing the survival, proliferation, differentiation, persistence, or effector function of the engineered cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will beobtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0022] FIG. l is a schematic of an illustrative engineered immune signaling construct of the disclosure. The construct comprises an extracellular region comprising a common gamma chainbinding domain, a transmembrane region, and an intracellular region comprising an intracellular signaling domain from a gamma chain family cytokine receptor subunit. The gamma chainbinding region can be a cytokine tethered to the transmembrane domain by a linker and / or hinge; such a construct which can be referred to as a tetherkine.

[0023] FIG. 2 is a schematic of an illustrative engineered immune signaling construct of the disclosure. The construct comprises an extracellular region comprising a common gamma chainbinding domain, a transmembrane region, and an intracellular region comprising an intracellular signaling domain from a gamma chain family cytokine receptor subunit. The gamma chainbinding region can be an antigen-binding fragment of an antibody (e.g., scFv or single domain antibody) that binds to gamma chain; such a construct can be referred to as a gripperkine.

[0024] FIG. 3 shows a schematic of a nucleic acid expression vector used to express an engineered immune signaling construct in cells.

[0025] FIG. 4 shows the transduction efficiency (% of cells expressing EGFP) and surface expression levels (% of cells stained positive for FLAG) of engineered immune signaling constructs in transfected primary human T-cells from two different donors (d8089 and d6995).

[0026] FIG. 5 provides scatter plots showing the correlation between the transduction efficiency (% of cells expressing EGFP) and surface expression levels (% of cells staining positive for FLAG) of engineered immune signaling constructs in transfected primary human T- cells from two different donors.

[0027] FIG. 6A provides scatter plots showing the effect of different extracellular domains (ECD) on the expression levels of engineered immune signaling constructs in transfected T-cells from one donor.

[0028] FIG. 6B provides scatter plots showing the effect of different intracellular domains (ICD) on the expression levels of engineered immune signaling constructs in transfected T-cells from one donor.

[0029] FIG. 7 is a timeline for an experiment to evaluate candidate engineered immune signaling constructs for expression and effects on signaling by gamma chain complexes (via assaying STAT5 phosphorylation) and cellular survival.

[0030] FIG. 8 provides scatter plots showing the expression profiles of engineered immune signaling constructs in transfected T-cells from two different donors.

[0031] FIG. 9. provides scatter plots for the percentage of cells exhibiting STAT5 phosphorylation, for cells that express an engineered immune signaling construct (EGFP+, left panel) or that lack the engineered immune signaling construct (EGFP-, right panel), for two donors.

[0032] FIG. 10 provides graphs plotting STAT5 phosphorylation for cells expressing engineered immune signaling constmcts with different intracellular domains (in transfected T- cells from two different donors).

[0033] FIG. 11 provides graphs plotting STAT5 phosphorylation for cells expressing engineered immune signaling constmcts, split out by different common gamma chain-binding extracellular domains (in transfected T-cells from two different donors).

[0034] FIG. 12 provides graphs showing STAT5 phosphorylation for bystander cells lacking engineered immune signaling constmct expression (EGFP-) co-cultured with cells that express engineered immune signaling constmcts, split out by different common gamma chain-binding extracellular domains.

[0035] FIG. 13 provides scatter plots showing a fold change in viable cells following IL-2 starvation as compared to untransduced control (Neg Ctrl) for cells that express engineered immune signaling constmcts (EGFP+ left chart), or for bystander cells lacking engineered immune signaling constmct expression co-cultured with cells that express engineered immune signaling constmcts (EGFP-, right chart).

[0036] FIG. 14 provides scatter plots showing the fold change in number of T-cells expressing engineered immune signaling constmcts (EGFP+) compared to bystander cells (EGFP-) for T- cells from two different donors.

[0037] FIG. 15 provides graphs showing the extent to which different engineered immune signaling constmcts affect STAT5 phosphorylation and survival (fold change in cell number) of transfected T-cells (EGFP+) and bystander cells (EGFP-) from two different donors.

[0038] FIG. 16 shows the memory phenotypes of T-cells from different donors that were transfected with illustrative engineered immune signaling constmcts following IL-2 starvation for 7 days.

[0039] FIG. 17 provides graphs showing the CD4 / CD8 ratios of T-cells from different donors that were transfected with illustrative engineered immune signaling constmcts following IL-2 starvation for 7 days.

[0040] FIG. 18 provides graphs comparing the memory subsets and CD4 / CD8 ratios of T- cells that were transfected with illustrative engineered immune signaling constmcts and starved of IL-2 for 7 days.DETAILED DESCRIPTIONI. ENGINEERED IMMUNE SIGNALING CONSTRUCT

[0041] Disclosed herein are engineered immune signaling constructs that can comprise, for example, an extracellular region, transmembrane region, and intracellular region. The extracellular region can comprise a common gamma chain-binding domain, and the intracellular region can comprise a common gamma chain family cytokine receptor / receptor subunit intracellular signaling domain. The engineered immune signaling construct can be configured such that the construct binds to common gamma chain and elicits constitutive signaling of a common gamma chain family cytokine receptor (e.g., via the heterodimer of the engineered immune signaling construct and common gamma chain) in cells that express common gamma chain (FIG. 1). The common gamma chain family cytokine receptor subunit can comprise, for example, IL-2Rb, IL-4Ra, IL-7Ra, IL-9Ra, IL-15Ra, or IL-21R. The signaling can be induced, for example, without requiring presence or administration of a soluble ligand (e.g., cytokine) to induce signaling.

[0042] Constitutive or ligand-independent gamma chain family receptor signaling induced by engineered immune signaling constructs disclosed herein can be useful for enhancing certain properties of cells that express common gamma chain. For example, the engineered immune signaling construct can be co-expressed with an exogenous antigen-recognition receptor (e.g., chimeric antigen receptor (CAR) or T cell receptor (TCR)) in an engineered cell. The exogenous antigen-recognition receptor can facilitate a response of the engineered cell to a target cell, for example, induce immune activation, proliferation, effector function, cytolytic activity, and / or pro-inflammatory cytokine production in response to the target cell, such as a cancer cell. In some embodiments, ligand-independent gamma chain receptor family signaling induced by an engineered immune signaling construct disclosed herein can enhance the survival, proliferation, differentiation, persistence, activation, effector function, cytolytic activity, cytokine production, or other properties of the engineered cell, for example, to improve the efficacy of a therapeutic regimen that utilizes the engineered cell. The gamma chain receptor family signaling can be signaling of, for example, IL-2R (e.g., IL-2Rbg), IL-4R, IL-7R, IL-9R, IL-15R, or IL-21R. In some embodiments, the gamma chain receptor family signaling can encompass signaling of related receptors that do not include gamma chain, such as TSLPR (for example, the extracellular region can comprise TSLP and can induce formation of a heterodimer of IL-7Ra and TSLPR, thereby inducing TSLP receptor signaling).

[0043] Gamma chain and common gamma chain can be used interchangeably herein. For example, gamma chain family and common gamma chain family can be used interchangeably,gamma chain family cytokine and common gamma chain family cytokine can be used interchangeably, and gamma chain family cytokine receptor and common gamma chain family cytokine receptor can be used interchangeably herein.A. Extracellular region

[0044] An engineered immune signaling construct disclosed herein can comprise an extracellular region that comprises a common gamma chain-binding domain.

[0045] In some embodiments, the common gamma chain-binding domain comprises a cytokine or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain family cytokine in the extracellular region is able to recruit components of the native receptor complex in cis (e.g., on the same cell, inducing autocrine-like signaling) and / or in trans (e.g., on an adjacent cell, inducing paracrine-like signaling). In some embodiments, recruitment of components of the native receptor complex in cis or in trans can facilitate advantageous biological effects of compositions, systems, and methods disclosed herein, for example, modulating survival, proliferation, differentiation, persistence, activation, effector function, cytolytic activity, cytokine production, or other properties of the cell expressing the engineered immune signaling construct and / or surrounding cells.

[0046] In some embodiments, the cytokine is a member of the common gamma chain family of cytokines or a gamma chain-binding domain thereof. For example, the common gamma chain-binding domain can be or can comprise, consist of, or consist essentially of IL-2, IL-4, IL- 7, IL-9, IL- 15, IL-21, or a gamma chain-binding domain thereof. Illustrative sequences of common gamma chain cytokine family members are provided in SEQ ID NOs: 1-22.

[0047] In some embodiments, the common gamma chain-binding domain comprises IL-2 or a gamma chain-binding domain thereof. In some embodiments, the common gamma chainbinding domain comprises IL-4 or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-7 or a gamma chainbinding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-9 or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-15 or a gamma chain-binding domain thereof. In some embodiments, the common gamma chain-binding domain comprises IL-21 or a gamma chain-binding domain thereof.

[0048] In some embodiments, an engineered immune signaling construct comprises a TSLPR (Thymic Stromal Lymphopoietin Protein Receptor) or IL-7Ra chain-binding domain. In some embodiments, the cytokine is TSLP or a TSLPR binding domain thereof. In some embodiments, the cytokine is TSLP or an IL-7Ra binding domain thereof. In some embodiments, the cytokineis IL-7 or a TSLPR binding domain thereof. In some embodiments, the cytokine is IL-7 or an IL-7Ra binding domain thereof. Illustrative sequences of TSLP, IL-7 and TSLPR-binding or IL- 7Ra-binding domains thereof are provided in SEQ ID NOs: 23-24 and 1-3.

[0049] Where the engineered immune signaling construct comprises an intracellular signaling domain of a common gamma chain family cytokine receptor subunit, the common gamma chain-binding domain can be heterologous with respect to the receptor subunit, for example, not be, contain a domain of, or be derived from, the receptor subunit. For example, the common gamma chain-binding domain can refer to domains that do not occur in a native or naturally- occurring version of the common gamma chain family cytokine receptor subunit.

[0050] The common gamma chain-binding domain can be heterologous with respect to IL- 9Ra, for example, not a domain of IL-9Ra or derived from IL-9Ra (e.g., for an engineered immune signaling construct that comprises an IL-9Ra intracellular signaling domain). The extracellular region or common gamma chain-binding domain can lack an extracellular domain of IL-9Ra.

[0051] The common gamma chain-binding domain can be heterologous with respect to IL- 2Rb, for example, not be a domain of IL-2Rb or derived from IL-2Rb (e.g., for an engineered immune signaling construct that comprises an IL-2Rb intracellular signaling domain). The extracellular region or common gamma chain-binding domain can lack an extracellular domain of IL-2Rb.

[0052] The common gamma chain-binding domain can be heterologous with respect to IL- 4Ra, for example, not be a domain of IL-4Ra or derived from IL-4Ra (e.g., for an engineered immune signaling construct that comprises an IL-4Ra intracellular signaling domain). The extracellular region or common gamma chain-binding domain can lack an extracellular domain of IL-4Ra.

[0053] The common gamma chain-binding domain can be heterologous with respect to IL- 7Ra, for example, not be a domain of IL-7Ra or derived from IL-7Ra (e.g., for an engineered immune signaling construct that comprises an IL-7Ra intracellular signaling domain). The extracellular region or common gamma chain-binding domain can lack an extracellular domain of IL-7Ra.

[0054] The common gamma chain-binding domain can be heterologous with respect to IL- 15Ra, for example, not be a domain of IL-15Ra or derived from IL-15Ra (e.g., for an engineered immune signaling construct that comprises an IL-15Ra intracellular signaling domain). The extracellular region or common gamma chain-binding domain can lack an extracellular domain ofIL-15Ra.

[0055] The common gamma chain-binding domain can be heterologous with respect to IL- 21R, for example, not be a domain of IL-21R or derived from IL-21R (e.g., for an engineered immune signaling construct that comprises an IL-21R intracellular signaling domain). The extracellular region or common gamma chain-binding domain can lack an extracellular domain of IL-21R.

[0056] The common gamma chain-binding domain can be heterologous with respect to IL- 2Ra, for example, not be a domain of IL-2Ra or derived from IL-2Ra (e.g., for an engineered immune signaling construct that comprises an IL-2Ra intracellular signaling domain). The extracellular region or common gamma chain-binding domain can lack an extracellular domain of IL-2Ra.

[0057] The cytokine can be a wild type cytokine or a gamma chain-binding domain thereof.

[0058] The cytokine can be a mutein cytokine or a gamma chain-binding domain thereof. For example, the cytokine can be a mutein that is modified to alter binding affinity of the cytokine for one or more receptor subunit(s).

[0059] Native IL-2 can bind to trimeric and dimeric IL-2 receptor (IL-2R). Trimeric IL-2R comprises IL-2Rot (CD25), IL-2RP (CD122), and the common gamma chain (ZL-2Ry, CD132), and can also be referred to as high affinity IL-2R or IL-2RaPy. Dimeric IL-2R comprises IL-2RP and IL-2Ry, and can also be referred to as IL-2RPy. IL-2RaPy can be present at high levels or predominantly expressed on immunosuppressive or immune tolerance-promoting regulatory T (Treg) cells, and accordingly in some embodiments an IL-2 comprises one or more modifications to reduce affinity for IL-2Ra and / or IL-2RaPy, e.g., to reduce IL-2 signaling in Tregs, or enhance affinity for IL-2Ra and / or ZL-2RaPy, e.g., to enhance IL-2 signaling in Tregs. IL-2RPy can be present at high levels or predominantly expressed on effector T cells, memory T cells, and NK cells, and accordingly in some embodiments an IL-2 comprises one or more modifications to increase affinity for IL-2RPy to increase IL-2 signaling in effector T cells, memory T cells, or NK cells, or to reduce affinity for IL-2RPy to reduce IL-2 signaling in effector T cells, memory T cells, or NK cells.

[0060] An illustrative example of a mutein cytokine is IL-2 non-alpha (or no alpha), which can be an IL-2 mutein engineered to disrupt interaction with IL-2Ra (CD25). Such an IL-2 mutein can have differential effects on certain T cell subsets, for example, preferentially induce IL-2 signaling on effector or memory T cells rather than regulatory T cells (Tregs).

[0061] IL-2 modifications can comprise, for example, modifications of residues T3, L18, D20, Q22, 128, N29, Y31, K35, T37, R38, F42, K43, Y45, K48, T51, E61, E62, V69, N71, L72, Q74, L80, R81, L85, 186, N88, 189, V91, 192, M104, C125, Q126, or S130 relative to SEQ ID NO: 6, or any combination thereof. In some embodiments, the modifications comprise a T3A, L18R,D20A, D20T, D20H, Q22E, I28T, N29S, Y31H, K35R, T37A, R38D, F42A, F42K, K43E, Y45A, Y45R, K48E, E61R, V69A, N71R, L72G, Q74P, L80F, R81D, L85V, I86V, N88D, N88R, N88G, I89V, V91D, V91K, I92F, M104V, C125A, C125S, Q126L, Q126F, Q126H, Q126T, or S130R substitution relative to SEQ ID NO: 6, or any combination thereof.

[0062] In some embodiments, an IL-15 is used comprising a modification of residue N72 relative to SEQ ID NO: 18 In some embodiments, an IL-15 is used comprising an N72D substitution relative to SEQ ID NO: 18.

[0063] In some embodiments, an IL-4 is used comprising a modification at position KI 17, T118, R121, E122, Y124, S125, S128, S 129, or a combination thereof relative to SEQ ID NO: 11. In some embodiments, an IL4 is used comprising a substitution that is KI 17R, T118V, R121Q, R121D, R121K, R121E, E122S, Y124W, Y124F, Y124D, S125F, S128G, S125R, SI 29 A, or a combination thereof relative to SEQ ID NO: 11.

[0064] The engineered immune signaling construct and / or extracellular region thereof can lack a ligand-binding domain of a receptor. For example, the engineered immune signaling construct can comprise a structure and configuration that facilitates signaling by the construct (e.g., IL-2R, such as IL-2Rbg, IL-4R, IL-7R, IL-9R, IL-15R, or IL-21R) when the cell expresses common gamma chain (or, e.g., TSLPR complex signaling when the cell expresses TSLPR or IL-7Ra), without requiring binding of an exogenous ligand. In some embodiments, the engineered immune signaling construct and / or extracellular region thereof lacks the ligand binding domain of an immune receptor, a cytokine receptor, or an immune inhibitory receptor. In some embodiments, the engineered immune signaling construct and / or extracellular region thereof lacks a ligand-binding domain of any receptor. In some embodiments, the engineered immune signaling construct and / or extracellular region thereof lacks a ligand-binding domain of a common gamma chain family cytokine receptor, for example, lacks the extracellular region of IL-2Ra, IL-2Rb, IL-4Ra, IL-7Ra, IL-9Ra, IL-15Ra, or IL-21R, or lacks the extracellular domain or ligand-binding domain of any one of SEQ ID NOs: 197-204. In some embodiments, the engineered immune signaling construct and / or extracellular region thereof lacks a domain that binds a soluble gamma chain family cytokine, such as soluble IL-2, IL-4, IL-7, IL-9, IL- 15, and / or IL-21. In some embodiments, the engineered immune signaling construct and / or extracellular region thereof does not bind or substantially does not bind any gamma chain family cytokine.

[0065] In some embodiments, the engineered immune signaling construct and / or extracellular region thereof lacks a ligand-binding domain of a TSLP receptor / receptor subunit, for example TSLPR or IL-7Ra. In some embodiments, the engineered immune signaling construct and / or extracellular region thereof lacks the extracellular region TSLP receptor / receptor subunit, forexample TSLPR or IL-7Ra. In some embodiments, the engineered immune signaling construct and / or extracellular region thereof does not bind or substantially does not bind soluble TSLP.

[0066] In some embodiments, the engineered immune signaling construct and / or extracellular region thereof comprises a ligand-binding domain of a receptor or a functional fragment thereof (e.g., of any one of SEQ ID NOs: 197-204). Such an engineered immune signaling construct can be configured to induce signaling of a gamma chain family receptor (e g., IL-2R (such as IL- 2Rbg), IL-4R, IL-7R, IL-9R, IL-15R, or IL-21R signaling) independent of soluble ligand. For example, in some embodiments, an engineered immune signaling construct comprises a gamma chain family cytokine, and an extracellular domain of a receptor of the gamma chain family cytokine, to promote constitutive binding of the cytokine to gamma chain and induction of signaling. For example, in some embodiments an engineered immune signaling construct comprises an extracellular region comprising IL-7, and further comprising an extracellular domain of IL-7Ra, such that binding of the tethered IL-7 to gamma chain and signaling induction (e.g., IL-2R (such as IL-2Rbg), IL-4R, IL-7R, IL-9R, IL-15R, or IL-21R signaling) is enhanced.

[0067] In some embodiments, use of an extracellular domain of a gamma chain family receptor (or a functional fragment thereof) in an engineered immune signaling construct can facilitate adoption of a conformation that more closely resembles a native heterodimeric complex formed between the cytokine, common gamma chain, and the additional receptor subunit, thereby facilitating enhanced binding affinity and / or signaling.

[0068] In some embodiments an engineered immune signaling construct comprises an extracellular region comprising IL-2, and further comprises an extracellular domain of IL-2Ra and / or IL-2Rb, such that binding of the tethered IL-2 to gamma chain and signaling induction (e.g., IL-2R (such as IL-2Rbg), IL-4R, IL-7R, IL-9R, IL-15R, or IL-21R signaling) is enhanced.

[0069] In some embodiments an engineered immune signaling construct comprises an extracellular region comprising IL-4, and further comprises an extracellular domain of IL-4Ra, such that binding of the tethered IL-4 to gamma chain and signaling induction (e g., IL-2R (such as IL-2Rbg), IL-4R, IL-7R, IL-9R, IL-15R, or IL-21R signaling) is enhanced.

[0070] In some embodiments an engineered immune signaling construct comprises an extracellular region comprising IL-9, and further comprises an extracellular domain of IL-9Ra, such that binding of the tethered IL-9 to gamma chain and signaling induction (e.g., IL-2R (such as IL-2Rbg), IL-4R, IL-7R, IL-9R, IL-15R, or IL-21R signaling) is enhanced.

[0071] In some embodiments an engineered immune signaling construct comprises an extracellular region comprising IL-15, and further comprises an extracellular domain of IL-15Ra, such that binding of the tethered IL-15 to gamma chain and signaling induction (e.g., IL- 2R (such as IL-2Rbg), IL-4R, IL-7R, IL-9R, IL-15R, or IL-21R signaling) is enhanced.

[0072] In some embodiments an engineered immune signaling construct comprises an extracellular region comprising IL-21, and further comprises an extracellular domain of IL-21R, such that binding of the tethered IL-21 to gamma chain and signaling induction (e.g., IL-2R (such as IL-2Rbg), IL-4R, IL-7R, IL-9R, IL-15R, or IL-21R signaling) is enhanced.

[0073] In some embodiments an engineered immune signaling construct comprises an extracellular region comprising TSLP, and further comprises an extracellular domain of IL7-Ra, such that binding of the tethered TSLP to TSLPR and signaling induction (e.g., TSLPR / IL-7Ra signaling) is enhanced.

[0074] In some embodiments an engineered immune signaling construct comprises an extracellular region comprising TSLP, and further comprises an extracellular domain of TSLPR, such that binding of the tethered TSLP to IL-7Ra and signaling induction (e.g., TSLPR / IL-7Ra signaling) is enhanced.

[0075] The common gamma chain-binding domain can comprise an antibody, antigen-binding fragment thereof, or other antigen-binding molecule. An antibody or antigen-binding fragment thereof that a common gamma chain-binding domain comprises can be or can comprise an immunoglobulin variable domain, a heavy chain variable (VH) domain, a light chain variable (VL) domain, Fab, Fab', F(ab')2, dimer or trimers of Fab conjugates, Fv, scFv, minibody, dia-, tria-, and tetrabody, linear antibody, heavy chain only antibody (HCAb), VHH, single domain antibody (sdAb), nanobody, or a combination thereof. The antibody or antigen-binding fragment thereof can be or comprise a fragment of a chimeric, humanized, or fully human antibody.

[0076] In some embodiments, the common gamma chain-binding domain comprises an scFv.

[0077] Illustrative common gamma chain-binding VH and VL domains are provided in SEQ ID NOs: 25-70, and illustrative common gamma chain-binding scFvs are provided in SEQ ID NOs: 71-91

[0078] An antibody or antigen-binding fragment thereof can be or can comprise a single domain antibody. The single domain antibody can be or can comprise a variable region of a heavy chain only antibody. Such a single domain antibody can also be known as a nanobody or VHH. The single domain antibody can be, for example, a variable region from or derived from a heavy chain only antibody from a camelid (e.g., camels: one-humped Camelus dromedaries and two-humped Camelus bactrianus; llamas: Lama glama, Lama guanicoe, and Lama vicugna; and alpacas: Vicugna pacos), a shark (e.g., a nurse shark), a wobbegong, or a spotted ratfish. Such animals have a special type of antibody called heavy chain Abs (HCAbs), that lack the entire light chain and the first heavy chain C region (CHI) compared to regular antibodies.

[0079] The variable (V) domain(s) of an antibody can mediate antigen binding and define the specificity of a particular antibody for an antigen. The variable domain can comprise relatively invariant sequences called framework regions, and hypervariable regions, which differ considerably in sequence among antibodies of different binding specificities. The variable domain can comprise four framework regions separated by three hypervariable regions. The variable domains can fold in a manner that brings the hypervariable regions together in close proximity to create an antigen binding site. The four framework regions can largely adopt an 13- sheet configuration, while the three hypervariable regions form loops connecting, and in some cases forming part of, the f3-sheet structure.

[0080] Within hypervariable regions are amino acid residues that primarily determine the binding specificity of the antibody in most cases. Sequences comprising these residues are known as complementarity determining regions (CDRs). One antigen binding site of an antibody with heavy and light chains or variable domains therefrom can comprise six CDRs, three in the hypervariable regions of the light chain variable domain, and three in the hypervariable regions of the heavy chain variable domain. The CDRs in the light chain are designated LI, L2, and L3, while the CDRs in the heavy chain are designated Hl, H2, and H3. CDRs can also be designated LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, or LC-CDR-1, LC-CDR-2, LC- CDR-3, HC-CDR-1, HC-CDR-2, and HC-CDR-3, respectively. The contribution of each CDR to antigen binding varies among antibodies, but in some embodiments heavy chain CDRs, in particular HCDR3, can contribute most to antigen-specific binding. CDRs can vary in length. For example, CDRs are often 5 to 14 residues in length, but CDRs as short as 0, 1, 2, or 3 residues and / or as long as 25 residues or longer exist.

[0081] Certain antibodies or antigen-binding domains contain less than six CDRs. For example, certain antibodies lack a light chain, and can be referred to as heavy chain only antibodies (HCAbs). HCAbs have three CDRs in a variable region that can be referred to as VHH. A single domain antibody, or nanobody, can be generated from such a VHH region of a heavy chain only antibody, for example.

[0082] The common gamma chain-binding domain can comprise complementarity determining regions (CDRs). For example, an antibody, antigen-binding fragment thereof, or antigen-binding domain can comprise CDRs. In some embodiments, the CDRs determine or substantially determine binding specificity and / or affinity for a surface molecule on a target cell. For example, the CDRs can be grafted onto a different suitable framework, or the framework region can be altered (e.g., via amino acid substitutions, deletions, and / or insertions), and the antigen-binding fragment or domain can retain binding for the target, and the extracellular binding domain remains functional despite the alterations outside of the CDRs. In someembodiments, one or more framework regions or amino acid sequences therein contribute to binding specificity and / or affinity.

[0083] An extracellular region or common gamma chain-binding domain of an engineered immune signaling construct disclosed herein can comprise CDRs. CDRs in or for use in an extracellular region or common gamma chain-binding domain can be identified by various methods, including but not limited to the Kabat method, the Chothia method, the IMGT method, the AHO method, the AbM method, the contact method, and the Paratome method. For example, CDRs can be identified from any VH, VL, and in some embodiments VHH domains disclosed herein using the Kabat method, the Chothia method, the IMGT method, the AHO method, the AbM method, the contact method, and / or the Paratome method.

[0084] Single domain antibodies can in some embodiments have longer CDR Hl and H3 loops compared with the respective classical CDRs and can require different methods to identify CDRs. Single domain antibody CDRs can be identified, for example, using the single domain antibody database (SAbDab), based on common sequence elements, or based on a sequence alignment to the Chothia numbering scheme (e.g., as described by Wilton, et al. (2018). sdAb- DB: the single domain antibody database. ACS Synthetic Biology 2018 7 (11), 2480-2484 DOI: 10.1021 / acssynbio.8b00407, which is incorporated herein by reference for such disclosure).

[0085] An extracellular region or gamma chain-binding region can comprise the CDR amino acid sequence(s) of any one or more of the CDR sequences of VH and / or VL domains disclosed herein. In some embodiments, a binding domain comprises an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequence selected from the CDR sequences disclosed herein. In some embodiments, a binding domain comprises an HCDR1, HCDR2, and HCDR3 amino acid sequence selected from the CDR sequences disclosed herein.

[0086] An extracellular region or gamma chain-binding domain can comprise one or more variant CDR sequences, e.g., each with at most one, at most two, or at most three amino acid substitution(s), insertion(s), or deletion(s) relative to any one of the CDR sequences disclosed herein. In some embodiments, a gamma chain-binding domain or extracellular region comprises one or more CDR sequences each with 0-1 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a gamma chain-binding domain or extracellular region comprises one or more CDR sequences each with 0-2 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a gamma chain-binding domain or extracellular region comprises one or more CDR sequences each with 0-3 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein.

[0087] In some embodiments, a gamma chain-binding domain or extracellular region comprises one or more variant CDR sequences each with 1-2 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a gamma chain-binding domain or extracellular region comprises one or more variant CDR sequences each with 1-3 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a gamma chain-binding domain or extracellular region comprises one or more variant CDR sequences each with 1-4 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein. In some embodiments, a gamma chain-binding domain or extracellular region comprises one or more variant CDR sequences each with 1-5 amino acid substitutions, insertions, and / or deletions relative to any one of the CDR sequences disclosed herein.

[0088] In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin constant domain, a light chain constant (CL) domain, a heavy chain constant domain (CH, such as CHI, CH2, CH3, and / or CH4), or an Fc domain, e.g., of IgA, IgD, IgE, IgG, IgM, IgGl, IgG2, IgG3, IgG4, IgAl, or IgA2.

[0089] In some embodiments, the common gamma chain-binding domain comprises an antigen-binding molecule (e.g., that is or is not an antibody or antigen-binding fragment thereof), for example, an ankyrin protein, ankyrin repeat protein, designed ankyrin repeat protein (DARPin), affibody, avimer, adnectin, anticalin, Fynomer, Kunitz domain, knottin, -hairpin mimetic, or an antigen-binding fragment thereof. In some embodiments, the antibody, antigenbinding fragment thereof, or other antigen-binding molecule is mammalian, camelid, humanized, chimeric, or human.

[0090] The common gamma chain-binding domain can be heterologous with respect to the common gamma chain family cytokine receptor subunit intracellular signaling domain in an engineered immune signaling construct, for example, not a domain of, or derived from, the common gamma chain family cytokine receptor subunit. The extracellular region, common gamma chain-binding domain, or engineered immune receptor can lack an extracellular domain of the common gamma chain family cytokine receptor subunit.

[0091] An extracellular region can comprise an additional extracellular domain or amino acid sequence that is or comprises a linker, hinge, and / or spacer (e.g., further comprise in addition to the common gamma chain binding domain). In some embodiments, an exogenous antigenrecognition receptor comprises a hinge, such as an IgG (e.g., IgGl or IgG4) hinge, CD28 hinge, or a CD8 (e.g., CD8a) hinge, or subdomain thereof. Illustrative hinge, linker, and spacer sequences and domains thereof are provided in SEQ ID NOs: 116-178.B. Transmembrane region

[0092] An engineered immune signaling construct disclosed herein can comprise a transmembrane region. The transmembrane region can be or can comprise, for example, a transmembrane domain from an immune receptor, such as from CD8a, CD28, or a receptor or receptor subunit for a common gamma chain family cytokine (e.g., IL-2Ra, IL-2Rb, IL-4Ra, IL- 7Ra, IL-9Ra, IL-15Ra, IL-21R, or IL-2Rg). A transmembrane region can anchor an engineered immune signaling construct in a membrane and can, for example, contribute to stability of dimerization and / or functionality of IL9R signaling when the engineered immune signaling construct binds to the common gamma chain (e.g., via the common gamma chain-binding domain).

[0093] In some embodiments, an engineered immune signaling construct comprises a transmembrane domain of IL-7Ra. In some embodiments, an engineered immune signaling construct comprises a transmembrane domain of IL-9Ra. In some embodiments, an engineered immune signaling construct comprises a transmembrane domain of IL-2Ra. In some embodiments, an engineered immune signaling construct comprises a transmembrane domain of IL-2Rb. In some embodiments, an engineered immune signaling construct comprises a transmembrane domain of IL-4Ra. In some embodiments, an engineered immune signaling construct comprises a transmembrane domain of IL-15Ra. In some embodiments, an engineered immune signaling construct comprises a transmembrane domain of IL-21R. In some embodiments, an engineered immune signaling construct comprises a transmembrane domain of IL-2Rg. In some embodiments, an engineered immune signaling construct comprises a transmembrane domain of CD8a. In some embodiments, an engineered immune signaling construct comprises a transmembrane domain of CD28. Illustrative transmembrane region / domain sequences are provided in SEQ ID NOs: 92-104.

[0094] An engineered immune signaling construct can comprise a transmembrane region or transmembrane domain of an immune receptor, an immune co-receptor (e.g., co-inhibitory or co-stimulatory immune co-receptor), a cytokine receptor, an Ig-superfamily member, an Fc receptor (e g., Fc gamma receptor), an NK receptor, or a receptor expressed by a lymphocyte (e.g., T cell, NK cell, or NKT cell).

[0095] An engineered immune signaling construct can comprise a transmembrane region or transmembrane domain of a cytokine receptor (e.g., a pro-inflammatory or anti-inflammatory cytokine receptor, or a chemokine receptor). An engineered immune signaling construct can comprise a transmembrane region or transmembrane domain of an interleukin receptor (e.g., a receptor of IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL- 16, IL-17, IL-18, IL-20, or IL-23) or a subunit thereof. In some embodiments the engineeredimmune signaling construct comprises a transmembrane domain of an interferon receptor (for example, a receptor of IFN-a, IFN-P, or IFN-y) or a subunit thereof. In some embodiments the engineered immune signaling construct comprises a transmembrane domain of a TNF receptor, e.g., TNFR1 or TNFR2 or a subunit thereof.C. Intracellular region

[0096] An engineered immune signaling construct disclosed herein can comprise an intracellular region, for example, that facilitates signaling. The engineered immune signaling construct can induce signaling via the intracellular region upon binding of the engineered immune signaling construct to gamma chain (or, e.g., IL-7Ra or TSLPR). For example, in a cell that expresses common gamma chain, the engineered immune signaling construct can be activated in a ligand-independent (e.g., soluble ligand-independent) manner, e.g., without requiring the presence of a soluble binding partner, such as a cytokine. In some embodiments, the engineered immune signaling construct is activated without requiring the presence of surface-expressed ligand on an adjacent cell. In some embodiments, the gamma chain-binding domain (e.g., cytokine) can interact with a surface expressed ligand or receptor (e.g., cytokine receptor) on an adjacent cell, thereby modulating signaling in one or both cells, and / or modulating the inflammatory milieu.

[0097] The ability of the common gamma chain-binding domain to constitutively bind common gamma chain when present can facilitate constitutive or substantially constitutive signaling (e.g., of a common gamma chain cytokine receptor, such as IL-2R (such as IL-2Rbg), IL-4R, IL-7R, IL-9R, IL-15R, IL-21R) in cells that express common gamma chain, e.g., without requiring an exogenous activation signal. Signaling in a ligand-independent manner or soluble ligand-independent manner can describe induction of signaling other than by binding of a soluble ligand, such as a soluble cytokine, to the engineered immune receptor. For example, induction of signaling in a ligand-independent or soluble ligand-independent manner can include binding of the common gamma chain-binding domain to common gamma chain, and can exclude binding of a soluble ligand (e.g. cytokine) to the engineered immune signaling construct and / or common gamma chain to induce signaling. In some embodiments, signaling in a liganddependent manner or soluble ligand-dependent manner excludes (e.g., does not refer to, include, or encompass) binding of the common gamma chain-binding domain to common gamma chain.

[0098] An engineered immune signaling construct can include an intracellular signaling domain of a type 1 cytokine receptor family member or subunit thereof. An engineered immune signaling construct can include an intracellular signaling domain of a common gamma chain cytokine receptor family member or subunit thereof.

[0099] An engineered immune signaling construct can include an intracellular signaling domain of the common gamma chain cytokine receptor subunit IL-9Ra. The native IL-9Ra subunit is characterized by four extracellular cysteines and the conserved WSXWS motif, while the intracellular domain contains a BOX1 consensus sequence and a serine rich region. IL-9Ra is found in both membrane bound and soluble forms, whereas the gamma chain subunit is observed only in a membrane bound form. IL-9 binding to native IL-9Ra results in the formation the IL-9R heterocomplex (comprising IL-9Ra and common gamma chain), resulting in IL-9R signaling. The IL-9R heterocomplex recruits Janus kinases (JAK) to mediate the phosphorylation of the receptor. Upon IL-9 binding to the receptor, a conformational change occurs in the IL-9R heterocomplex, which allows JAK molecules to bind to the proline rich BOX1 motif in the membrane-proximal region of IL-9Ra. JAK1 associates with IL-9Ra, whereas JAK3 binds to gamma chain. Phosphorylated JAK1 and JAK3 then mediate the phosphorylation of receptor tyrosine residues. Phosphorylated tyrosine residues act as docking sites for the downstream Src homology 2 (SH2) domain containing signaling molecules such as Signal Transducer and Activator of Transcription (STAT) transcription factors, insulin receptor substrate (IRS), and the adaptors of the Mitogen-Activated Protein Kinase (MAPK) pathways. IL-9R signaling results in various properties that can be advantageous to engineered cells, including modulation of proliferation, differentiation, persistence, activation, effector function, cytolytic activity, cytokine production, and other properties, which can be harnessed in compositions, systems, and methods disclosed herein (e.g., without relying on the presence of a ligand or soluble ligand, such as IL-9).

[0100] An engineered immune signaling construct can include an intracellular signaling domain of the common gamma chain cytokine receptor subunit IL-2Rb. The native IL-2Rb subunit is characterized by two extracellular cysteines and the conserved WSXWS motif, while the intracellular domain contains a BOX1 consensus sequence and a serine rich region. IL-2 binding to native IL-2Rb can result in the formation an IL-2R complex comprising IL-2Rb and common gamma chain (IL-2Rbg), resulting in IL-2R signaling. An alternative IL-2R complex further comprises IL-2Ra (IL-2Rabg). The IL-2R complex recruits Janus kinases (JAK) to mediate the phosphorylation of the receptor. Upon IL-2 binding to the receptor, a conformational change occurs in the IL-2R complex, which allows JAK molecules to bind to the proline rich BOX1 motif in the membrane-proximal region of IL-2Rb. JAK1 associates with IL- 2Rb, whereas JAK3 binds to gamma chain. Phosphorylated JAK1 and JAK3 then mediate the phosphorylation of receptor tyrosine residues. Phosphorylated tyrosine residues act as docking sites for the downstream Src homology 2 (SH2) domain containing signaling molecules such as Signal Transducer and Activator of Transcription (STAT) transcription factors, insulin receptorsubstrate (IRS), and the adaptors of the Mitogen-Activated Protein Kinase (MAPK) pathways. IL-2R signaling results in various properties that can be advantageous to engineered cells, including modulation of proliferation, differentiation, persistence, activation, effector function, cytolytic activity, cytokine production, and other properties, which can be harnessed in compositions, systems, and methods disclosed herein (e.g., without relying on the presence of a ligand or soluble ligand, such as IL-2).

[0101] An engineered immune signaling construct can include an intracellular signaling domain of the common gamma chain cytokine receptor subunit IL-4Ra. The native IL-4Ra subunit is characterized by four extracellular cysteines and the conserved WSXWS motif, while the intracellular domain contains a BOX1 consensus sequence and a serine rich region. IL-4Ra is found in both membrane bound and soluble forms, whereas the gamma chain subunit is observed only in a membrane bound form. IL-4 binding to native IL-4Ra results in the formation the IL-4R heterocomplex (comprising IL-4Ra and common gamma chain) and IL-4R signaling. The IL-4R heterocomplex recruits Janus kinases (JAK) to mediate the phosphorylation of the receptor. Upon IL-4 binding to the receptor, a conformational change occurs in the IL-4R heterocomplex, which allows JAK molecules to bind to the proline rich BOX1 motif in the membrane-proximal region of IL-4Ra. JAK1 associates with IL-4Ra, whereas JAK3 binds to gamma chain. Phosphorylated JAK1 and JAK3 then mediate the phosphorylation of receptor tyrosine residues. Phosphorylated tyrosine residues act as docking sites for the downstream Src homology 2 (SH2) domain containing signaling molecules such as Signal Transducer and Activator of Transcription (STAT) transcription factors, insulin receptor substrate (IRS), and the adaptors of the Mitogen-Activated Protein Kinase (MAPK) pathways. IL-4R signaling results in various properties that can be advantageous to engineered cells, including modulation of proliferation, differentiation, persistence, activation, effector function, cytolytic activity, cytokine production, and other properties, which can be harnessed in compositions, systems, and methods disclosed herein (e.g., without relying on the presence of a ligand or soluble ligand, such as IL-4).

[0102] An engineered immune signaling construct can include an intracellular signaling domain of the common gamma chain cytokine receptor subunit IL-7Ra. The native IL-7Ra subunit is characterized by four extracellular cysteines and the conserved WSXWS motif, while the intracellular domain contains a BOX1 consensus sequence and a serine rich region. IL-7Ra is found in both membrane bound and soluble forms, whereas the gamma chain subunit is observed only in a membrane bound form. IL-7 binding to native IL-7Ra can result in the formation an IL-7R heterocomplex (comprising IL-7Ra and common gamma chain), and IL-7R signaling. IL-7 binding to native TSLPR can also result in formation of a complex of IL-7Ra andTSLPR, and TSLPR signaling. The IL-7R heterocomplex recruits Janus kinases (JAK) to mediate the phosphorylation of the receptor. Upon IL-7 binding to the receptor, a conformational change occurs in the IL-7R heterocomplex, which allows JAK molecules to bind to the proline rich B0X1 motif in the membrane-proximal region of IL-7Ra. JAK1 associates with IL-7Ra, whereas JAK3 binds to gamma chain. Phosphorylated JAK1 and JAK3 then mediate the phosphorylation of receptor tyrosine residues. Phosphorylated tyrosine residues act as docking sites for the downstream Src homology 2 (SH2) domain containing signaling molecules such as Signal Transducer and Activator of Transcription (STAT) transcription factors, insulin receptor substrate (IRS), and the adaptors of the Mitogen-Activated Protein Kinase (MAPK) pathways. IL-7R signaling results in various properties that can be advantageous to engineered cells, including modulation of proliferation, differentiation, persistence, activation, effector function, cytolytic activity, cytokine production, and other properties, which can be harnessed in compositions, systems, and methods disclosed herein (e.g., without relying on the presence of a ligand or soluble ligand, such as IL-7 or TSLP).

[0103] An engineered immune signaling construct can include an intracellular signaling domain of the common gamma chain cytokine receptor subunit IL-15Ra and / or IL-15Rb. The native IL-15Ra subunit is characterized by four extracellular cysteines and an extracellular Sushi domain. IL-15Ra is found in both membrane bound and soluble forms. Native membrane bound IL-15Ra comprises a short cytoplasmic tail and does not directly initiate intracellular signaling cascades. Instead, upon IL- 15 binding, native membrane bound IL-15Ra forms a heterocomplex with IL-15Rb (also known as IL-2Rb) and / or common gamma chain, resulting in IL-15R signaling. The native IL-15Rb subunit is characterized by a WSXWS motif and an intracellular Box 1 motif. IL- 15 binds with high affinity to IL-15Ra, which then associates with a complex composed of the IL-2Rb / IL-15Rb and common gamma-chain subunits, expressed either on the same cell (cis-presentation) or on a different cell (trans-presentation). The IL-15R heterocomplex recruits Janus kinases (JAK) to mediate the phosphorylation of the receptor. Upon IL- 15 binding to the receptor, a conformational change occurs in the IL-15R heterocomplex, which allows JAK molecules to bind to the proline rich BOX1 motif in the membrane-proximal region of IL-15Rb. JAK1 associates with IL-15Rb, whereas JAK3 binds to gamma chain. Phosphorylated JAK1 and JAK3 then mediate the phosphorylation of receptor tyrosine residues. Phosphorylated tyrosine residues act as docking sites for the downstream Src homology 2 (SH2) domain containing signaling molecules such as Signal Transducer and Activator of Transcription (STAT) transcription factors, insulin receptor substrate (IRS), and the adaptors of the Mitogen- Activated Protein Kinase (MAPK) pathways. IL-15R signaling results in various properties that can be advantageous to engineered cells, including modulation ofproliferation, differentiation, persistence, activation, effector function, cytolytic activity, cytokine production, and other properties, which can be harnessed in compositions, systems, and methods disclosed herein (e g , without relying on the presence of a ligand or soluble ligand, such as IL-15).

[0104] An engineered immune signaling construct can include an intracellular signaling domain of the common gamma chain cytokine receptor subunit IL-21R (equivalent to alpha chain of other members of the gamma chain cytokine family). The native IL-21R subunit is characterized by four extracellular cysteines and the conserved WSXWS motif, while the intracellular domain contains a BOX1 consensus sequence and a serine rich region. IL-21 binding to native IL-21R results in the formation the IL-21R heterocomplex comprising the IL- 21R subunit and common gamma chain. The IL-21R heterocomplex recruits Janus kinases (JAK) to mediate the phosphorylation of the receptor. Upon IL-21 binding to the receptor, a conformational change occurs in the IL-21R heterocomplex, which allows JAK molecules to bind to the proline rich BOX1 motif in the membrane-proximal region of IL-21R. JAK1 associates with IL-21R, whereas JAK3 binds to gamma chain. Phosphorylated JAK1 and JAK3 then mediate the phosphorylation of receptor tyrosine residues. Phosphorylated tyrosine residues act as docking sites for the downstream Src homology 2 (SH2) domain containing signaling molecules such as Signal Transducer and Activator of Transcription (STAT) transcription factors, insulin receptor substrate (IRS), and the adaptors of the Mitogen- Activated Protein Kinase (MAPK) pathways. IL-21R signaling results in various properties that can be advantageous to engineered cells, including modulation of proliferation, differentiation, persistence, activation, effector function, cytolytic activity, cytokine production, and other properties, which can be harnessed in compositions, systems, and methods disclosed herein (e.g., without relying on the presence of a ligand or soluble ligand, such as IL-21).

[0105] In some embodiments, an engineered immune signaling construct can include an intracellular signaling domain of the common gamma chain receptor subunit IL-2Ra. The native IL-2Ra subunit is characterized by four extracellular cysteines and an extracellular Sushi domain. IL-2Ra is found in both membrane bound and soluble forms. Native membrane bound IL-2Ra comprises a short cytoplasmic tail and does not directly initiate intracellular signaling cascades. Instead, upon IL-2 binding, native membrane bound IL-2Ra forms a heterocomplex with IL-2Rb and / or common gamma chain. The IL-2R heterocomplex recruits Janus kinases (JAK) to mediate the phosphorylation of the receptor. Upon IL-2 binding to the receptor, a conformational change occurs in the IL-2R heterocomplex, which allows JAK molecules to bind to the proline rich BOX1 motif in the membrane-proximal region of IL-2Rb. JAK1 associates with IL-2Rb, whereas JAK3 binds to gamma chain. Phosphorylated JAK1 and JAK3 thenmediate the phosphorylation of receptor tyrosine residues. Phosphorylated tyrosine residues act as docking sites for the downstream Src homology 2 (SH2) domain containing signaling molecules such as Signal Transducer and Activator of Transcription (STAT) transcription factors, insulin receptor substrate (IRS), and the adaptors of the Mitogen-Activated Protein Kinase (MAPK) pathways. IL-2R signaling results in various properties that can be advantageous to engineered cells, including modulation of proliferation, differentiation, persistence, activation, effector function, cytolytic activity, cytokine production, and other properties, which can be harnessed in compositions, systems, and methods disclosed herein (e.g., without relying on the presence of a ligand or soluble ligand, such as IL-2).

[0106] In some embodiments, the intracellular region of an engineered immune signaling construct comprises an intracellular signaling domain or a functional fragment or derivative thereof of a common gamma chain family cytokine receptor subunit, for example, that comprises a BOX1 motif.

[0107] The intracellular region can comprise an IL-9 receptor alpha (IL-9Ra) intracellular signaling domain or a functional fragment or derivative thereof, for example, that comprises a JAK1 binding motif and / or a BOX1 motif.

[0108] The intracellular region can comprise an IL-2 receptor alpha (IL-2Ra) intracellular signaling domain or a functional fragment or derivative thereof.

[0109] The intracellular region can comprise an IL-2 receptor beta (IL-2Rb / IL- 15Rb) intracellular signaling domain or a functional fragment or derivative thereof, for example, that comprises a JAK1 binding motif and / or a BOX1 motif.

[0110] The intracellular region can comprise an IL-4 receptor alpha (IL-4Ra) intracellular signaling domain or a functional fragment or derivative thereof, for example, that comprises a JAK1 binding motif and / or a BOX1 motif.[OHl] The intracellular region can comprise an IL-7 receptor alpha (IL-7Ra) intracellular signaling domain or a functional fragment or derivative thereof, for example, that comprises a JAK1 binding motif and / or a BOX1 motif.

[0112] The intracellular region can comprise an IL-15 receptor alpha (IL-15Ra) intracellular signaling domain or a functional fragment or derivative thereof.

[0113] The intracellular region can comprise an IL-21 receptor (IL-21R) intracellular signaling domain or a functional fragment or derivative thereof, for example, that comprises a JAK1 binding motif and / or a BOX1 motif.

[0114] Illustrative intracellular signaling domains are provided in SEQ ID NOs: 105-115 and 533-540.D. Configuration

[0115] An engineered immune signaling construct disclosed herein can comprise, consist essentially of, or consist of, from N-to-C terminus, (i) an extracellular region, (ii) a transmembrane region, and / or (iii) an intracellular region. In some embodiments, an engineered immune signaling construct disclosed herein comprises, consists essentially of, or consists of, from N-to-C terminus, (i) an extracellular region, (ii) a transmembrane region, and (iii) an intracellular region. In some embodiments, an engineered immune signaling construct disclosed herein comprises, consists essentially of, or consists of, from C-to-N terminus, (i) an extracellular region, (ii) a transmembrane region, and (iii) an intracellular region. In some embodiments, an engineered immune signaling construct disclosed herein comprises, from N-to- C terminus, (i) an extracellular region, and (ii) an intracellular region. In some embodiments, an engineered immune signaling construct disclosed herein comprises, from C-to-N terminus, (i) an extracellular region, and (ii) an intracellular region.

[0116] In some embodiments, an engineered immune signaling construct comprises, consists essentially of, or consists of, from N-to-C terminus, or from C-to-N terminus, (i) an extracellular region that that comprises a common gamma chain-binding domain (e.g., a cytokine, an antigenbinding fragment of an antibody, an scFv, or gamma chain-binding fragment thereof), (ii) a transmembrane region (e.g., from an immune receptor or a gamma chain family cytokine receptor, such as IL-7Ra or IL-9Ra), and (iii) an intracellular region comprising a signaling domain (e.g., of a common gamma chain family cytokine receptor subunit, such as an IL-9 receptor alpha (IL-9Ra), IL-2Rb, IL-7Ra, IL-4Ra, or IL-21R intracellular signaling domain).

[0117] A protein or polypeptide (e.g., engineered immune signaling construct or exogenous antigen-recognition receptor) can comprise one or more linkers, for examplejoining two domains, such as an extracellular region or domain to a transmembrane domain, a first extracellular domain to a second extracellular domain, a transmembrane domain to an intracellular or cytoplasmic domain, or a first intracellular domain to a second intracellular domain.

[0118] A linker can be a chemical bond, for example, a covalent bond or a non-covalent bond. A linker as described herein can include a flexible or rigid linker. A linker can be a peptide.

[0119] A linker can be selected to achieve a desired functionality of the engineered immune signaling construct or exogenous antigen-recognition receptor. For example, various linkers can be tested to identify a configuration of one or more linkers that allow an engineered immune signaling construct of the disclosure to exhibit low background activity, for example, low induction of signaling in the absence of common gamma chain. Various linkers can be tested to identify a configuration of one or more linkers that allow an engineered immune signalingconstruct of the disclosure to exhibit an appropriate level of signaling induction in the presence of common gamma chain.

[0120] A linker can comprise a linker sequence, for example, a linker peptide sequence. The length a linker can be adjusted to allow for proper folding or to increase or decrease biological activity of the engineered immune signaling construct. A linker sequence can be, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, or about 70 amino acid residues in length. In some cases, a linker sequence can be, for example at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, or at least about 50 amino acids in length. In some cases, a linker sequence can be, for example, at most about 2, at most about 3, at most about 4, at most about 5, at most about 6, at most about 7, at most about 8, at most about 9, at most about 10, at most about 15, at most about 20, at most about 30, at most about 40, at most about 50, at most about 60, at most about 70, at most about 80, or at most about 100 amino acids in length. In some cases, a linker is 5-20 amino acids in length. In some cases, a linker is 10-20 amino acids in length.

[0121] A flexible linker can have a sequence containing glycine residues. The small size of the glycine residues can provide flexibility, and allow for mobility of the connected protein domains. The incorporation of serine or threonine can maintain the stability of the linker in aqueous conditions by forming hydrogen bonds with the water molecules, thereby reducing unfavorable interactions between the linker and protein moieties. In some cases flexible linkers can also contain additional amino acids, such as threonine and alanine, to maintain flexibility, and / or polar amino acids such as lysine and glutamine, to improve solubility.

[0122] A rigid linker can have, for example, an alpha helix-structure. An alpha-helical rigid linker can act as a spacer between protein domains. A rigid linker can have a proline-rich sequence, (XP)n, with X designating alanine, lysine, glutamine, or any amino acid, and n designating a number of repeats. The presence of proline in non-helical linkers can increase stiffness, and allow for effective separation of protein domains.

[0123] A linker can comprise a hinge region, for example an amino acid sequence derived from a hinge region of an antibody or immune receptor. In some embodiments, a linker comprises a hinge region from CD28, CD8a, IgGl, or IgG4.

[0124] Examples of linkers and domains thereof include, but are not limited to, those disclosed in SEQ ID NOs: 116-178, or repeats thereof, which can be used to link any portion (e.g., domain) of an engineered immune signaling construct disclosed herein to any other portion(e.g., domain) of an engineered immune signaling construct disclosed herein. A linker can comprise any one or more of SEQ ID NOs: 116-178, or repeats thereof, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of any one of SEQ ID NOs: 116-178.

[0125] In some embodiments, a linker comprises a combination of linkers, hinges, and / or spacers disclosed herein, for example, a combination of a short flexible linker and a rigid alpha helical linker, or a combination of a short flexible linker and a hinge region.

[0126] A protein or polypeptide disclosed herein can comprise an N-terminal methionine. A protein or polypeptide disclosed herein can lack an N-terminal methionine.

[0127] A protein or polypeptide disclosed herein can comprise a signal sequence. A protein or polypeptide disclosed herein can lack a signal sequence, for example a mature peptide can lack a signal peptide after processing and secretion or membrane insertion.

[0128] Polypeptides disclosed herein can comprise chemical modifications, such as glycosylation, fucosylation, sialylation, and / or pegylation.

[0129] A polynucleotide encoding a polypeptide of the disclosure can encode a signal peptide. In some cases, a polypeptide of the disclosure comprises a signal peptide. A signal peptide can be cleaved off during processing of the protein (e.g., secretion or membrane localization), thus in some cases a mature polypeptide disclosed herein does not contain a signal peptide.

[0130] A signal peptide at the N-terminus of a protein can be involved in transport of the protein to or through a membrane, transport to different a membranous cellular compartment, or secretion of the protein from the cell. A polynucleotide encoding a polypeptide of the disclosure can encode a signal peptide to facilitate secretion of the polypeptide. A signal peptide can be selected for its ability to facilitate ER processing and secretion of the polypeptide. Any suitable signal peptide can be used. A signal peptide can be about 10 to about 40 amino acids in length. In some cases, a signal peptide is at least about 10, 15, 16, 20, 21, 22, 25, or 30 amino acids in length, or more. In some cases, a signal peptide is at most about 15, 16, 20, 21, 22, 25, or 30 amino acids in length, or less. In some cases, a signal peptide is about 16-30 amino acids in length.

[0131] A polypeptide or domain (e.g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) disclosed herein can comprise, consist essentially of, or consist of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to any one of SEQ ID NOs: 1-540.

[0132] A polypeptide or domain (e.g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) disclosed herein can comprise, consist essentially of, or consist of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to at least 100 consecutive amino acids of any one of SEQ ID NOs: 1-540

[0133] A polypeptide or domain (e.g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) disclosed herein can comprise, consist essentially of, or consist of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to least 200 consecutive amino acids any one of SEQ ID NOs: 1-540

[0134] A polypeptide or domain (e.g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) disclosed herein can comprise, consist essentially of, or consist of an amino acid sequence with at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at leastabout 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, or about 100% sequence identity or sequence similarity to least 300 consecutive amino acids any one of SEQ ID NOs: 1-540

[0135] A polypeptide or domain (e.g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) disclosed herein can comprise, consist essentially of, or consist of an amino acid sequence with at most about 70%, at most about 71%, at most about 72%, at most about 73%, at most about 74%, at most about 75%, at most about 76%, at most about 77%, at most about 78%, at most about 79%, at most about 80%, at most about 81%, at most about 82%, at most about 83%, at most about 84%, at most about 85%, at most about 86%, at most about 87%, at most about 88%, at most about 89%, at most about 90%, at most about 91%, at most about 92%, at most about 93%, at most about 94%, at most about 95%, at most about 95.5%, at most about 96%, at most about 96.5%, at most about 97%, at most about 97.5%, at most about 98%, at most about 98.5%, at most about 99%, or at most about 99.5% sequence identity or sequence similarity to any one of SEQ ID NOs: 1-540.

[0136] In some embodiments, a polypeptide or domain (e.g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) comprises, consists essentially of, or consists of an amino acid sequence with about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, or about 99.5% or about 100% sequence identity or sequence similarity to any one of SEQ ID NOs: 1- 540

[0137] In some embodiments, the polypeptide or domain (e.g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 1-540.

[0138] In some embodiments, the polypeptide or domain (e g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) comprises an amino acid sequence with one or more insertions, deletions, and / or substitutions relative to any one of SEQ ID NOs: 1-540.

[0139] For example, the polypeptide or domain (e.g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) can comprise an amino acid sequence with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid insertions relative to any one of SEQ ID NOs: 1-540.

[0140] In some embodiments, the polypeptide or domain (e g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) comprises an amino acid sequence with at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid insertions relative to any one of SEQ ID NOs: 1-540.

[0141] In some embodiments, the polypeptide or domain (e g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid insertions relative to any one of SEQ ID NOs: 1-540.

[0142] The one or more insertions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The one or more insertions can be contiguous, noncontiguous, or a combination thereof.

[0143] In some embodiments, the polypeptide or domain (e g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) comprises an amino acid sequence with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid deletions relative to any one of SEQ ID NOs: 1-540.

[0144] In some embodiments, the polypeptide or domain (e g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) comprises an amino acid sequence with at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid deletions relative to any one of SEQ ID NOs: 1-540.

[0145] In some embodiments, the polypeptide or domain (e g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid deletions relative to any one of SEQ ID NOs: 1-540.

[0146] The one or more deletions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The one or more deletions can be contiguous, noncontiguous, or a combination thereof.

[0147] In some embodiments, the polypeptide or domain (e g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) comprises an amino acid sequence with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid substitutions relative to any one of SEQ ID NOs: 1-540.

[0148] In some embodiments, the polypeptide or domain (e g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) comprises an amino acid sequence with at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions relative to any one of SEQ ID NOs: 1-540.

[0149] In some embodiments, the polypeptide or domain (e g., an engineered immune signaling construct, extracellular region, common gamma chain-binding domain, transmembrane region, intracellular region, hinge, linker, spacer, or domain thereof) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid substitutions relative to any one of SEQ ID NOs: 1-540.

[0150] The one or more substitutions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The one or more substitutions can be contiguous, non-contiguous, or a combination thereofH. POLYNUCLEOTIDES AND VECTORS

[0151] Compositions and methods disclosed herein can comprise a polynucleotide, for example, that encodes an engineered immune signaling construct. In some embodiments, the compact size of engineered immune signaling constructs disclosed herein relative to alternative constructs designed to provide immunomodulatory (e.g., constitutive) signaling allows the construct to be efficiently packaged in a vector, for example, facilitating simpler delivery in a single vector together with an exogenous antigen recognition receptor (e.g., CAR).

[0152] A polynucleotide can comprise, for example, one or more expression regulatory regions (e.g., a promoter, enhancer, intron, and / or exon), one or more transgenes (e.g., encoding an engineered immune signaling construct), a polyadenylation signal, or a combination thereof.

[0153] In some embodiments, a polynucleotide comprises a first nucleotide sequence that encodes an engineered immune signaling construct, and a second nucleotide sequence that encodes an exogenous antigen-recognition receptor, for example, a chimeric antigen receptor (CAR), T cell receptor (TCR), single-chain TCR (scTCR), or single-chain T cell receptor variable fragment (scTv). In some embodiments, an engineered immune signaling construct and an exogenous antigen-recognition receptor are encoded by separate polypeptides.

[0154] A polynucleotide can be a substance whose molecules comprise or consist essentially of nucleotides linked in a chain. Non-limiting examples of the polynucleotide include a circular nucleic acid, a DNA, a single stranded DNA, a double stranded DNA, a genomic DNA, a plasmid, a nanoplasmid, a plasmid DNA, a viral DNA, a mini circle (e.g., lacking a bacterial origin of replication), and an RNA.

[0155] In some embodiments, a polynucleotide encodes two or more polypeptides linked by one or more 2A linkers or self-cleaving peptides, which can be processed into separate polypeptides co-translationally or after translation (e.g., P2A, T2A, F2A, E2A). Examples of 2A linkers that can be used are provided SEQ ID NOs: 179-194. Inclusion of a 2A linker can increase the likelihood that an appropriate ratio of components are produced (e.g., a 1 : 1, 1:2, 1 :3, 1 :4, or 1 :5 ratio of two components). In some cases, inclusion of a 2A linker can increase the likelihood that equal or close to equal levels of two components of the engineered immune signaling construct or exogenous antigen-recognition receptor are produced. For example, if a polynucleotide encodes a TCR alpha chain constant region and TCR beta chain constant region, inclusion of a 2A linker can increase the likelihood that equal or close to equal levels of a TCRalpha chain constant region and TCR beta chain are produced. In some embodiments an exogenous antigen-recognition receptor can comprise a TCR gamma chain constant region and TCR delta chain constant region, and inclusion of a 2A linker can increase the likelihood that equal or close to equal levels of or TCR gamma chain and TCR delta chain, are produced. In some cases, use of a 2A linker can allow for fewer components in a system for transgene expression and / or genome modification, e.g., inclusion of multiple components in one vector rather than separate vectors.

[0156] An expression construct or polynucleotide disclosed herein can be or can comprise DNA. An expression construct or polynucleotide disclosed herein can be or can comprise double stranded DNA. For example, an expression construct or polynucleotide disclosed herein can be or comprise a plasmid, such as a nanoplasmid. In some embodiments, an expression construct or polynucleotide disclosed herein is or comprises a minicircle, a midge, a MIP, or a doggy bone. In some embodiments, an expression construct or polynucleotide comprises an R6K origin of replication. In some embodiments an expression construct or polynucleotide lacks an origin of replication. An expression construct or polynucleotide disclosed herein can be or can comprise a circular polynucleotide. An expression construct or polynucleotide disclosed herein can be or can comprise a linear polynucleotide. An expression construct or polynucleotide disclosed herein can comprise one or more transgenes or open reading frames.

[0157] A polynucleotide can be or be present in an expression construct. A polynucleotide or expression construct can comprise a promoter, enhancer, or combination thereof that drive or upregulate expression of a transgene, for example, that encodes an engineered immune signaling construct or exogenous antigen-recognition receptor. The transgene can be operatively linked to and / or under regulatory control of the promoter or enhancer.

[0158] A promoter disclosed herein can be a mammalian promoter or derived from a mammalian promoter. A promoter disclosed herein can be a human promoter or derived from a human promoter. The promoter can be a promoter as found in a naturally-occurring genome. In some embodiments, a promoter is not found in a naturally-occurring genome. In some embodiments, the promoter is a synthetic or engineered promoter. The promoter can be a minimal promoter. A promoter can be a constitutive, viral, inducible, or tissue-specific promoter.

[0159] A promoter can be an immune-cell selective promoter, for example, a promoter that results in preferential expression in immune cells as compared to non-immune cells. An immune cell-selective promoter can result in preferential expression in, for example, lymphocytes, T cells, CD4+ T cells, CD8+ T cells, alpha-beta T cells, gamma-delta T cells, T regulatory cells (Tregs), cytotoxic T lymphocytes, Thl cells, Th2 cells, Thl7 cells, Th9 cells, naive T cells,memory T cells, effector T cells, effector-memory T cells (TEM), central memory T cells (TCM), resident memory T cells (TRM), follicular helper T cells (TFH), Natural killer T cells (NKTs), tumor-infiltrating lymphocytes (TILs), Natural killer cells (NKs), Innate Lymphoid Cells (ILCs), ILC1 cells, ILC2 cells, ILC3 cells, lymphoid tissue inducer (LTi) cells, B cells, Bl cells, Bia cells, Bib cells, B2 cells, plasma cells, B regulatory cells, memory B cells, marginal zone B cells, follicular B cells, germinal center B cells, antigen presenting cells (APCs), monocytes, macrophages, Ml macrophages, M2 macrophages, tissue-associated macrophages, dendritic cells, plasmacytoid dendritic cells, neutrophils, mast cells, basophils, eosinophils, common myeloid progenitors, common lymphoid progenitors, or any combination thereof. The expression can be preferential compared to control cells, such as fibroblasts, neurons, epithelial cells, keratinocytes, or hepatocytes, etc.

[0160] A promoter can be a T-cell selective promoter, for example, a promoter that results in preferential expression in T cells as compared to non-T cells. In some embodiments, a T cell- selective promoter can limit off-target effects, e.g., limit off target effects resulting from expression of an engineered immune signaling construct in non-T cells. Non-limiting examples of T-cell selective promoters include promoters that natively drive expression of CD3 (e.g., CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta), CD4, CD8, CD28, TCRB, or TRAC.

[0161] In some embodiments, a polynucleotide disclosed herein comprises RNA, for example, mRNA.

[0162] In some embodiments, an expression construct or polynucleotide disclosed herein is or comprises single stranded DNA. In some embodiments, an expression construct or polynucleotide disclosed herein comprises a component of a viral genome or a viral packaging element, for example, a 5' and / or 3' inverted terminal repeat (ITR). In some embodiments, an expression construct or polynucleotide disclosed herein is not single stranded DNA. In some embodiments, an expression construct or polynucleotide disclosed herein lacks a component of a viral genome or lacks a viral packaging element, for example, lacks a 5' and / or 3' inverted terminal repeat (ITR).

[0163] In some embodiments, an expression construct or polynucleotide disclosed herein is integrating, e.g., integrates into the genome of an engineered cell. In some embodiments, an expression construct or polynucleotide disclosed herein is non-integrating, e.g., does not integrate into the genome of an engineered cell.

[0164] A polynucleotide can include one or more homology arms, for example, comprising sequences that are complementary to a genomic DNA sequence to be targeted for insertion (e.g., via homologous recombination or homology directed repair). A polynucleotide can comprise one or more promoter regions, barcodes, restriction sites, cleavage sites, endonucleaserecognition sites, primer binding sites, selectable markers, unique identification sequences, resistance genes, linker sequences, or any combination thereof. In some aspects, these sites may be useful for enzymatic digestion, amplification, sequencing, targeted binding, purification, providing resistance properties (e.g., antibiotic resistance for selection), or any combination thereof. A polynucleotide may also include transcriptional or translational regulatory sequences, for example, one or more promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A linkers and / or polyadenylation signals.

[0165] A polynucleotide can be assembled by a variety of methods, e.g., by automated solidphase synthesis. A polynucleotide can be constructed using standard solid-phase DNA / RNA synthesis. A polynucleotide can also be constructed using a synthetic procedure. A polynucleotide can be synthesized manually or in a fully automated fashion. A polynucleotide can be a recombinant nucleic acid. In some cases, a synthetic procedure may comprise 5'- hydroxyl oligonucleotides that can be initially transformed into corresponding 5'-H-phosphonate mono esters, subsequently oxidized in the presence of imidazole to activated 5'- phosphorimidazolidates, and finally reacted with pyrophosphate on a solid support. This procedure may include a purification step after the synthesis such as PAGE, HPLC, MS, or any combination thereof. Polynucleotides can be purchased commercially.

[0166] In some embodiments, a polynucleotide or expression construct disclosed herein comprises natural, synthetic, and / or artificial nucleotide analogues or bases. In some embodiments, the synthetic or artificial nucleotide analogues or bases comprise modifications at one or more of a deoxyribose moiety, ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.

[0167] In some embodiments, a nucleotide analogue or artificial nucleotide base comprises a nucleic acid with a modification at a 2' hydroxyl group of the ribose moiety. In some instances, the modification includes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Illustrative alkyl moieties include, but are not limited to, halogens, sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols and oxygen. In some instances, the alkyl moiety further comprises a modification. In some instances, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, a nitrile group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some instances, the alkyl moiety further comprises a hetero substitution. In some instances, the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur. In some instances, the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino.

[0168] A polynucleotide, gene editing component, or other cargo can be delivered to a cell by any suitable method, for example, using any suitable vector.

[0169] In some embodiments a composition or method utilizes a vector comprising any of the polynucleotides described herein.

[0170] A vector can be or can comprise a viral vector, a gamma-retroviral vector, a lentiviral vector, an adeno-associated viral vector, a transposon, and the like. Any vector systems can be used including, but not limited to, DNA vectors, RNA vectors, ribonucleoprotein vectors, hybrid DNA-RNA vectors, plasmid vectors, minicircle vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors and adeno-associated virus vectors, etc. Non-viral vector delivery systems can include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems can include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. In some cases, one vector is used. In some cases, two vectors are used. In some cases, three or more vectors are used.

[0171] In some cases, the vector is a viral vector, such as a lentiviral vector, a y-retroviral vector, or an adeno-associated virus (AAV) vector. In some embodiments, the vector is a non- viral vector, for example, a plasmid, nanoplasmid, minicircle, a midge, a MIP, or a doggybone, a lipid-based nanoparticle, a liposome, a circular polynucleotide (e.g., DNA or RNA), a linear polynucleotide (e.g., a DNA or RNA), or a combination thereof.

[0172] In some cases, a polynucleotide, gene editing component, or other can be delivered to cells without the use of vectors. In some cases, one or more polynucleotides, gene editing components, or other cargos of the disclosure can be delivered to cells via vectors, and one or more polynucleotides, gene editing components, or other cargos can be delivered without the use of vectors.

[0173] In some embodiments, a vector is or comprises a poloxamer, nanoparticle, polyplex, or dendrimer.

[0174] A vector can be a nanoparticle, for example, an inorganic nanoparticle, such as a gold, silica, iron oxide, titanium, calcium phosphate, PLGA, poly(B-amino ester) (PBAE, e.g., PBAE- 447), or hydrogel nanoparticle. In some embodiments a vector is not a nanoparticle, e.g., is not an inorganic nanoparticle.

[0175] Nucleic acids can be encapsulated in particles through electrostatic association and physical entrapment. To prevent or slow the disassociation of cargo nucleic acids from nanoparticles following systemic administration, a polymerizable conjugate with a degradable, disulfide linkage can be employed. Nanoparticles can be encapsulated with a lipid coating to improve oral bioavailability, minimize enzymatic degradation and cross blood brain barrier. Thenanoparticle surface can also be PEGylated to improve water solubility, circulation in vivo, and stealth properties.

[0176] A vector can be a polyplex, for example, a complex of one or more polymers and nucleic acids. A polyplex can comprise cationic polymers. Fabrication of a polyplex can be based on self-assembly by ionic interactions. A polyplex can comprise polyethyleneimine, chitosan, poly(beta-amino esters), and / or polyphosphoramidate. In some embodiments a vector is not a polyplex.

[0177] A vector can be a dendrimer. A dendrimer can be a highly branched macromolecule with a spherical shape. The surface of dendrimer particles can be functionalized such as, for example, with positive surface charges (cationic dendrimers), which can be employed for the delivery of nucleic acids. Dendrimer-nucleic acid complexes are taken into a cell via endocytosis. In some embodiments a vector is not a dendrimer.

[0178] A vector disclosed herein can be a non-viral, lipid-based vector. A non-viral, lipid- based vector can be, for example, a liposome, a lipoplex, a lipid nanoparticle, a vesicle, or a micelle.HL CELLS

[0179] Also provided herein are cells (e.g., engineered cells) that comprise, encode, and / or are capable of expressing an engineered immune signaling construct disclosed herein. An engineered cell can comprise an engineered immune signaling construct disclosed herein or a polynucleotide encoding the engineered immune signaling construct, and can further comprise an exogenous antigen-recognition receptor or a polynucleotide encoding the exogenous antigenrecognition receptor.

[0180] The exogenous antigen-recognition receptor can allow the cell to respond to a target cell, for example, elicit an anti-cancer immune response against a cancer cell, and the engineered immune signaling construct can modulate properties of the engineered cell and its response.

[0181] A cell can be an immune cell or a precursor thereof. A cell can be a peripheral blood mononuclear cell (PBMC). A cell can be a lymphoid cell. A cell can be a lymphocyte. A cell can be a T cell. A cell can be a B cell. A cell can be a natural killer (NK) cell. A cell can be a Natural Killer T (NKT) cell. A cell can be a mammalian cell. A cell can be a human cell.

[0182] Non-limiting examples of cells that can be used include lymphocytes, T cells, CD4+ T cells, CD8+ T cells, alpha-beta T cells, gamma-delta T cells, T regulatory cells (Tregs), cytotoxic T lymphocytes, Thl cells, Th2 cells, Thl7 cells, Th9 cells, naive T cells, memory T cells, effector T cells, effector-memory T cells (TEM), central memory T cells (TCM), resident memory T cells (TRM), follicular helper T cells (TFH), tumor-infiltrating lymphocytes (TILs),Innate Lymphoid Cells (ILCs), ILC1 cells, ILC2 cells, ILC3 cells, lymphoid tissue inducer (LTi) cells, B cells, Bl cells, Bia cells, Bib cells, B2 cells, plasma cells, B regulatory cells, memory B cells, marginal zone B cells, follicular B cells, germinal center B cells, antigen presenting cells (APCs), monocytes, macrophages, Ml macrophages, M2 macrophages, tissue-associated macrophages, dendritic cells, plasmacytoid dendritic cells, neutrophils, mast cells, basophils, eosinophils, precursors thereof, and combinations thereof. In some cases, compositions, systems, and methods of the disclosure comprise T cells. In some cases, compositions, systems, and methods of the disclosure comprise primary human T cells (e.g., primary human T cells that are autologous or allogeneic with respect to a subject).

[0183] A cell can be an alpha beta T cell. A cell can be a gamma delta T cell. In some embodiments, a cell comprises a disruption or deletion of one or more TCR-encoding genes, such as TRAC, TRB, TRG, and / or TRD. In some embodiments, a cell comprises a disruption or deletion of a variable region of one or more TCR-encoding genes, such as a disruption or deletion in TRAC, TRB, TRG, and / or TRD.

[0184] A cell can be a primary cell. A cell can be an immortalized cell. A cell can be of a cell line. A cell can be differentiated from a stem cell, for example, an induced pluripotent stem cell (iPSC), embryonic stem cell, hematopoietic stem cell (HSC), or the like.

[0185] Cells can be engineered to comprise or be capable of expressing an engineered immune signaling construct. For example, cell engineering techniques disclosed herein and / or known to a skilled person can be used to modify cells to comprise a polynucleotide that encodes an engineered immune signaling construct of the disclosure, thereby generating engineered cells (such as engineered T cells).

[0186] Cells can be engineered to comprise or be capable of expressing an engineered immune signaling construct, an additional polypeptide disclosed herein (e.g., a CAR), and / or to reduce expression of an endogenous TCR gene. The methods can comprise contacting a cell with a polynucleotide, or with a vector that comprises the polynucleotide, under conditions that permit uptake of the polynucleotide by the cell. A polynucleotide can comprise a nucleotide sequence that encodes an engineered immune signaling construct disclosed herein or a component thereof. In some cases, a polynucleotide is utilized to alter a genome of a cell.

[0187] An engineered cell can be generated by a method that comprises contacting a cell with a polynucleotide or vector disclosed herein.

[0188] For targeted integration, a polynucleotide sequence to be inserted can be flanked by homology arms comprising sequences that are complementary to a genomic DNA sequence to be targeted for insertion (e.g., via homologous recombination and / or homology-directed repair, HDR). A double stranded break can be introduced at a target site in the genome, and thehomology arms can promote insertion of the polynucleotide. In some cases, a polynucleotide can be excised from a vector, such as a nanoplasmid (e.g., via a nuclease), and inserted into the genome of the cell.

[0189] A polynucleotide can be inserted in a safe harbor locus. A safe harbor can comprise a genomic location where a polynucleotide can integrate and function without substantially perturbing endogenous activity, for example, with a relatively low impact on local or global gene expression. For example, one or more polynucleotides can be inserted into any one of HPRT, an AAVS site (E.G., AAVS1, AAVS2, etc.), CCR5, hROSA26, and / or any combination thereof. A polynucleotide can be inserted in an intergenic region. A polynucleotide can be inserted in a non-coding region. A polynucleotide can be inserted within a gene. In some cases, a polynucleotide can disrupt a gene it is inserted into (e.g., reduce or eliminate expression of the disrupted gene). A disrupted gene can be for example, an endogenous TCR gene (e.g., TRAC, TCRB, TCRBC1, TRBC2, TRG, TRD), or an immune checkpoint gene (e.g., PD-1, CTLA-4). A polynucleotide can be inserted adjacent to or near to a promoter.

[0190] In some cases, one or more polynucleotides of the disclosure can be inserted randomly into the genome of a cell. For instance, a polynucleotide can encode its own promoter or can be inserted into a position where it is under the control of an endogenous promoter. Alternatively or additionally, a polynucleotide can be inserted into a gene, such as an intron of a gene, an exon of a gene, a promoter, or a non-coding region.

[0191] A variety of enzymes can catalyze generation of a double- stranded break in the genome and / or insertion of foreign DNA into a host genome. Non-limiting examples of gene editing tools and techniques include CRISPR systems, CRISPR-associated polypeptide (Cas), TALEN, zinc finger nuclease (ZFN), zinc finger associate gene regulation polypeptide, meganuclease, Mega-TAL, transposon-based systems, natural master transcription factors, epigenetic modifying enzymes, recombinase, flippase, transposase, RNA-binding proteins (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

[0192] A CRISPR system can be utilized to facilitate insertion of a polynucleotide encoding an engineered immune signaling construct or a component thereof into a cell genome. For example, a CRISPR system can introduce a double stranded break at a target site in a genome or a random site of a genome.

[0193] In some cases, a CRISPR system comprises CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated(Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, or type VI CRISPR- associated (Cas) polypeptides a derivative, variant, or functional fragment thereof

[0194] In some embodiments, a CRISPR system comprises a Class I system or endonuclease (e.g., Type I, Type III or Type IV Cas proteins). A class I system can be of the I-A, I-B, I-C, I- U, I-D, I-E, I-F, IV-A, IV-B, III-A, III-D, III-C, or III-B subtype.

[0195] In some embodiments, a CRISPR system comprises a Class II system or endonuclease (e.g., Type II, Type V, or Type VI). A class II, Type II system can be of the II- A, II-B, II-C1, or II-C2 subtype. A class II, Type V systems can of the V-A, V-Bl, V-B2, V-C, V-D, V-E, V-Fl, V-F1(V-U3), V-F2, V-F3, V-G, V-H, V-I, V-K (V-U5), V-Ul, V-U2, or V-U4 subtype. A Class II, Type IV systems can be of the: VI- A, VI-B1, VI-B2, VI-C, or VI-D subtype.

[0196] In some embodiments, a Cas protein used in a method disclosed herein is a class II endonuclease. In some embodiments, a Cas protein used in a method disclosed herein is a class II, type V Cas endonuclease. In some embodiments, a Cas protein used in a method disclosed herein is a class II, type V-A Cas endonuclease.

[0197] Non-limiting examples of Cas proteins that can be used in the CRISPR systems include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Cpfl, c2cl, c2c3, Cas9HiFi, homologues thereof, and modified versions thereof. An unmodified CRISPR enzyme can have DNA cleavage activity, such as Cas9. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and / or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. A Cas protein can be a high-fidelity Cas protein. Alternatives to S. pyogenes Cas9 may include RNA-guided endonucleases from the Cpfl family that display cleavage activity in mammalian cells.

[0198] In some embodiments, a gene editing system comprises a Cas protein, and the system further comprises a guide RNA (gRNA) which complexes with the Cas protein. In some embodiments, the gene editing moiety comprises an RNA-binding protein (RBP) complexed with a gRNA which is able to form a complex with a Cas protein.

[0199] In some cases, a dual nickase approach may be used to introduce a double stranded break. Cas proteins can be mutated at certain amino acids within either nuclease domains, thereby deleting activity of one nuclease domain and generating a nickase Cas protein capable of generating a single strand break. A nickase along with two distinct guide RNAs targetingopposite strands may be utilized to generate a DSB within a target site (often referred to as a “double nick” or “dual nickase” CRISPR system).

[0200] A transposon-based system can be utilized for insertion of a polynucleotide encoding an engineered immune signaling construct of the disclosure or a component thereof into a genome. A transposon can comprise a polynucleotide that can be inserted into a DNA sequence. A class I transposon can be transcribed into an RNA intermediate, then reverse transcribed and inserted into a DNA sequence. A class II transposon can comprise a DNA sequence that is excised from one DNA sequence and / or inserted into another DNA sequence. A class II transposon system can comprise (i) a transposon vector that contains a sequence (e.g., comprising a transgene) flanked by inverted terminal repeats, and (ii) a source for the transposase enzyme. A transposon system (e.g., class II transposon system) can direct the integration of a polynucleotide sequence encoding an engineered immune signaling constmct or a component thereof, while leaving behind the rest of the vector. A transposon and a transposase can be introduced into a cell. In some cases, a vector that encodes a transposase and comprises a polynucleotide is introduced into a cell, and the transposase is expressed and mediates insertion of the transposon into the genome.

[0201] Examples of transposon-based systems that can be used include, but are not limited to, sleeping beauty (e.g., derived from the genome of salmonid fish); piggyback (e.g., derived from lepidopteran cells and / or the Myotis lucifugus); mariner (e.g., derived from Drosophila); frog prince (e.g., derived from Rana pipiens); Tol2 (e.g., derived from medaka fish); and spinON.

[0202] In some embodiments an engineered immune signaling construct or other polypeptide can be expressed in an engineered cell without genomic integration of a polynucleotide that encodes the engineered immune signaling construct or other polypeptide. For example, an engineered immune signaling construct or other polypeptide can be expressed from an episomal vector, such as a DNA, RNA, circular DNA, circular RNA, minicircle, or the like. An engineered immune signaling construct or other polypeptide can be transiently expressed. For example, expression of an engineered immune signaling construct or other polypeptide can be reduced as a nucleic acid that encodes it is degraded. One method of generating engineered cells is through the use of a ribonucleic acid (RNA) system, e.g., a system that involves delivering one or more polynucleotides as an RNA. In some cases, the use of RNA can minimize DNA- induced toxicity and immunogenicity sometimes observed with the use of DNA.

[0203] Cells can be genetically engineered to comprise a polynucleotide that encodes an engineered immune signaling construct ex vivo. For example, cells can be taken from a subject in one or more blood draws and / or apheresis procedures, modified ex vivo, optionally selectedand / or expanded before and / or after genetic modification, and optionally re-introduced into the subject or a different subject by infusion or injection.

[0204] In some cases, cells are genetically engineered to comprise an engineered immune signaling construct of the disclosure in vivo. For example, a vector can be used to deliver gene editing components to cells in a subject without removing the cells from the subject. Vectors can be delivered in vivo by administration to an individual subject, for example, by parenteral administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application.

[0205] Methods to introduce gene editing components into a cell include, but are not limited to, electroporation, sonoporation, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viral vectors including adenoviral, AAV, and retroviral vectors.

[0206] Electroporation using, for example, the Neon® Transfection System (ThermoFisher Scientific), the Xenon Electroporation System (ThermoFisher Scientific), or the AMAXA® Nucleofector (AMAXA® Biosystems) can also be used for delivery of nucleic acids into a cell. Electroporation parameters may be adjusted to optimize transfection efficiency and / or cell viability. Electroporation devices can have multiple electrical wave form pulse settings such as exponential decay, time constant and square wave. Every cell type has a unique optimal Field Strength (E) that is dependent on the pulse parameters applied (e.g., voltage, capacitance and resistance). Application of optimal field strength causes electropermeabilization through induction of transmembrane voltage, which allows nucleic acids to pass through the cell membrane. In some cases, the electroporation pulse voltage, the electroporation pulse width, number of pulses, cell density, and tip type may be adjusted to optimize transfection efficiency and / or cell viability.

[0207] Cells can be selected or enriched for having or not having one or more given factors (e.g., cells may be separated based on the presence or absence of one or more factors). Selection techniques include positive selection and negative selection techniques, e.g., fluorescent activated cell sorting (FACS) or magnetic activated cell sorting (MACS). In some cases, cells can be selected before gene editing, for example, to enrich for a population of cells disclosed herein (e.g., immune cells, such as T cells or a T cell subset disclosed herein, such as gamma delta T cells or alpha beta T cells). Cells can be selected after gene editing, for example, to enrich for a population of cells disclosed herein (e.g., engineered cells that express an engineered immune signaling construct or additional polypeptide). Engineered cells can be selected or enriched based on a tag or marker, such as an epitope tag. The tag or marker can be appended to the engineered immune signaling construct. In some embodiments, the tag ormarker is not appended to the engineered immune signaling construct. The tag or marker can be co-expressed with the engineered immune signaling construct as disclosed herein. The tag or marker can comprise a reporter gene, such as a fluorescent protein.

[0208] Cells can be selected, enriched, or expanded on the basis of being positive or negative for a given factor. In some embodiments, cells are selected, enriched, or expanded on the basis of being positive for two or more factors. In some embodiments, cells can be selected, enriched, or expanded on the basis of being positive for one or more factors, and negative for one or more factors.

[0209] In some cases, a selectable marker is introduced to a cell, e.g., together with or as part of a polynucleotide encoding an engineered immune signaling construct, so that cells that comprise the engineered immune signaling construct or modification express the selectable marker and can be selected, enriched, or expanded. In some cases, a selectable marker is an antibiotic resistance gene, and cells that do not express the antibiotic resistance gene can be killed by treatment with the antibiotic (e.g., to select or enrich for cells that comprise an engineered immune signaling construct). In some embodiments, the selectable marker is an epitope tag. In some embodiments the selectable marker is a DHFR resistance gene

[0210] Expression of an engineered immune signaling construct can be quantified, for example, by qPCR, RNA sequencing, western blot, or flow cytometry.

[0211] In some embodiments, selected cells can be expanded ex vivo and / or in vitro before gene editing or delivery of a polynucleotide, after gene editing or delivery of a polynucleotide, before selection, after selection, before expansion, after expansion, or a combination thereof. In some embodiments, selected cells can be expanded ex vivo and / or in vitro before gene editing or delivery of a polynucleotide. In some embodiments, selected cells can be expanded ex vivo and / or in vitro after gene editing or delivery of a polynucleotide. In some embodiments, selected cells can be expanded ex vivo and / or in vitro before selection and / or enrichment. In some embodiments, selected cells can be expanded ex vivo and / or in vitro after selection and / or enrichment. In some embodiments, selected cells can be expanded ex vivo and / or in vitro before expansion. In some embodiments, selected cells can be expanded ex vivo and / or in vitro after expansion.

[0212] Cells of the disclosure can be cryopreserved, e.g., frozen in the presence of a cryopreservative such as DMSO, and stored at a low temperature (e g., at -80°C or in liquid nitrogen). Cells of the disclosure can be cryopreserved before gene editing, after gene editing, before selection, after selection, before expansion, after expansion, or a combination thereof.E. Exogenous antigen-recognition receptor

[0213] A cell (e.g., engineered cell) can comprise an engineered immune signaling construct disclosed herein or a polynucleotide encoding the engineered immune signaling construct, and can further comprise an exogenous antigen-recognition receptor or a polynucleotide encoding the exogenous antigen-recognition receptor.

[0214] An exogenous antigen-recognition receptor can be or can comprise a chimeric antigen receptor (CAR). An exogenous antigen-recognition receptor can be or can comprise a T cell receptor (TCR). An exogenous antigen-recognition receptor can be or can comprise a singlechain TCR (scTCR) or single-chain T cell receptor variable fragment (scTv). An exogenous antigen-recognition receptor can comprise an extracellular domain (including an extracellular binding domain), a transmembrane domain, and a cytoplasmic signaling domain.

[0215] An exogenous antigen-recognition receptor can be expressed by a cell (e.g., immune cell) and configured to induce activation of and / or signaling in the cell upon contacting a target cell that expresses or presents a cell surface molecule. A target cell can be a cell that is associated with a disease or condition. A target cell can be a cancer cell. A target cell can be an immune cell. A target cell can be a hematologic cancer cell. A target cell can be a solid tumor cell. A target cell can be a leukemia cell. A target cell can be a lymphoma cell. A target cell can be a myeloma cell. A target cell can be a B cell. A target cell can be a CD19+ cell. A target cell can be a T cell. A target cell can be a cell that is associated with an autoimmune or inflammatory disease. A target cell can be a fibrotic cell, e.g., a fibroblast.

[0216] In some embodiments, an exogenous antigen-recognition receptor is a chimeric antigen receptor (CAR). In some embodiments, an exogenous antigen-recognition receptor is a first, second, third, fourth, or fifth generation CAR. A first generation CAR can contain a single CD3 zeta cytoplasmic signaling domain (e.g., and lack a co-stimulatory cytoplasmic signaling domain). A second generation CAR can comprise a CD3 zeta cytoplasmic signaling domain and a co-stimulatory cytoplasmic signaling domain, such as a CD28 or 4 IBB costimulatory domain. A third generation CAR can comprise a CD3 zeta cytoplasmic signaling domain and two co- stimulatory cytoplasmic signaling domains, for example, two of CD28, 41BB, and 0X40. A fourth generation CAR can comprise a CD3 zeta cytoplasmic signaling domain and a co- stimulatory cytoplasmic signaling domain (such as a CD28 or 4 IBB costimulatory domain), and a protein, such as interleukin 12 (IL-12), that is constitutively or inducibly expressed upon CAR activation. A fourth generation CAR can be, for example, a T cell redirected for universal cytokine-mediated killing (TRUCK). A fifth generation CAR can be based on a second generation CAR and contain a truncated cytoplasmic IL-2 receptor -chain domain with a binding site for the transcription factor STAT3.

[0217] In some embodiments, an exogenous antigen-recognition receptor is a universal CAR, for example, an extracellular binding domain can be combined with amino acid sequence(s) from one or more components of a TCR signaling complex and / or a chimeric antigen receptor (CAR) to generate a “universal” exogenous antigen-recognition receptor that can be armed and disarmed based on the presence of adapter molecule(s). An adapter molecule can direct an immune cell expressing the exogenous antigen-recognition receptor to a target cell (e.g., a cancer cell), and upregulate activation of the immune cell upon encountering the target cell (e g., leading to a cytotoxic response against the target cell). A universal CAR can be capable of binding to various adapter molecules that can confer target specificity. Adapter molecules can comprise small molecules, binding fragments of a receptor or receptor ligand, small molecules, antibodies or antigen-binding fragments thereof, or a combination thereof.

[0218] In some embodiments, an exogenous antigen-recognition receptor is a dual CAR, a split CAR, or an inducible split CAR. A dual CAR can comprise two CARs with different extracellular binding domains, and thus signal induction based on two target antigens. A split CAR can comprise two CARs with different extracellular binding domains and separation of costimulatory domains (e.g., CD28 and 4 IBB) from CD3zeta on the distinct CAR polypeptides, thereby requiring engagement of both CARs for T cell activation.

[0219] An exogenous antigen-recognition receptor can comprise a component of a TCR signaling complex, for example, an extracellular domain, transmembrane domain, and / or cytoplasmic domain of a TCR signaling complex, such as a human TCR signaling complex.

[0220] In some embodiments, an exogenous antigen-recognition receptor that comprises a component of a TCR signaling complex comprises two TCR chains (e.g., an alpha chain and a beta chain, or a gamma chain and a delta chain).

[0221] In some embodiments, an exogenous antigen-recognition receptor that comprises a component of a TCR signaling complex is a TCR, e.g., comprises an extracellular binding domain that comprises TCR variable regions and TCR CDRs. In some embodiments, an exogenous antigen-recognition receptor that comprises a component of a TCR signaling complex is not TCR, for example, comprises a non-TCR extracellular binding domain, and comprises a component of a TCR signaling complex.

[0222] An exogenous antigen-recognition receptor disclosed herein can comprise an extracellular domain. The extracellular domain can comprise an extracellular binding domain that can specifically bind to a cell surface molecule on a target cell, thereby modulating signaling by the exogenous antigen-recognition receptor.

[0223] In some embodiments, an extracellular binding domain of an exogenous antigenrecognition receptor binds to CD19, ACE2, an Fc domain, APRIL, BAFFR, B7H6, B7H3,BCMA, CA9, CAIX, carcinoembryonic antigen, CD 133, CD 16, CD 174, CD22, CD23, CD27, CD274, CD276, CD33, CD38, CD44, CD5, CD70, CEACAM5, CSPG4, CTLX, DNAM-1, Dsg3, EBY IL13, E3 adnectin, EGFR, EGFRvIII, Envs, EPCAM, EPHA2, EPHB4, EPHRIN B2, ErbB, ERBB2, FAP, fibroblast activation protein, FLT3, FLT3L, F0LH1, F0LR1, FSH, FSHR, GD2, glycoprotein B, glycoprotein E2, GMCSF, GMR, gpl20, gp41, GPC3, GPNMB, HBsAg, HER2, ICAM-I, IL10, IL10R, IL11, ILl lRa, IL13Ra2, IL1RAP, IL3RA, Insulin-B chain, Islet-specific glucose-6-phosphatase catalytic subunit-related protein, KDR, L1CAM, LFA-1, M2e, mesothelin, MET, MICA, MICB, MPL, MS4A1, MSLN, MUC1, myelin oligodendrocyte glycoprotein, NCAM1, Nectin-2, NKG2D, NKp30, PDCD1, PSCA, PSMA, PVR, R0R1, SARS-CoV2 S protein, SDC1, SLAMF7, SSTR, TIE, TACI, TEM1, TNFRSF17, TNFRSF8, TPO, transmembrane form of IgE, TriPRIL, ULBP1, ULBP1-6, ULBP2, or VEGFR2. In some embodiments, an extracellular binding domain of an exogenous antigenrecognition receptor binds to CD19. In some embodiments, the extracellular binding domain of an exogenous antigen-recognition receptor comprises an FMC63 scFv. In some embodiments, an extracellular binding domain of an exogenous antigen-recognition receptor binds to a surface molecule on a T cell, for example, a human T cell.

[0224] In some embodiments, an extracellular binding domain of an exogenous antigenrecognition receptor is or comprises a component of a receptor or a receptor ligand, for example, utilizes the naturally occurring specificity of a receptor or ligand. For example, an extracellular binding domain of an exogenous antigen-recognition receptor can comprise a receptor-binding domain or ligand-binding domain of B7H6, an Fc domain, APRIL, BCMA, CD16, CD27, CD70, CTLX, DNAM-1, EI3Y IL13, E3 adnectin, EGFR, EPHB4, EPHRIN B2, ErbBl, ErbB2, ErbB3, ErbB4, FLT3, FLT3L, FSH, FSHR, GMCSF, GMR, ICAM-I, IL10, IL10R, IL11, ILl lRa, IL13Ra2, LFA-1, MICA, MICB, MPL, Nectin-2, or NKG2D.

[0225] In some embodiments, an extracellular binding domain of an exogenous antigenrecognition receptor is or comprises an autoantigen targeted by immune cells in an autoimmune disorder, or an epitope thereof. For example, an extracellular binding domain can comprise an autoantibody target, such as DSG3, factor VIII (FVIII), or an epitope thereof. The exogenous antigen-recognition receptor can be, for example, a chimeric autoantibody receptor (CAAR).

[0226] In some embodiments, an extracellular binding domain of an exogenous antigenrecognition receptor binds to an autoimmunity-associated target, for example, muscle-associated receptor tyrosine kinase (MuSK), insulin peptide-major histocompatibility complex (MHC) class II complex or insulin.

[0227] An extracellular binding domain of an exogenous antigen-recognition receptor can utilize one or more antigen-binding domains, for example, an antigen-binding domain of orderived from an antibody. In some embodiments, an extracellular binding domain of an exogenous antigen-recognition receptor disclosed herein comprises an antigen-binding domain or fragment from an antibody, such as an scFv or a nanobody.

[0228] An extracellular binding domain of an exogenous antigen-recognition receptor of the disclosure can comprise complementarity determining regions (CDRs). For example, an antibody, antigen-binding fragment thereof, or antigen-binding domain can comprise CDRs. In some embodiments, the CDRs determine or substantially determine binding specificity and / or affinity for a surface molecule on a target cell. For example, the CDRs can be grafted onto a different suitable framework, or the framework region can be altered (e.g., via amino acid substitutions, deletions, and / or insertions), and the antigen-binding fragment or domain can retain binding for the target, and the extracellular binding domain remains functional despite the alterations outside of the CDRs. In some embodiments, one or more framework regions or amino acid sequences therein contribute to binding specificity and / or affinity.

[0229] CDRs (e.g., in or for use in an extracellular binding domain of an exogenous antigen recognition receptor) can be identified by various methods, including but not limited to the Kabat method, the Chothia method, the IMGT method, the AHO method, and the Paratome method.

[0230] An extracellular binding domain of an exogenous antigen-recognition receptor can comprise an antibody fragment, antigen-binding domain, or antigen-binding fragment of an antibody. Non-limiting examples of antibody fragments, antigen-binding fragments, and antigen-binding domains include Fab, Fab', F(ab')2, dimers and trimers of Fab conjugates, Fv, scFv, nanobodies, minibodies, dia-, tria-, and tetrabodies, and linear antibodies.

[0231] The extracellular binding domain of an exogenous antigen-recognition receptor can be or can comprise a single domain antibody. The single domain antibody can be or can comprise a variable region of a heavy chain only antibody. Such a single domain antibody can also be known as a nanobody or VHH. The single domain antibody can be, for example, a variable region from or derived from a heavy chain only antibody from a camelid or other animal disclosed herein. An extracellular binding domain of an exogenous antigen-recognition receptor can comprise an antigen-binding domain or fragment of a chimeric, humanized, or fully human antibody.

[0232] In addition to antibodies and antigen-binding fragments or domains thereof, other compounds can also comprise antigen-binding domains that can be used in compositions, systems, and methods of the disclosure, such as in an extracellular binding domain of an exogenous antigen-recognition receptor. Non-limiting examples of non-antibody antigenbinding compounds include ankyrin proteins, ankyrin repeat proteins, designed ankyrin repeatproteins (DARPins), affibodies, avimers, adnectins, anticalins, Fynomers, Kunitz domains, knottins, -hairpin mimetics, and receptors and derivatives thereof.

[0233] The exogenous antigen-recognition receptor can comprise one or more additional extracellular domains as well as the extracellular binding domain.

[0234] In some embodiments, an exogenous antigen-recognition receptor comprises an additional extracellular domain or amino acid sequence that is a linker or spacer. In some embodiments, an exogenous antigen-recognition receptor comprises a hinge, such as an IgG hinge or a CD8 hinge.

[0235] An exogenous antigen-recognition receptor can comprise a transmembrane domain. Any suitable transmembrane domain can be used. In some embodiments, the exogenous antigenrecognition receptor comprises a transmembrane domain of CD8. In some embodiments, the exogenous antigen-recognition receptor comprises a transmembrane domain of CD28. The transmembrane domain can be a transmembrane domain of an immune receptor or TCR signaling complex component disclosed herein, for example, of a mammalian or a human TCR signaling complex. The transmembrane domain can comprise, for example, a transmembrane domain of TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta. In some embodiments, a transmembrane domain is not from an immune receptor or is not from a TCR signaling complex component.

[0236] An exogenous antigen-recognition receptor can comprise a cytoplasmic domain or a mutant, variant, or derivative thereof. A cytoplasmic domain can comprise a cytoplasmic signaling domain or a mutant, variant, or derivative thereof. The cytoplasmic signaling domain can contribute to the ability of the exogenous antigen-recognition receptor to elicit a response. For example, the cytoplasmic signaling domain can contribute to induction of signaling and / or immune cell activation upon of binding of the exogenous antigen-recognition receptor (e.g., an extracellular binding domain thereof) to a surface molecule of a target cell. In some cases, the cytoplasmic signaling domain can contribute to the induction of a pro-inflammatory response, an anti-cancer immune response, TCR signaling, T cell activation, T cell proliferation, cytokine production, a cytotoxic response against the target cell, or a combination thereof. A cytoplasmic signaling domain of an exogenous antigen-recognition receptor can enhance the proliferation, survival, and / or function of an engineered cell, and / or development of effector and / or memory immune responses (e.g., memory T cells). In some embodiments, signaling (e.g., IL-2R, IL-4R, IL-7R, IL-9R, IL-15R, IL-21R, or TSLPR signaling) by an engineered immune signaling construct augments that of the exogenous antigen recognition receptor to further enhance functional properties of the cell, as disclosed herein.

[0237] A cytoplasmic signaling domain of an exogenous antigen-recognition receptor can partake in an immune cell activation pathway that involves, for example, phosphorylation, dephosphorylation, calcium release, ubiquitination, de-ubiquitination, proteolytic cleavage, protein-protein interactions, a transcriptional response, or a combination thereof. An immune cell activation pathway can comprise, for example, an innate, adaptive, STING, NFkB, inflammasome, TCR, BCR, JAK / STAT, TLR, NLR, RLR, co-stimulatory, co-inhibitory, cytokine, or chemokine signaling pathway. A cytoplasmic signaling domain of an exogenous antigen-recognition receptor can comprise one or more immunoreceptor tyrosine-based activation motifs (IT AMs).

[0238] In some embodiments, the exogenous antigen-recognition receptor contains a cytoplasmic signaling domain of CD3 zeta or a functional fragment thereof. In some embodiments, the exogenous antigen-recognition receptor contains a cytoplasmic signaling domain of CD3 zeta with 1, 2, or 3 functional or active IT AMs. In some embodiments, the exogenous antigen-recognition receptor contains a cytoplasmic signaling domain of CD3 zeta with one inactivated IT AM or two inactivated IT AMs. In some embodiments, the exogenous antigen-recognition receptor does not contain a cytoplasmic signaling domain of CD3 zeta or a functional fragment thereof.

[0239] An exogenous antigen-recognition receptor can comprise a cytoplasmic signaling domain of a T cell signal two costimulatory signaling domain, or a functional fragment thereof. In some embodiments, an exogenous antigen-recognition receptor does not contain a cytoplasmic signaling domain of a T cell signal two costimulatory signaling domain.

[0240] A cytoplasmic domain or cytoplasmic signaling domain of an exogenous antigenrecognition receptor can be derived from and / or interact with a kinase, (e.g., a protein kinase, a tyrosine kinase or a serine / threonine kinase, a receptor tyrosine kinase, a lipid kinase, a phosphoinositide kinase, a carbohydrate kinase, or a combination thereof), a phosphatase, a ubiquitin ligase, a caspase, an adapter protein, a transcription factor, an ion channel, or a combination thereof. A cytoplasmic domain or cytoplasmic signaling domain of an exogenous antigen-recognition receptor can contribute to interaction of the exogenous antigen-recognition receptor with additional proteins or factors (e.g., members of a complex and / or signal transduction pathway).

[0241] An exogenous antigen-recognition receptor can comprise a cytoplasmic signaling domain of a costimulatory immune receptor, or a functional fragment thereof. Non-limiting examples of costimulatory immune receptors include CD28, 2B4 (CD244, SLAMF4), 4- IBB (CD137), CD2 (LFA2, 0X34), CD21, CD226 (DNAM1), CD27 (TNFRSF7), CD30 (TNFRSF8), CD4, CD40, CD8, CD84 (SLAMF5), CRACC (CD319, BLAME), CRTAM(CD355), DcR3, DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS (CD278), LIGHT, LT R (TNFRSF3), Lyl08 (NTBA, CD352, SLAMF6), Ly9 (CD229,SLAMF3), 0X40 (CD 134), SLAM (CD 150, SLAMF1), TIM1 (HAVCR1, KIMI), and TIM2. In some embodiments, an exogenous antigen-recognition receptor does not contain a cytoplasmic signaling domain of a costimulatory immune receptor.

[0242] An exogenous antigen-recognition receptor can comprise a cytoplasmic signaling domain of an activating NK receptor, or a functional fragment thereof. An exogenous antigenrecognition receptor can comprise a cytoplasmic signaling domain of a tumor necrosis factor receptor superfamily member, or a functional fragment thereof. An exogenous antigenrecognition receptor can comprise a cytoplasmic signaling domain of an immunoglobulin superfamily member, or a functional fragment thereof.

[0243] An exogenous antigen-recognition receptor can comprise a cytoplasmic signaling domain of an Fey receptor (FcyR), an Fee receptor (FceR), an Fea receptor (FcaR), an Fcp receptor (FcpR), neonatal Fc receptor (FcRn), CD4, CD5, CD8, CD21, CD22, CD27, CD28, CD32, CD40, CD45, CD66d, CD79a, CD79b, CD80, CD86, CD278 (ICOS), CD247< CD247r], 41BB, DAP10, DAP12, FYN, LAT, Lek, MAPK, MHC complex, NF AT, NF-KB, PLC-y, iC3b, C3dg, C3d, Zap70, MyD88, a functional fragment thereof, or a combination thereof.

[0244] An exogenous antigen-recognition receptor can comprise a cytoplasmic signaling domain that is a component of a TCR signaling complex, for example, of a mammalian or a human TCR signaling complex. The cytoplasmic signaling domain of an exogenous antigenrecognition receptor can comprise, for example, a cytoplasmic signaling domain of CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, a functional fragment thereof, or a combination thereof.

[0245] In some cases, an exogenous antigen-recognition receptor of the disclosure does not contain a cytoplasmic signaling domain, but can nonetheless elicit an immune cell activation signal, for example, via a cytoplasmic signaling domain in another protein that can associate with the exogenous antigen-recognition receptor. In some embodiments, an exogenous antigenrecognition receptor of the disclosure that comprises constant regions from one or more TCR chains can transmit an immune cell activation signal via associated CD3 proteins that comprise cytoplasmic signaling domains (e.g., CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 eta, or a combination thereof).

[0246] An exogenous antigen-recognition receptor can comprise one or more cytoplasmic signaling domains or mutants, variants, or derivatives thereof. An exogenous antigenrecognition receptor can comprise, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cytoplasmic signaling domains or mutants, variants, or derivatives thereof. An exogenous antigen-recognition receptor can comprise at least one, at least two, at least three, at least four,or at least five cytoplasmic signaling domains. An exogenous antigen-recognition receptor can comprise at most one, at most two, at most three, at most four, at most five, or at most ten cytoplasmic signaling domains.

[0247] A cytoplasmic signaling domain can be from a mammalian protein. In some cases, a cytoplasmic signaling domain is from a murine (mouse) protein. In some cases, a cytoplasmic signaling domain is from a human protein.

[0248] In some cases, a cytoplasmic signaling domain can comprise modifications compared to a wild type sequence or a sequence disclosed herein (for example, one or more insertions, deletions, and / or substitutions). Mutations can be introduced, for example, to alter (e g., increase or decrease) the stability of an interaction between the cytoplasmic signaling domain and an interacting partner (e.g., signaling adaptor protein or enzyme). Mutations can be introduced, for example, to alter (e.g., increase or decrease) the intensity of an immune activation signal upon binding of the exogenous antigen-recognition receptor to a binding partner.

[0249] An exogenous antigen-recognition receptor of the disclosure can comprise one or more linkers for example, between different domains of the protein. A linker can be a chemical bond, for example, a covalent bond or a non-covalent bond. A linker as described herein can include a flexible or rigid linker. A linker can be a peptide. A linker can be a hinge. A linker can be a linker disclosed herein.

[0250] Components of an exogenous antigen-recognition receptor of the disclosure can be configured such that when the exogenous antigen-recognition receptor is expressed in an engineered immune cell, contacting the immune cell with a target cell induces an immune cell activation signal (e.g., upon binding of the extracellular binding domain of the exogenous antigen-recognition receptor to a surface molecule on the target cell). An exogenous antigenrecognition receptor of the disclosure can comprise one or more extracellular domains, one or more transmembrane domains, and one or more cytoplasmic domains configured such that when the exogenous antigen-recognition receptor is expressed in an engineered immune cell, contacting the immune cell with a target cell induces an immune cell activation signal.

[0251] In some embodiments, an exogenous antigen-recognition receptor is a chimeric antigen receptor that is associated with, known as, or within an engineered cell or construct known as Tisagenlecleucel (Kymriah®), Axicabtagene Ciloleucel (Yescarta®), Brexucabtagene Autoleucel (Tecartus™), Lisocabtagene maraleucel, Idecabtagene Vicleucel, or KTE-X19.IV. PHARMACEUTICAL COMPOSITIONS

[0252] Pharmaceutical compositions of the present disclosure can comprise a composition disclosed herein and a pharmaceutically acceptable excipient. A pharmaceutical composition cancomprise, for example, a pharmaceutically acceptable excipient, vehicle, carrier, or diluent, and an expression construct or polynucleotide, vector, and / or an engineered cell disclosed herein. A pharmaceutical composition can be formulated, for example, for systemic, local, parenteral, intratumoral, intravenous, intraperitoneal, subcutaneous, transdermal, or intramuscular administration. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like. Sterile phosphate- buffered saline is one example of a pharmaceutically suitable excipient.

[0253] A pharmaceutical composition can comprise engineered cells in a unit dosage form. A pharmaceutical composition can comprise a polynucleotide or vector comprising the polynucleotide in a unit dosage form.

[0254] In some cases, unit dosage forms, include, but are not limited to, sterile or substantially sterile parenteral solutions or suspensions, tablets, capsules, pills, powders, granules, oral solutions or suspensions, and oil water emulsions.

[0255] A formulation or composition described herein can be an aqueous solution. Compositions in some examples herein are provided as sterile or substantially sterile liquid preparations, e g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Compositions described herein can also comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof. Sterile injectable solutions containing the engineered cells can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.

[0256] Formulations of the engineered cells can include those for systemic, local, parenteral, intratumoral, intravenous, intraperitoneal, subcutaneous, transdermal, or intramuscular administration.V. KITS AND SYSTEMS

[0257] In some aspects, provided herein is a system comprising: (a) an engineered immune signaling construct disclosed herein; and (b) an exogenous antigen-recognition receptor disclosed herein. The system can be used to treat a condition in a subject in need thereof. For example, the exogenous antigen-recognition receptor can facilitate a response of the engineered cell to a target cell, for example, induce immune activation, proliferation, effector function, cytolytic activity, and / or pro-inflammatory cytokine production in response to the target cell, such as a cancer cell. The engineered immune signaling construct can modulate (e.g., furtherenhance) the immune activation, survival, proliferation, effector function, cytolytic activity, persistence, differentiation, and / or pro-inflammatory cytokine production of the engineered cell.

[0258] In some aspects, the present disclosure provides a kit comprising an engineered cell, polynucleotide, and / or vector disclosed herein. In some embodiments the kit comprises instructions.

[0259] In some embodiments, the kit comprises a polynucleotide and / or vector disclosed herein, and instructions for generating an engineered cell that comprises the engineered immune signaling construct or a polynucleotide encoding the engineered immune signaling construct. In some embodiments, the kit comprises one or more polynucleotides and / or vectors that encode the engineered immune signaling construct and exogenous antigen-recognition receptor, and instructions for generating an engineered cell that comprises the engineered immune signaling construct or a polynucleotide encoding the engineered immune signaling construct, and that comprises the exogenous antigen-recognition receptor or a polynucleotide encoding the exogenous antigen-recognition receptor.VI. METHODS

[0260] Engineered immune signaling constructs, exogenous antigen-recognition receptors, polynucleotides encoding the engineered immune signaling constructs and / or exogenous antigen-recognition receptors, engineered cells expressing the engineered immune signaling constructs and / or exogenous antigen-recognition receptors, and other compositions disclosed herein can be used in methods. For example, an engineered cell that comprises or expresses (i) an engineered immune signaling construct disclosed herein, and (ii) an exogenous antigenrecognition receptor, can be administered to a subject. In some embodiments, the engineered cell is administered as part of a pharmaceutical composition. In some embodiments, one or more expression constructs, polynucleotides, systems, and / or vectors (e.g., encoding the exogenous antigen-recognition receptor, engineered immune signaling construct, or both) are administered to the subject, such that the engineered cell is generated in vivo.

[0261] The exogenous antigen-recognition receptor can bind to a surface molecule on a target cell (e g., cancer cell, infected cell, cell that mediates an autoimmune disease, or fibrotic cell), thereby facilitating a response of the engineered cell to the target cell, such as immune activation, proliferation, effector function, cytolytic activity, and / or pro-inflammatory cytokine production. In an example, the exogenous antigen-recognition receptor is a CAR or TCR, the target cell is a cancer cell, and binding of the CAR to the surface molecule on the target cell elicits immune activation, proliferation, effector function, cytolytic activity, pro-inflammatory cytokine production, or a combination thereof by the engineered cell.

[0262] The engineered immune signaling construct can be utilized in the method, for example, to enhance immune activation, proliferation, effector function, cytolytic activity, pro- inflammatory cytokine production, survival, persistence, and / or differentiation (e.g., memory and / or effector differentiation) of the cell.

[0263] Disclosed herein are methods that can comprise treating a subject in need thereof. The subject can have a disease or condition, such as a cancer. The cancer can be a solid tumor. The cancer can be a liquid tumor. The cancer can be a hematologic tumor. The cancer can be an immune cell cancer. The cancer can be a B cell cancer. The cancer can be a T cell cancer. The cancer can be a myeloid cell cancer. The cancer can be a leukemia. The cancer can be a lymphoma. The cancer can be a myeloma. The cancer can be a carcinoma. The cancer can be a sarcoma. The cancer can be an adenoma. The cancer can be, for example, B-cell lymphoma, mantle cell lymphoma, multiple myeloma, acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma, T cell lymphoblastic leukemia, follicular lymphoma, anaplastic large cell lymphoma (ALCL), peripheral T cell lymphoma-not otherwise specified (PTCL-NOS), angioimmunoblastic T-cell lymphoma (AITL), cutaneous T-cell lymphoma (CTCL), adult T-cell lymphoma and leukemia (ATLL), or T-cell acute lymphoblastic leukemia or lymphoma (T- ALL / LBL).

[0264] In some embodiments, the disease or condition is an infectious disease. In some embodiments, a method disclosed herein can be used for treating an infectious disease, for example, a viral, bacterial, or parasitic (e.g., eukaryotic parasitic) infection.

[0265] In some embodiments, the disease or condition is an autoimmune condition or disorder. In some embodiments, a method disclosed herein can be used for treating an autoimmune disorder.

[0266] In some embodiments, the disease or condition is a fibrotic condition or disorder. In some embodiments, a method disclosed herein can be used for treating, reducing, or preventing fibrosis, such as cardiac fibrosis (e.g., with a CAR targeting FAP).

[0267] Disclosed methods can comprise administering to a subject an engineered cell, expression construct, polynucleotide, system, vector, and / or pharmaceutical composition disclosed herein.

[0268] Illustrative methods of treatment can include administration of an engineered cell, expression construct, polynucleotide, system, and / or vector disclosed herein, including as part of a pharmaceutical composition. The engineered cell, expression construct, polynucleotide, system, and / or vector can be administered in an amount effective to treat or prevent a disease or condition. “Treatment” (and grammatical variations thereof such as “treat” or “treating”) can describe clinical intervention in an attempt to alter the natural course of the individual (subject)being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment can include preventing occurrence or recurrence of disease, alleviation of signs and / or symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0269] An engineered cell, expression construct, polynucleotide, system, vector, and / or pharmaceutical composition can be administered to an individual subject, for example, by parenteral administration or topical application. An engineered cell, expression construct, polynucleotide, system, vector, and / or pharmaceutical composition can be administered to an individual subject, for example, by intravenous, intraperitoneal, intramuscular, subdermal, intracerebral, intracerebroventricular, intra-articular, intraarterial, intrathecal, intracapsular, subcapsular, intraorbital, intracardiac, intradermal, subcutaneous, subarachnoid, or intracranial injection or infusion. The administration can be via localized injection or infusion. The administration can be via systemic injection or infusion. The administration can be via intravenous injection or infusion. The administration can be via intratumoral injection or infusion. The administering can be local. The administering can be systemic.

[0270] Various dosing schedules can be used. In some embodiments, an engineered cell, expression construct, polynucleotide, system, and / or vector is administered to a subject once. In some embodiments, an engineered cell, expression construct, polynucleotide, system, and / or vector is administered to a subject two or more times.

[0271] In some embodiments, provided is a composition, method, or system for generating an engineered cell or a population thereof that comprises, encodes, and / or is capable of expressing an exogenous antigen-recognition receptor disclosed herein. Methods disclosed herein can comprise contacting a cell or a population of cells with a composition or system disclosed herein (e.g., a vector comprising a polynucleotide), thereby expressing the exogenous antigenrecognition receptor and generating an engineered immune cell.

[0272] In some embodiments, an engineered immune signaling construct disclosed herein increases signaling of a common gamma chain family cytokine receptor (e.g., IL-2R (such as IL- 2Rbg), IL-4R, IL-7R, IL-9R, IL-15R, or IL-21R) or TSLPR by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold in a cell expressing the engineered immune signaling construct, as compared to a corresponding control cell that lacks the engineered immune signaling construct.

[0273] In some embodiments, an engineered immune signaling construct disclosed herein expressed in a first cell increases signaling of a common gamma chain family cytokine receptor (e.g., IL-2R (such as IL-2Rbg), IL-4R, IL-7R, IL-9R, IL-15R, or IL-21R) or TSLPR by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold in a bystander cell that lacks the engineered immune signaling construct, as compared to a corresponding control condition in which both the first cell and the bystander cell lack the engineered immune signaling construct. The signaling can be as determined by a suitable assay, for example, to measure phosphorylation of a JAK (e.g., JAK1, JAK3) or a STAT (e.g., STAT5), or a reporter assay.

[0274] In some embodiments, an engineered immune signaling construct disclosed herein increases survival by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold for a population of cells expressing the engineered immune signaling construct, as compared to a corresponding population of control cells that lack the engineered immune signaling construct.

[0275] In some embodiments, an engineered immune signaling construct disclosed herein expressed in a first population of cells increases survival by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold for a population of bystander cells that lack the engineered immune signaling construct, as compared to a corresponding control condition in which both the first population of cells and the population of bystander cell lacks the engineered immune signaling construct. The survival can be as determined by a suitable assay, for example, a cytotoxicity assay, LDH assay, dye exclusion assay, and / or flow cytometric staining. The survival can be determined in a stress condition, for example, following IL-2 starvation.

[0276] In some embodiments, an engineered immune signaling construct disclosed herein increases persistence by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold fora population of cells expressing the engineered immune signaling construct, as compared to a corresponding population of control cells that lack the engineered immune signaling construct.

[0277] In some embodiments, an engineered immune signaling construct disclosed herein expressed in a first population of cells increases persistence by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold for a population of bystander cells that lack the engineered immune signaling construct, as compared to a corresponding control condition in which both the first population of cells and the population of bystander cell lacks the engineered immune signaling construct. The persistence can be as determined by a suitable assay, for example, measuring the number of cells in peripheral blood a given amount of time after administration of the cells to a subject.

[0278] In some embodiments, an engineered immune signaling construct disclosed herein increases proliferation by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold of a cell expressing the engineered immune signaling construct, as compared to a corresponding control cell that lacks the engineered immune signaling construct.

[0279] In some embodiments, expressing an engineered immune signaling construct disclosed herein in a first cell increases proliferation by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2- fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold of a bystander cell that lacks the engineered immune signaling construct, as compared to a corresponding control condition in which both the first cell and the bystander cell lack the engineered immune signaling construct. The proliferation can be as determined by a suitable assay, for example, a lymphoproliferation assay.

[0280] In some embodiments, an engineered immune signaling construct disclosed herein increases effector function (e.g., cytolytic activity or cytokine production) by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold of a cell expressing the engineered immunesignaling construct, as compared to a corresponding control cell that lacks the engineered immune signaling construct.

[0281] In some embodiments, expressing an engineered immune signaling construct disclosed herein in a first cell increases effector function (e.g., cytolytic activity or cytokine production) by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold of a bystander cell that lacks the engineered immune signaling construct, as compared to a corresponding control condition in which both the first cell and the bystander cell lack the engineered immune signaling construct. The effector can be as determined by a suitable assay, for example, a killing assay, intracellular cytokine production, or ELISA following co-incubation of engineered cells expressing the engineered immune signaling construct and exogenous antigen recognition receptor with target cells.

[0282] In some embodiments, an engineered immune signaling construct disclosed reduces exhaustion by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold for a population of cells expressing the engineered immune signaling construct, as compared to a corresponding population of control cells that lack the engineered immune signaling construct.

[0283] In some embodiments, expressing an engineered immune signaling construct disclosed herein in a first population of cells reduces exhaustion by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold for a population of bystander cells that lacks the engineered immune signaling construct, as compared to a corresponding control condition in which both the first population of cells and the population of bystander cells lack the engineered immune signaling construct. The exhaustion can be as determined by a suitable assay, for example, staining for exhaustion markers, or evaluating loss of cytolytic activity after multiple exposure to target cells.

[0284] In some embodiments, a population of cells expressing an engineered immune signaling construct disclosed herein improves control of tumor volume or tumor burden by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold compared to a corresponding population of control cells that lack the engineered immune signaling construct.

[0285] In some embodiments, a population of cells expressing an engineered immune signaling construct disclosed herein improves average survival duration by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 50 fold, at least 100 fold, at least 250 fold, at least 500 fold, or at least 1000 fold compared to a corresponding population of control cells that lack the engineered immune signaling construct.VII. ILLUSTRATIVE EMBODIMENTS

[0286] Embodiment 1. A method of inducing signaling of a common gamma chain family cytokine receptor in a cell, the method comprising expressing an engineered immune signaling construct in the cell, wherein the engineered immune signaling construct comprises: (a) an extracellular region that comprises a common gamma chain-binding domain; (b) a transmembrane region; and (c) an intracellular region comprising an intracellular signaling domain of a cytokine receptor subunit, wherein the cytokine receptor subunit is a common gamma chain family cytokine receptor subunit; wherein the common gamma chain-binding domain is heterologous to the cytokine receptor subunit, and the signaling is induced in a soluble ligand-independent manner.

[0287] Embodiment 2. The method of embodiment 1, wherein the signaling is constitutively induced if the cell expresses the common gamma chain.

[0288] Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the extracellular region or the gamma chain binding domain consists essentially of (i) the common gamma chain-binding domain, and (ii) optionally a hinge, linker, and / or spacer.

[0289] Embodiment 4. The method of embodiment 1 or embodiment 2, wherein the extracellular region or the gamma chain binding domain consists of (i) the common gamma chain-binding domain, and (ii) optionally a hinge, linker, and / or spacer.

[0290] Embodiment 5. The method of any one of embodiments 1-4, wherein the extracellular region or the gamma chain-binding domain comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1-91.

[0291] Embodiment 6. The method of any one of embodiments 1-4, wherein the extracellular region or the gamma chain-binding domain comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 1-91.

[0292] Embodiment 7. The method of any one of embodiments 1-6, wherein the common gamma chain-binding domain comprises a cytokine or a gamma chain-binding domain thereof.

[0293] Embodiment 8. The method of any one of embodiments 1-7, wherein the common gamma chain-binding domain comprises a wild type cytokine or a gamma chain-binding domain thereof.

[0294] Embodiment 9. The method of any one of embodiments 1-7, wherein the common gamma chain-binding domain comprises a mutein cytokine or a gamma chain-binding domain thereof.

[0295] Embodiment 10. The method of any one of embodiments 1-9, wherein the common gamma chain-binding domain comprises IL-7 or a gamma chain -binding domain thereof.

[0296] Embodiment 11. The method of any one of embodiments 1-9, wherein the common gamma chain-binding domain comprises IL-2 or a gamma chain -binding domain thereof.

[0297] Embodiment 12. The method of any one of embodiments 1-9, wherein the common gamma chain-binding domain comprises IL-4 or a gamma chain -binding domain thereof.

[0298] Embodiment 13. The method of any one of embodiments 1-9, wherein the common gamma chain-binding domain comprises IL-9 or a gamma chain -binding domain thereof.

[0299] Embodiment 14. The method of any one of embodiments 1-9, wherein the common gamma chain-binding domain comprises IL-15 or a gamma chain-binding domain thereof.

[0300] Embodiment 15. The method of any one of embodiments 1-9, wherein the common gamma chain-binding domain comprises IL-21 or a gamma chain-binding domain thereof.

[0301] Embodiment 16. The method of any one of embodiments 1-9, wherein the common gamma chain-binding domain comprises TSLP or a TSLP receptor (TSLPR) chain-binding domain thereof.

[0302] Embodiment 17. The method of any one of embodiments 1-6, wherein the common gamma chain-binding domain comprises an antibody or antigen-binding fragment thereof.

[0303] Embodiment 18. The method of any one of embodiments 1-6, wherein the common gamma chain-binding domain comprises a single chain variable fragment (scFv).

[0304] Embodiment 19. The method of any one of embodiments 1-6, wherein the common gamma chain-binding domain comprises a fragment antigen-binding (Fab).

[0305] Embodiment 20. The method of any one of embodiments 1-6, wherein the common gamma chain-binding domain comprises a single domain antibody.

[0306] Embodiment 21. The method of any one of embodiments 1-20, wherein the transmembrane region comprises a transmembrane domain of an immune receptor.

[0307] Embodiment 22. The method of any one of embodiments 1-21, wherein the transmembrane region comprises a transmembrane domain of a common gamma chain family cytokine receptor.

[0308] Embodiment 23. The method of any one of embodiments 1-22, wherein the transmembrane region comprises a transmembrane domain of IL-2Ra, IL-2Rb, IL-4Ra, IL-7Ra, IL-9Ra, IL-15Ra, or IL-21R.

[0309] Embodiment 24. The method of any one of embodiments 1-22, wherein the transmembrane region comprises an IL-7Ra transmembrane domain.

[0310] Embodiment 25. The method of any one of embodiments 1-21, wherein the transmembrane region comprises a CD8a transmembrane domain.

[0311] Embodiment 26. The method of any one of embodiments 1-21, wherein the transmembrane region comprises a CD28 transmembrane domain.

[0312] Embodiment 27. The method of any one of embodiments 1-26, wherein the transmembrane region comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 92-104.

[0313] Embodiment 28. The method of any one of embodiments 1-26, wherein the transmembrane region comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 92-104.

[0314] Embodiment 29. The method of any one of embodiments 1-28, wherein the intracellular signaling domain comprises a JAK1 binding domain.

[0315] Embodiment 30. The method of any one of embodiments 1-29, wherein the intracellular signaling domain comprises a BOXl motif.

[0316] Embodiment 31. The method of any one of embodiments 1-30, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-2Rb, optionally wherein the signaling is IL-2R signaling.

[0317] Embodiment 32. The method of any one of embodiments 1-30, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-7Ra, optionally wherein the signaling is IL-7R signaling.

[0318] Embodiment 33. The method of any one of embodiments 1-30, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-9Ra, optionally wherein the signaling is IL-9R signaling.

[0319] Embodiment 34. The method of any one of embodiments 1-30, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-4Ra, optionally wherein the signaling is IL-4R signaling.

[0320] Embodiment 35. The method of any one of embodiments 1-30, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-21R, optionally wherein the signaling is IL-21R signaling.

[0321] Embodiment 36. The method of any one of embodiments 1-35, wherein the intracellular region or the intracellular signaling domain comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 105-115 and 533-540.

[0322] Embodiment 37. The method of any one of embodiments 1-35, wherein the intracellular region or the intracellular signaling domain comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 105-115 and 533-540.

[0323] Embodiment 38. The method of any one of embodiments 1-37, wherein the engineered immune signaling construct consists essentially of the extracellular region, the transmembrane region, and the intracellular region.

[0324] Embodiment 39. The method of any one of embodiments 1-37, wherein the engineered immune signaling construct consists of the extracellular region, the transmembrane region, and the intracellular region.

[0325] Embodiment 40. The method of any one of embodiments 1-39, wherein the extracellular region lacks a ligand-binding domain of a cytokine receptor.

[0326] Embodiment 41. The method of any one of embodiments 1-40, wherein the extracellular region lacks a ligand-binding domain of an immune inhibitory receptor.

[0327] Embodiment 42. The method of any one of embodiments 1-41, wherein the method increases signaling of the common gamma chain family cytokine receptor in the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct.

[0328] Embodiment 43. The method of any one of embodiments 1-42, wherein the method increases signaling of the common gamma chain family cytokine receptor in a bystander cell that lacks the engineered immune signaling construct by at least 5%, as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signaling construct.

[0329] Embodiment 44. The method of embodiment 42 or embodiment 43, wherein the increase in signaling is as determined by a STAT5 phosphorylation assay.

[0330] Embodiment 45. The method of any one of embodiments 1-44, wherein the method enhances survival or persistence of the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct.

[0331] Embodiment 46. The method of any one of embodiments 1-45, wherein the method enhances survival or persistence of a bystander cell that lacks the engineered immune signaling construct by at least 5% as compared to a control condition in which both the cell and the bystander cell lack the engineered immune signaling construct.

[0332] Embodiment 47. The method of embodiment 45 or embodiment 46, wherein the survival is as determined by an IL-2 starvation assay.

[0333] Embodiment 48. The method of any one of embodiments 1-47, wherein the method enhances proliferation of the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct.

[0334] Embodiment 49. The method of any one of embodiments 1-48, wherein the method enhances proliferation of a bystander cell that lacks the engineered immune signaling construct by at least 5% as compared to a control condition in which both the cell and the bystander cell lack the engineered immune signaling construct.

[0335] Embodiment 50. The method of embodiment 48 or embodiment 49, wherein the proliferation is as determined by a lymphoproliferation assay.

[0336] Embodiment 51. An engineered immune signaling construct comprising: (a) an extracellular region comprising a common gamma chain-binding domain, wherein the extracellular region lacks a soluble ligand-binding domain of a receptor; (b) a transmembrane region; and (c) an intracellular region comprising an intracellular signaling domain of a cytokine receptor subunit, wherein the cytokine receptor subunit is a common gamma chain family cytokine receptor subunit and is heterologous to the common gamma chain-binding domain.

[0337] Embodiment 52. The engineered immune signaling construct of embodiment 51, wherein the extracellular region consists essentially of (i) the common gamma chain-binding domain, and (ii) optionally a hinge, linker, and / or spacer.

[0338] Embodiment 53. The engineered immune signaling construct of embodiment 51, wherein the extracellular region consists of (i) the common gamma chain-binding domain, and (ii) optionally a hinge, linker, and / or spacer.

[0339] Embodiment 54. The engineered immune signaling construct of any one of embodiments 51-53, wherein the extracellular region or the gamma chain-binding domain comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1-91.

[0340] Embodiment 55. The engineered immune signaling construct of any one of embodiments 51-54, wherein the extracellular region or the gamma chain-binding domain comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 1-91.

[0341] Embodiment 56. The engineered immune signaling construct of any one of embodiments 51-55, wherein the common gamma chain-binding domain comprises a cytokine or a gamma chain-binding domain thereof.

[0342] Embodiment 57. The engineered immune signaling construct of any one of embodiments 51-56, wherein the common gamma chain-binding domain comprises a wild type cytokine or a gamma chain-binding domain thereof.

[0343] Embodiment 58. The engineered immune signaling construct of any one of embodiments 51-56, wherein the common gamma chain-binding domain comprises a mutein cytokine or a gamma chain-binding domain thereof.

[0344] Embodiment 59. The engineered immune signaling construct of any one of embodiments 51-58, wherein the common gamma chain-binding domain comprises IL-7 or a gamma chain-binding domain thereof.

[0345] Embodiment 60. The engineered immune signaling construct of any one of embodiments 51-58, wherein the common gamma chain-binding domain comprises IL-2 or a gamma chain-binding domain thereof.

[0346] Embodiment 61. The engineered immune signaling construct of any one of embodiments 51-58, wherein the common gamma chain-binding domain comprises IL-4 or a gamma chain-binding domain thereof.

[0347] Embodiment 62. The engineered immune signaling construct of any one of embodiments 51-58, wherein the common gamma chain-binding domain comprises IL-9 or a gamma chain-binding domain thereof.

[0348] Embodiment 63. The engineered immune signaling construct of any one of embodiments 51-58, wherein the common gamma chain-binding domain comprises IL-15 or a gamma chain-binding domain thereof.

[0349] Embodiment 64. The engineered immune signaling construct of any one of embodiments 51-58, wherein the common gamma chain-binding domain comprises IL-21 or a gamma chain-binding domain thereof.

[0350] Embodiment 65. The engineered immune signaling construct of any one of embodiments 51-58, wherein the common gamma chain-binding domain comprises TSLP or a TSLP receptor (TSLPR) chain-binding domain thereof.

[0351] Embodiment 66. The engineered immune signaling construct of any one of embodiments 51-55, wherein the common gamma chain-binding domain comprises an antibody or antigen-binding fragment thereof.

[0352] Embodiment 67. The engineered immune signaling construct of any one of embodiments 51-55, wherein the common gamma chain-binding domain comprises a single chain variable fragment (scFv).

[0353] Embodiment 68. The engineered immune signaling construct of any one of embodiments 51-55, wherein the common gamma chain-binding domain comprises a fragment antigen-binding (Fab).

[0354] Embodiment 69. The engineered immune signaling construct of any one of embodiments 51-55, wherein the common gamma chain-binding domain comprises a single domain antibody.

[0355] Embodiment 70. The engineered immune signaling construct of any one of embodiments 51-69, wherein the transmembrane region comprises a transmembrane domain of an immune receptor.

[0356] Embodiment 71. The engineered immune signaling construct of any one of embodiments 51-70, wherein the transmembrane region comprises a transmembrane domain of a cytokine receptor.

[0357] Embodiment 72. The engineered immune signaling construct of any one of embodiments 51-71, wherein the transmembrane region comprises a transmembrane domain of a common gamma chain family cytokine receptor.

[0358] Embodiment 73. The engineered immune signaling construct of any one of embodiments 51-72, wherein the transmembrane region comprises a transmembrane domain of IL-2Ra, IL-2Rb, IL-4Ra, IL-7Ra, IL-9Ra, IL-15Ra, or IL-21R.

[0359] Embodiment 74. The engineered immune signaling construct of any one of embodiments 51-73, wherein the transmembrane region comprises an IL-7Ra transmembrane domain.

[0360] Embodiment 75. The engineered immune signaling construct of any one of embodiments 51-70, wherein the transmembrane region comprises a CD8a transmembrane domain.

[0361] Embodiment 76. The engineered immune signaling construct of any one of embodiments 51-70, wherein the transmembrane region comprises a CD28 transmembrane domain.

[0362] Embodiment 77. The engineered immune signaling construct of any one of embodiments 51-76, wherein the transmembrane region comprises, consists essentially of, orconsists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 92-104.

[0363] Embodiment 78. The engineered immune signaling construct of any one of embodiments 51-76, wherein the transmembrane region comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 92-104.

[0364] Embodiment 79. The engineered immune signaling construct of any one of embodiments 51-78, wherein the intracellular signaling domain comprises a JAK1 binding domain.

[0365] Embodiment 80. The engineered immune signaling construct of any one of embodiments 51-79, wherein the intracellular signaling domain comprises a BOX1 motif.

[0366] Embodiment 81. The engineered immune signaling construct of any one of embodiments 1-80, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-2Rb, optionally wherein the signaling is IL-2R signaling.

[0367] Embodiment 82. The engineered immune signaling construct of any one of embodiments 1-80, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-7Ra, optionally wherein the signaling is IL-7R signaling.

[0368] Embodiment 83. The engineered immune signaling construct of any one of embodiments 1-80, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-9Ra, optionally wherein the signaling is IL-9R signaling.

[0369] Embodiment 84. The engineered immune signaling construct of any one of embodiments 1-80, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-4Ra, optionally wherein the signaling is IL-4R signaling.

[0370] Embodiment 85. The engineered immune signaling construct of any one of embodiments 1-80, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-21R, optionally wherein the signaling is IL-21R signaling.

[0371] Embodiment 86. The engineered immune signaling construct of any one of embodiments 51-85, wherein the intracellular region or the intracellular signaling domain comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 105-115 and 533-540.

[0372] Embodiment 87. The engineered immune signaling construct of any one of embodiments 51-85, wherein the intracellular region or the intracellular signaling domain comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 105-115 and 533-540.

[0373] Embodiment 88. The engineered immune signaling construct of any one of embodiments 51-87, wherein the engineered immune signaling construct consists essentially of the extracellular region, the transmembrane region, and the intracellular region.

[0374] Embodiment 89. The engineered immune signaling construct of any one of embodiments 51-87, wherein the engineered immune signaling construct consists of the extracellular region, the transmembrane region, and the intracellular region.

[0375] Embodiment 90. The engineered immune signaling construct of any one of embodiments 51-89, wherein the extracellular region lacks a soluble ligand-binding domain.

[0376] Embodiment 91. A polynucleotide encoding the engineered immune signaling construct of any one of embodiments 51-90.

[0377] Embodiment 92. A vector comprising the polynucleotide of embodiment 91.

[0378] Embodiment 93. The vector of embodiment 92, wherein the vector is a viral vector.

[0379] Embodiment 94. The vector of embodiment 92, wherein the vector is a non -viral vector.

[0380] Embodiment 95. A method of making an engineered cell, the method comprising contacting a cell with the polynucleotide of embodiment 91 or the vector of any one of embodiments 92-94.

[0381] Embodiment 96. The method of embodiment 95, wherein the contacting is in vitro or ex vivo.

[0382] Embodiment 97. The method of embodiment 95, wherein the contacting is in vivo.

[0383] Embodiment 98. A cell expressing the engineered immune signaling construct of any one of embodiments 51-90.

[0384] Embodiment 99. The cell of embodiment 98, wherein the cell is an immune cell.

[0385] Embodiment 100. The cell of embodiment 98, wherein the cell is a lymphocyte.

[0386] Embodiment 101. The cell of embodiment 98, wherein the cell is a T cell.

[0387] Embodiment 102. The cell of embodiment 98, wherein the cell is an NK cell.

[0388] Embodiment 103. The cell of any one of embodiments 98-103, wherein the cell expresses an exogenous antigen-recognition receptor.

[0389] Embodiment 104. The cell of embodiment 103, wherein the exogenous antigenrecognition receptor is a chimeric antigen receptor.

[0390] Embodiment 105. The cell of embodiment 103, wherein the exogenous antigenrecognition receptor is a T cell receptor.

[0391] Embodiment 106. The cell of any one of embodiments 98-105, wherein if the cell expresses common gamma chain, the engineered immune signaling construct constitutively induces signaling of the intracellular signaling domain in the cell.

[0392] Embodiment 107. The cell of any one of embodiments 98-106, wherein the cell expresses common gamma chain.

[0393] Embodiment 108 The cell of embodiment 107, wherein the engineered immune signaling construct constitutively binds to the common gamma chain.

[0394] Embodiment 109. The cell of any one of embodiments 98-108, wherein the cell expresses common gamma chain, and the engineered immune signaling construct constitutively induces signaling of the intracellular signaling domain in the cell.

[0395] Embodiment 110. The cell of any one of embodiments 98-109, wherein the engineered immune signaling construct increases signaling of a common gamma chain family cytokine receptor comprising the cytokine receptor subunit and common gamma chain in the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct.

[0396] Embodiment 111. The cell of any one of embodiments 98-110, wherein the engineered immune signaling construct increases signaling of a gamma chain family cytokine receptor in a bystander cell that lacks the engineered immune signaling construct by at least 5%, as compared to a control condition in which both the cell and the bystander cell lack the engineered immune signaling construct.

[0397] Embodiment 112. The cell of embodiment 110 or embodiment 111, wherein the increase in signaling is as determined by a STAT5 phosphorylation assay.

[0398] Embodiment 113. The cell of any one of embodiments 98-112, wherein the engineered immune signaling construct enhances survival or persistence of the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct.

[0399] Embodiment 114. The cell of any one of embodiments 98-113, wherein the engineered immune signaling construct enhances survival or persistence of a bystander cell that lacks the engineered immune signaling construct by at least 5% as compared to a control condition in which both the cell and the bystander cell lack the engineered immune signaling construct.

[0400] Embodiment 115. The cell of embodiment 113 or embodiment 114, wherein the survival is as determined by an IL-2 starvation assay.

[0401] Embodiment 116. The cell of any one of embodiments 98-115, wherein the engineered immune signaling construct enhances proliferation of the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct.

[0402] Embodiment 117. The cell of any one of embodiments 98-116, wherein the engineered immune signaling construct enhances proliferation of a bystander cell that lacks the engineered immune signaling construct by at least 5% as compared to a control condition in which both the cell and the bystander cell lack the engineered immune signaling construct.

[0403] Embodiment 118. The cell of embodiment 116 or embodiment 117, wherein the proliferation is as determined by a lymphoproliferation assay.

[0404] Embodiment 119 A pharmaceutical composition comprising: (a) the polynucleotide of embodiment 91, the vector of any one of embodiments 92-94, or the cell of any one of embodiments 98-118, and (b) a pharmaceutically-acceptable excipient, vehicle, carrier, or diluent.

[0405] Embodiment 120. A method of treating a condition in a subject in need thereof, the method comprising administering to the subject the polynucleotide of embodiment 91, the vector of any one of embodiments 92-94, the cell of any one of embodiments 98-118, or the pharmaceutical composition of embodiment 119.

[0406] Embodiment 121. The method of embodiment 120, wherein the condition comprises or is cancer.

[0407] Embodiment 122. The method of embodiment 120, wherein the condition comprises or is an infectious disease.

[0408] Embodiment 123. The method of embodiment 120, wherein the condition comprises or is an autoimmune disease.

[0409] Embodiment 124. A method of enhancing the efficacy of a therapeutic regimen that comprises an engineered cell, the method comprising expressing the engineered immune signaling construct of any one of embodiments 51-90 in the engineered cell, thereby enhancing the efficacy of the therapeutic regimen.

[0410] Embodiment 125. A method of enhancing survival, proliferation, differentiation, persistence, or effector function of an engineered cell, the method comprising expressing the engineered immune signaling construct of any one of embodiments 51-90 in the engineered cell, thereby enhancing the survival, proliferation, differentiation, persistence, or effector function of the engineered cell.VIII. EXAMPLESF. EXAMPLE 1: design of illustrative engineered immune signaling constructs

[0411] This example demonstrates design of illustrative engineered immune signaling constructs. Engineered immune signaling constructs were designed that comprise an extracellular region comprising a common gamma chain-binding domain (cytokine), a transmembrane region, and an intracellular region comprising an intracellular signaling domain of a common gamma chain family cytokine receptor / receptor subunit.

[0412] SEQ ID NO: 205 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-7 as a common gamma chain-binding domain and comprises a short flexible linker, IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0413] SEQ ID NO: 206 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 as a common gamma chain-binding domain and comprises a short flexible linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0414] SEQ ID NO: 207 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 (non alpha) as a common gamma chain-binding domain and comprises a short flexible linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0415] SEQ ID NO: 208 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-4 as a common gamma chain-binding domain and comprises a short flexible linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0416] SEQ ID NO: 209 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL- 15 as a common gamma chain-binding domain and comprises a short flexible linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0417] SEQ ID NO: 210 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-9 as a common gamma chain-binding domain and comprises a short flexible linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0418] SEQ ID NO: 211 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-7 as a common gamma chain-binding domain and comprises a rigid alpha helical linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0419] SEQ ID NO: 212 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 as a common gamma chain-binding domain and comprises a rigid alpha helical linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0420] SEQ ID NO: 213 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 (non alpha) as a common gamma chain-binding domain and comprises a rigid alpha helical linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0421] SEQ ID NO: 214 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-4 as a common gamma chain-binding domain and comprises a rigid alpha helical linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0422] SEQ ID NO: 215 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL- 15 as a common gamma chain-binding domain and comprises a rigid alpha helical linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0423] SEQ ID NO: 216 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-9 as a common gamma chain-binding domain and comprises a rigid alpha helical linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0424] SEQ ID NO: 217 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-7 as a common gamma chain-binding domain and comprises a CD8 hinge domain, CD8 transmembrane domain, and an IL-9Ra intracellular domain.

[0425] SEQ ID NO: 218 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 as a common gamma chain-binding domain and comprises a CD8 hinge domain, CD8 transmembrane domain, and an IL-9Ra intracellular domain.

[0426] SEQ ID NO: 219 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 as a common gamma chain-binding domain and comprises a CD8 hinge domain, CD8 transmembrane domain, and an IL-9Ra intracellular domain.

[0427] SEQ ID NO: 220 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-4 as a common gamma chain-binding domain and comprises a CD8 hinge domain, CD8 transmembrane domain, and an IL-9Ra intracellular domain.

[0428] SEQ ID NO: 221 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL- 15 as a common gamma chain-binding domain and comprises a CD8 hinge domain, CD8 transmembrane domain, and an IL-9Ra intracellular domain.

[0429] SEQ ID NO: 222 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-9 as a common gamma chain-binding domain and comprises a CD8 hinge domain, CD8 transmembrane domain, and an IL-9Ra intracellular domain.

[0430] SEQ ID NO: 223 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-7 as a common gamma chain-binding domain and comprises a CD28 hinge domain, CD28 transmembrane domain, and an IL-9Ra intracellular domain.

[0431] SEQ ID NO: 224 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 as a common gamma chain-binding domain and comprises a CD28 hinge domain, CD28 transmembrane domain, and an IL-9Ra intracellular domain.

[0432] SEQ ID NO: 225 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 (non alpha) as a common gamma chain-binding domain and comprises a CD28 hinge domain, CD28 transmembrane domain, and an IL-9Ra intracellular domain.

[0433] SEQ ID NO: 226 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-4 as a common gamma chain-binding domain and comprises a CD28 hinge domain, CD28 transmembrane domain, and an IL-9Ra intracellular domain.

[0434] SEQ ID NO: 227 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL- 15 as a common gamma chain-binding domain and comprises a CD28 hinge domain, CD28 transmembrane domain, and an IL-9Ra intracellular domain.

[0435] SEQ ID NO: 228 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-9 as a common gamma chain-binding domain and comprises a CD28 hinge domain, CD28 transmembrane domain, and an IL-9Ra intracellular domain.

[0436] SEQ ID NO: 229 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-7 as a common gamma chain-binding domain and comprises a long flexible linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0437] SEQ ID NO: 230 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 as a common gamma chain-binding domain and comprises a long flexible linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0438] SEQ ID NO: 231 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 (non alpha) as a common gamma chain-binding domain and comprises a long flexible linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0439] SEQ ID NO: 232 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-4 as a common gamma chain-binding domain and comprises a long flexible linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0440] SEQ ID NO: 233 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL- 15 as a common gamma chain-binding domain and comprises a long flexible linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0441] SEQ ID NO: 234 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-9 as a common gamma chain-binding domain and comprises a long flexible linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0442] SEQ ID NO: 235 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-7 as a common gamma chain-binding domain and comprises a combined linker with flexible and rigid parts, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0443] SEQ ID NO: 236 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 as a common gamma chain-binding domain and comprises a combined linker with flexible and rigid parts, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0444] SEQ ID NO: 237 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 (non alpha) as a common gamma chain-binding domain andcomprises a combined linker with flexible and rigid parts, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0445] SEQ ID NO: 238 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-4 as a common gamma chain-binding domain and comprises a combined linker with flexible and rigid parts, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0446] SEQ ID NO: 239 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL- 15 as a common gamma chain-binding domain and comprises a combined linker with flexible and rigid parts, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0447] SEQ ID NO: 240 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-9 as a common gamma chain-binding domain and comprises a combined linker with flexible and rigid parts, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0448] SEQ ID NO: 241 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-7 as a common gamma chain-binding domain and comprises a flexible linker, a CD8a hinge, a CD8a transmembrane domain, and an IL-9Ra intracellular domain.

[0449] SEQ ID NO: 242 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 as a common gamma chain-binding domain and comprises a flexible linker, a CD8a hinge, a CD8a transmembrane domain, and an IL-9Ra intracellular domain.

[0450] SEQ ID NO: 243 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 (non alpha) as a common gamma chain-binding domain and comprises a flexible linker, a CD8a hinge, a CD8a transmembrane domain, and an IL-9Ra intracellular domain.

[0451] SEQ ID NO: 244 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-4 as a common gamma chain-binding domain and comprises a flexible linker, a CD8a hinge, a CD8a transmembrane domain, and an IL-9Ra intracellular domain.

[0452] SEQ ID NO: 245 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL- 15 as a common gamma chain-binding domain and comprises a flexible linker, a CD8a hinge, a CD8a transmembrane domain, and an IL-9Ra intracellular domain.

[0453] SEQ ID NO: 246 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-9 as a common gamma chain-binding domain and comprises a flexible linker, a CD8a hinge, a CD8a transmembrane domain, and an IL-9Ra intracellular domain.

[0454] SEQ ID NO: 247 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-7 as a common gamma chain-binding domain and comprises a flexible linker, a CD28 hinge, a CD28 transmembrane domain, and an IL-9Ra intracellular domain

[0455] SEQ ID NO: 248 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 as a common gamma chain-binding domain and comprises a flexible linker, a CD28 hinge, a CD28 transmembrane domain, and an IL-9Ra intracellular domain.

[0456] SEQ ID NO: 249 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-2 (non alpha) as a common gamma chain-binding domain and comprises a flexible linker, a CD28 hinge, a CD28 transmembrane domain, and an IL-9Ra intracellular domain.

[0457] SEQ ID NO: 250 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-4 as a common gamma chain-binding domain and comprises a flexible linker, a CD28 hinge, a CD28 transmembrane domain, and an IL-9Ra intracellular domain.

[0458] SEQ ID NO: 251 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL- 15 as a common gamma chain-binding domain and comprises a flexible linker, a CD28 hinge, a CD28 transmembrane domain, and an IL-9Ra intracellular domain.

[0459] SEQ ID NO: 252 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-9 as a common gamma chain-binding domain and comprises a flexible linker, a CD28 hinge, a CD28 transmembrane domain, and an IL-9Ra intracellular domain.

[0460] SEQ ID NO: 253 provides an illustrative sequence of an engineered immune signaling construct that utilizes IL-7 as a common gamma chain-binding domain and comprises a short flexible linker, an IL-7Ra transmembrane domain, and an IL-9Ra intracellular domain.

[0461] SEQ ID NO: 254 provides an illustrative sequence of an engineered immune signaling construct with a common gamma chain-binding domain scFv, a rigid alpha helical linker, an IL- 7Ra transmembrane domain, and an intracellular signaling domain of IL-9Ra.

[0462] SEQ ID NO: 255 provides an illustrative sequence of an engineered immune signaling construct with a common gamma chain-binding domain scFv, a CD8a hinge domain, a CD8a transmembrane domain, and an intracellular signaling domain of IL-9Ra.

[0463] SEQ ID NO: 256 provides an illustrative sequence of an engineered immune signaling construct with a common gamma chain-binding domain scFv, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular signaling domain of IL-9Ra.

[0464] SEQ ID NO: 257 provides an illustrative sequence of an engineered immune signaling construct with a common gamma chain-binding domain scFv, an IL-7Ra transmembrane domain, and an intracellular signaling domain of IL-9Ra.

[0465] SEQ ID NOs: 258-532 provide additional illustrative sequences of engineered immune signaling constructs, combining in various combinations: TSLP or a gamma chain-binding domain (e.g., scFv, IL-7, IL-2, IL-2 (non-alpha), IL-4, IL-15, IL-9, or IL-21), an intracellular signaling domain (e.g., of IL-9Ra, IL-2Rb, IL-7Ra, IL-4Ra, IL-21R), a transmembrane domain (e.g., of IL-7Ra, CD8a, or CD28), and linkers / and / or hinges disclosed herein.G. EXAMPLE 2: Effect of engineered immune signaling constructs on immune cell activation, signaling, cytokine production, and proliferation

[0466] Primary human T cells are transduced with lentiviral constructs encoding engineered immune signaling constructs disclosed herein. Untransduced and empty vector controls are also included.

[0467] In a first experimental setup, the cells are stimulated with anti-CD3 / anti-CD28 microbeads, or plate bound anti-CD3 antibody. At suitable time point(s) (e.g., after 8-24 hours of stimulation), supernatants are collected and a panel of cytokines and chemokines are quantified via a multiplex immunoassay. The measured cytokines and chemokines can include, e.g., CCL2, CCL5, CCL7, CCL22, Eotaxin, Fractalkine, GM-CSF, IFNy, IL-10, IL-2, IL-3, IL- 4, IL-5, IL-6, IL-7, IL-9, IL-8, IL-10, IL-12 (p70), IL-13, IL-17A, IL-17B, IL-21, IL-23, IP-10, IT AC, M-CSF, MIG, MIP-la, MIP-10, MIP-3a, and / or TNF-a.

[0468] At suitable time point(s) (e.g., after 8-24 hours of stimulation), the cells are stained and processed for flow cytometric evaluation for extracellular and / or intracellular markers associated with T cell activation, proliferation, and function, which can include, e g., CD3, CD4, CD8, CD25, CD39, CD45RA, CD69, CD62L, CD107a, CD134 (0X40), CD137 (41BB), CXCR3, CXCR5, CCR4, CCR7, HLA-DR, CTLA4, and / or PD1

[0469] In an additional assay, phospho-specific flow cytometry is used to evaluate activation of signaling pathways (for example, with antibodies specific for phosphorylated Akt, Erkl / 2, JNK, Lek, NF-KB p65, p38, STAT3, STAT5, ZAP70, and / or Rb). At suitable time points after treatment with anti-CD3 / anti-CD28 microbeads (e.g., treatment for about 15 or 30 minutes), cells are fixed, permeabilized (e.g., with ice-cold 50% methanol for 30 min on ice), then washed, stained, and processed for flow cytometry.

[0470] The effects of the engineered immune signaling construct on immune cell activation, function, and cytokine production are evaluated.

[0471] A further assay is conducted to evaluate cellular proliferation The transduced cells and untransduced controls are stained with CFSE or a similar dye prior to stimulation with the anti- CD3 / anti-CD28 microbeads. 48 hours after stimulation, the effect of the engineered immune signaling construct on cellular proliferation is evaluated by flow cytometry.H. EXAMPLE 3: Effects of engineered immune signaling construct on CAR-T cells

[0472] Primary human T cells are transduced with lentiviral constructs encoding (1) a chimeric antigen receptor (e.g., a second or third generation CAR specific for HER2); and / or (2) an engineered immune signaling construct disclosed herein. Untransduced and empty vector controls are also included.

[0473] HER2-overexpressing cells are used as target cells, and a HER2 -negative cell line is used as a control.

[0474] The T cells are co-cultured with target cells. Controls without the engineered immune signaling construct are also included. The cells are co-cultured at suitable effectortarget ratios (e.g., 10:1, 5: 1, 3:1, 2:1, 1: 1, 1 :2, 1:3, 1 :5, and / or 1: 10).

[0475] At suitable time point(s) (e.g., after 8-24 hours of co-incubation), supernatants are collected and a panel of cytokines and chemokines are quantified via a multiplex immunoassay. The measured cytokines and chemokines can include, e.g., CCL2, CCL5, CCL7, CCL22, Eotaxin, Fractalkine, GM-CSF, JFNy, IL-ip, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-8, IL- 10, IL-12 (p70), IL-13, IL-17A, IL-17B, IL-21, IL-23, IP-10, ITAC, M-CSF, MIG, MIP-la, MIP-ip, MIP-3a, and / or TNF-a.

[0476] At suitable time point(s) (e.g., after 8-24 hours of co-incubation), the cells are stained and processed for flow cytometric evaluation for extracellular and / or intracellular markers associated with T cell activation, proliferation, and function, which can include, e.g., CD3, CD4, CD8, CD25, CD39, CD45RA, CD69, CD62L, CD107a, CD134 (0X40), CD137 (41BB), CXCR3, CXCR5, CCR4, CCR7, HLA-DR, CTLA4, and / or PD1.

[0477] In an additional assay, phospho-specific flow cytometry is used to evaluate activation of signaling pathways (for example, with antibodies specific for phosphorylated Akt, Erkl / 2, JNK, Lek, NF-KB p65, p38, STAT3, STAT5, ZAP70, and / or Rb). At suitable time point(s) after co-incubation (e.g., for about 15 or 30 minutes), cells are fixed, permeabilized (e.g., with ice- cold 50% methanol for 30 min on ice), then washed, stained, and processed for flow cytometry.

[0478] The effects of the engineered immune signaling construct on immune cell activation, function, and cytokine production are evaluated.

[0479] A further assay is conducted to evaluate cellular proliferation. The transduced T cells and untransduced controls are stained with CFSE or a similar dye prior to co-incubation with the target cells. 48 hours later, the effect of the engineered immune signaling construct on cellular proliferation is evaluated by flow cytometry.

[0480] Killing assays are performed to evaluate the effect of the engineered immune signaling construct on CAR-T target cell killing. The target cells (HER2-overexpressing) are transduced to express (or alternatively labelled with) a suitable fluorescent protein, such as mCherry or a redfluorescent protein. Following co-culture at suitable effectortarget ratios, target cell death is measured in an Incucyte assay, for example, with live cell imaging for cell counts or total fluorescent object integrated intensity (a measure of target cell volume / area), and / or with an incucyte Caspase-3 / 7 reagent or incucyte Annexin V reagent. Incucyte Cell-By-Cell Analysis Software is used to quantify both the labeled target cell population and the effector cell population. The assay can be performed in the presence of IL-2.

[0481] Assays are also performed to evaluate the impact of the engineered immune signaling construct on markers associated with T cell exhaustion, memory, activation, and function. The CAR-T cells are stimulated with target cells or anti-CD3 / anti-CD28, and optionally rested then restimulated. Illustrative stimulation and rest periods can include: stimulation for 3, 7, 11, 14, 18, and 24 days, and optionally rested for 3, 5, 7, 11, or 14 days.

[0482] Before and after (re)-stimulation, the T cells are stained and processed for mass and / or flow cytometry to evaluate markers associated with T cell exhaustion, memory, activation, and function, e g., CD4, CD8, CD25, CD27, CD28, CD39, CD45RA, CD45RO, CD62L, CD69, CD95, CD107a, CD197, PD1, PDL1, 0X40, 2B4, TIM3, CXCR3, 41BB, BLIMP1, T-BET, CTLA4, BTLA, LAG3, CXCR3, CXCR5, CCR4, CCR7, IL7R, KI67, and / or EOMES. The impact of the engineered immune signaling construct is assessed, including, for example, for markers associated with exhaustion (e.g., PD-1, TIM-3, LAG-3, CTLA-4, BTLA, 2B4, and CD39) and memory (e.g., CD45RA, IL-7R, CD27, CD197).I. EXAMPLE 4: Effects of engineered immune signaling construct on CAR-T cells in vivo

[0483] Immunodeficient NOD.Cg-Prkdcscid I12rgtmlWjl / SzI (NSG) animals are used that accept human xenografts. 1 xlO 6 luciferase-expressing Ieko-1 cells are injected intravenously into each recipient. In a second study, the cells are injected subcutaneously.

[0484] Tumor progression is monitored twice a week until measurable radiance is observed in the body of each animal. -7-10 days post-tumor inoculation, animals undergo randomized group assignment based on body weight and tumor burden. T cells expressing CARs with or without an engineered immune signaling construct are administered to tumor bearing mice at two dose levels.

[0485] CAR-T cells are infused via tail vein injection or intratumoral injection to treatment groups. Peripheral blood is taken every two weeks and mouse survival monitored (e.g., over 60 days). Tumor burden is assessed by luciferase bioluminescent imaging. Mouse weights are recorded weekly in conjunction with imaging. Circulating engineered T cell persistence isenumerated and phenotyped (e.g., for expression of CD69, CD25, Ki67) using flow cytometry at weeks 1, 3, 5, and 7 post transfer.

[0486] Maximal tumor burden is based on the parameters of mouse body condition score, general appearance, and respiration, all of which are given a score based on observation. Tumor size is monitored over time for animals with solid tumors. Animals that reach a tumor burden score of 40 or 20% loss of original body weight are euthanized. Survival is monitored.

[0487] At sacrifice, spleen weight is recorded and major organs (heart, liver, lungs, kidneys, gut) preserved in optimum cutting temperature compound for histological analysis examining immune infiltrate and tissue damage. Cells are harvested from the blood, spleen, and bone marrow. Samples are assayed for phenotype as well as markers of activation (e.g., LAG3, CD25, HLA-DR) and functionality (e.g., granzyme B, ZFNy) by flow cytometry.J. EXAMPLE 5: Expression of engineered immune signaling constructs in primary human T cells

[0488] Expression constructs encoding the 323 engineered immune signaling constructs provided herein were synthesized (e.g., with sequences from SEQ ID NOs: 205-532). The expression constructs contained elements shown in FIG. 3, including a hEFl / HTLV promoter to drive expression, a signal peptide, an extracellular FLAG tag, the engineered immune signaling construct, a P2A sequence, an EGFP reporter, a T2A sequence, and a human DHFR resistance gene.

[0489] These constructs were then transfected individually into lentiviral production HEK293 cell lines, and 72 hours post transfection, culture medium from the respective viral production cell lines was harvested and filtered to obtain lentiviral supernatant.

[0490] One day after initiation of lentiviral production, frozen primary human T cells from two donors (d8089 and d6995) were thawed and cultured at 50,000 cells per well in 96-well plates containing X-VIVO15 medium with 5% human AB serum and 50 lU / ml IL-2. One well of T cells per donor was prepared per construct, as well as additional wells for untransduced cells and staining controls.

[0491] One day post cell thawing, T cells were activated using CD3 / CD28 dynabeads in the presence of IL-2 to prepare for viral transduction.

[0492] Two days post T cell thawing, filtered lentiviral supernatant from the 323 constructs was individually transferred into wells containing the activated T cells.

[0493] Two days post viral transduction, cells were additionally grown in Methotrexate (MTX) for selection of cells expressing the DHFR resistance gene.

[0494] At time points post transduction, an aliquot of the transduced cell culture was stained with anti -FLAG antibodies for flow cytometric analysis. FLAG versus GFP expression was used as a proxy for surface expression compared to transduction efficiency, respectively.

[0495] At day 5 post-transduction, the percentage of FLAG+ cells and GFP+ cells were measured (FIG. 4). Higher expression of GFP was observed, potentially reflecting difficulty of staining, differences in surface versus intracellular levels of markers, and / or sensitivity of the assays. However, analysis indicated that the percentage of GFP+ and FLAG+ cells were highly correlated, indicating surface expression correlated with transduction rates (FIG. 5). For one of the donors, expression was also analyzed based on the construct extracellular domain (FIG. 6A) or intracellular domain (FIG. 6B) to evaluate potential effects of each on surface expression.K. EXAMPLE 6: Engineered immune signaling constructs can elicit gamma chain signaling and enhance survival of primary human T cells in cis and / or in trans

[0496] To compare the ability of engineered immune signaling constructs to stimulate T cells expressing the construct (in cis) and other T cells not expressing the construct (in trans), an assay was performed to measure phosphorylation of STAT5, which is one of the main signaling proteins downstream of the common gamma-chain(gc) receptors.

[0497] A schematic of the experiment is provided in FIG. 7. Primary human donor T cells were transduced, and methotrexate selection was conducted as described for EXAMPLE 5. For the pSTAT5 and survival assays, 50,000 EGFP+ cells for each assay were expanded and transduced wells of cells were mixed with 50,000 fresh untransduced (UTD) cells labelled with CellTrace Violet in the absence of IL-2. Depending on the transduction rate and cellular expansion, the amount of EGFP- cells added to the assay could vary across samples to some extent. After the cells were mixed, fresh culture medium (without IL-2) was added to a final volume of 200pL per well. 24h post-assay set up, the cells were resuspended, and 50pl (out of total 200pl) of cells + media was removed per well to run the pSTAT5 intracellular staining. The rest of the culture was kept for another 6 days to obtain the final survival data and stain for memory phenotypes. All laboratory analysis was performed via flow cytometry.

[0498] EGFP reporter expression was correlated across the two donors, and a positive correlation of EGFP and FLAG tag expression was observed (FIG. 8).

[0499] At day 14 post-transduction, pSTAT5 positive cells were measured to evaluate effects of the engineered immune signaling constructs in cis (GFP+; were also CellTrace Violet negative) and in trans (GFP-; were also CellTrace Violet positive).

[0500] STAT5 phosphorylation generally correlated between the two donors (FIG. 9).

[0501] STAT5 phosphorylation elicited in cis is shown for the two donors grouped by the intracellular signaling domain in FIG. 10, and by extracellular (e.g., common gamma chainbinding) domain in FIG. 11.

[0502] The percent of cells exhibiting positive staining for STAT5 phosphorylation was highest for constructs with IL2Rb or IL7R intracellular signaling domains on average, however this varied between individual constructs, and STAT5 phosphorylation higher than the untransduced control (dashed line) was observed for some constructs in each group (FIG. 10). Similarly, when grouped by extracellular (gamma chain-binding) domain, average STAT5 phosphorylation varied between groups, with certain constructs showing favorable STAT5 phosphorylation from each group (FIG. 11).

[0503] 7ra« -stimulation above untransduced control background was observed for constructs with various tethered cytokines, but not for constructs comprising the gamma chain-binding scFv (ycFab) (FIG. 12). STAT5 phosphorylation for tethered cytokines varied between constructs, and without wishing to be bound by theory, may be dependent on other construct design elements (e g., linker, hinge, and / or transmembrane domain).

[0504] Illustrative constructs eliciting high STAT5 phosphorylation in cis are shown in TABLE 1. Illustrative constructs eliciting high STAT5 phosphorylation in trans are shown in TABLE 2. Illustrative constructs showing high STAT5 phosphorylation both in cis and in trans are bolded in the tables.

[0505] Survival was evaluated on day 20 following 7 days of IL-2 starvation to evaluate the ability of the engineered immune signaling constructs to enhance T cell survival in IL-2 starvation conditions. Data were processed into a fold change in viable cells as compared to untransduced control (Neg Ctrl). As shown in FIG. 13, a number of engineered immune signaling constructs enhanced survival of primary human T cells in cis (left panel) or in trans (right panel).

[0506] Illustrative constructs enhancing survival in cis are shown in TABLE 3. Illustrative constructs enhancing survival in trans are shown in TABLE 4. Illustrative constructs enhancing survival both in cis and in trans are bolded in the tables.

[0507] Data were plotted to compare the fold change in survival between GFP+ and GFP- cells for each construct (FIG. 14). The results suggest, for example, that certain constructs enhanced survival from IL-2 starvation both in cis and in trans (e.g., upper right quadrant), or preferentially in cis rather than in trans (e g., lower right quadrant).

[0508] Illustrative constructs enhancing survival both in cis and in trans for donor 6995 are shown in TABLE 5. Illustrative constructs enhancing survival both in cis and in trans for donor 8089 are shown in TABLE 6. Illustrative constructs enhancing survival both in cis and in trans for both donors are bolded in the tables.

[0509] When STAT5 phosphorylation and fold change in survival were compared, a number of engineered immune signaling constructs were shown to induce both pSTAT5 upregulation and enhanced survival (FIG. 15).

[0510] Illustrative constructs enhancing survival and pSTAT5 in cis are shown in TABLE 7. Illustrative constructs enhancing survival and pSTAT5 in trans are shown in TABLE 8.Illustrative constructs enhancing survival and pSTAT5 for both donors either in cis or in trans are bolded in their respective tables.L. EXAMPLE 7: Effector / memory phenotypes and CD4 / CD8 ratio for cells expressing illustrative engineered immune signaling constructs following IL-2 starvation

[0511] Following the IL-2 starvation assay described in EXAMPLE 6, select samples were stained for CD4, CD8, CD62L, and CD45RA, and evaluated by flow cytometry.

[0512] T cell effector / memory subsets were identified in the flow cytometry data based on the following gating strategy: stem cell memory (Tscm; CD62L+ CD45RA+), central memory (Tcm; CD62L+, CD45RA-), effector memory (Tem; CD62L-, CD45RA-) and terminal effector memory CD45RA+ (Temra; CD62L-, CD45RA+).

[0513] As shown in FIG. 16, different proportions of effector / memory subsets were observed for cells expressing different engineered immune signaling constructs following the IL-2 starvation assay, suggesting that certain constructs may influence effector / memory phenotype.

[0514] As shown in FIG. 17, different ratios of CD4+ to CD8+ were observed for cells expressing different engineered immune signaling constructs following the IL-2 starvation assay, suggesting that certain constructs may favor survival or expansion of CD4+ and / or CD8+ T cells.

[0515] Additional data for certain constructs are shown in FIG. 18.

[0516] Domains of the constructs depicted in FIG. 17 are shown in TABLE 9 and TABLE10IX. ILLUSTRATIVE SEQUENCES

[0517] TABLE 11: illustrative sequences. The sequences in table 11 can each be a synthetic construct.-Ill-

[0518] The degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs designed for this purpose, such as global or local alignment algorithms. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, Needle (EMBOSS), Stretcher (EMBOSS), GGEARCH2SEQ, Water (EMBOSS), Matcher (EMBOSS), LALIGN, SSEARCH2SEQ, or another suitable method or algorithm. A global alignment algorithm, such as a Needleman and Wunsch algorithm, can be used to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps Default settings can be used.

[0519] To generate similarity scores for two amino acid sequences, scoring matrices can be used that assign positive scores for some non-identical amino acids (e g., amino acids withsimilar physio-chemical properties and / or amino acids that exhibit frequent substitutions in orthologs, homologs, or paralogs), Non -limiting examples of scoring matrices include PAM30, PAM70, PAM250, BLOSUM45, BLOSUM50, BLOUM62, BLOSUM80, and BLOSUM90.

[0520] Amino acids can include genetically encoded and non-genetically encoded occurring amino acids. Amino acids can include naturally occurring and non-naturally occurring amino acids. Amino acids can be L forms or D forms. Substitutions disclosed herein can include conservative and / or non-conservative amino acid substitutions. A conservative amino acid substitution can be a substitution of one amino acid for another amino acid of similar biochemical properties (e.g., charge, size, and / or hydrophobicity). A non-conservative amino acid substitution can be a substitution of one amino acid for another amino acid with different biochemical properties (e.g., charge, size, and / or hydrophobicity). A conservative amino acid change can be, for example, a substitution that has minimal effect on the secondary or tertiary structure of a polypeptide. A conservative amino acid change can be an amino acid change from one hydrophilic amino acid to another hydrophilic amino acid. Hydrophilic amino acids can include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gin (Q), Asp (D), Lys (K) and Arg (R). A conservative amino acid change can be an amino acid change from one hydrophobic amino acid to another hydrophilic amino acid. Hydrophobic amino acids can include He (I), Phe (F), Vai (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), and Pro (P). A conservative amino acid change can be an amino acid change from one acidic amino acid to another acidic amino acid. Acidic amino acids can include Glu (E) and Asp (D). A conservative amino acid change can be an amino acid change from one basic amino acid to another basic amino acid. Basic amino acids can include His (H), Arg (R) and Lys (K). A conservative amino acid change can be an amino acid change from one polar amino acid to another polar amino acid. Polar amino acids can include Asn (N), Gin (Q), Ser (S) and Thr (T). A conservative amino acid change can be an amino acid change from one nonpolar amino acid to another nonpolar amino acid. Nonpolar amino acids can include Leu (L), Val(V), He (I), Met (M), Gly (G) and Ala (A). A conservative amino acid change can be an amino acid change from one aromatic amino acid to another aromatic amino acid. Aromatic amino acids can include Phe (F), Tyr (Y) and Trp (W). A conservative amino acid change can be an amino acid change from one aliphatic amino acid to another aliphatic amino acid. Aliphatic amino acids can include Ala (A), Vai (V), Leu (L) and He (I). In some embodiments, a conservative amino acid substitution is an amino acid change from one amino acid to another amino acid within one of the following groups: Group I: Ala, Pro, Gly, Gin, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Vai, lie, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of inducing signaling of a common gamma chain family cytokine receptor in a cell, the method comprising expressing an engineered immune signaling construct in the cell, wherein the engineered immune signaling construct comprises:(a) an extracellular region that comprises a common gamma chain-binding domain;(b) a transmembrane region; and(c) an intracellular region comprising an intracellular signaling domain of a cytokine receptor subunit, wherein the cytokine receptor subunit is a common gamma chain family cytokine receptor subunit; wherein the common gamma chain-binding domain is heterologous to the cytokine receptor subunit, and the signaling is induced in a soluble ligand-independent manner.

2. The method of claim 1, wherein the signaling is constitutively induced if the cell expresses the common gamma chain.

3. The method of claim 1, wherein the extracellular region or the gamma chain binding domain consists essentially of (i) the common gamma chain-binding domain, and (ii) optionally a hinge, linker, and / or spacer.

4. The method of claim 1, wherein the extracellular region or the gamma chain binding domain consists of (i) the common gamma chain-binding domain, and (ii) optionally a hinge, linker, and / or spacer.

5. The method of claim 1, wherein the extracellular region or the gamma chain-binding domain comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1-91.

6. The method of claim 1, wherein the extracellular region or the gamma chain-binding domain comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 1-91.

7. The method of claim 1, wherein the common gamma chain-binding domain comprises a cytokine or a gamma chain-binding domain thereof.

8. The method of claim 1, wherein the common gamma chain-binding domain comprises a wild type cytokine or a gamma chain-binding domain thereof.

9. The method of claim 1, wherein the common gamma chain-binding domain comprises a mutein cytokine or a gamma chain-binding domain thereof.

10. The method of claim 1, wherein the common gamma chain-binding domain comprises IL- 7 or a gamma chain-binding domain thereof.

11. The method of claim 1, wherein the common gamma chain-binding domain comprises IL- 2 or a gamma chain-binding domain thereof.

12. The method of claim 1, wherein the common gamma chain-binding domain comprises IL- 4 or a gamma chain-binding domain thereof.

13. The method of claim 1, wherein the common gamma chain-binding domain comprises IL- 9 or a gamma chain-binding domain thereof.

14. The method of claim 1, wherein the common gamma chain-binding domain comprises IL- 15 or a gamma chain-binding domain thereof.

15. The method of claim 1, wherein the common gamma chain-binding domain comprises IL- 21 or a gamma chain-binding domain thereof.

16. The method of claim 1, wherein the common gamma chain-binding domain comprises TSLP or a TSLP receptor (TSLPR) chain-binding domain thereof.

17. The method of claim 1, wherein the common gamma chain-binding domain comprises an antibody or antigen-binding fragment thereof.

18. The method of claim 1, wherein the common gamma chain-binding domain comprises a single chain variable fragment (scFv).

19. The method of claim 1, wherein the common gamma chain-binding domain comprises a fragment antigen-binding (Fab).

20. The method of claim 1, wherein the common gamma chain-binding domain comprises a single domain antibody.

21. The method of claim 1, wherein the transmembrane region comprises a transmembrane domain of an immune receptor.

22. The method of claim 1, wherein the transmembrane region comprises a transmembrane domain of a common gamma chain family cytokine receptor.

23. The method of claim 1, wherein the transmembrane region comprises a transmembrane domain of IL-2Ra, IL-2Rb, IL-4Ra, IL-7Ra, IL-9Ra, IL-15Ra, or IL-21R.

24. The method of claim 1, wherein the transmembrane region comprises an IL-7Ra transmembrane domain.

25. The method of claim 1, wherein the transmembrane region comprises a CD8a transmembrane domain.

26. The method of claim 1, wherein the transmembrane region comprises a CD28 transmembrane domain.

27. The method of claim 1, wherein the transmembrane region comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 92-104.

28. The method of claim 1, wherein the transmembrane region comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 92-104.

29. The method of claim 1, wherein the intracellular signaling domain comprises a JAK1 binding domain.

30. The method of claim 1, wherein the intracellular signaling domain comprises a BOX1 motif.

31. The method of claim 1, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-2Rb, optionally wherein the signaling is IL-2R signaling.

32. The method of claim 1, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-7Ra, optionally wherein the signaling is IL-7R signaling.

33. The method of claim 1, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-9Ra, optionally wherein the signaling is IL-9R signaling.

34. The method of claim 1, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-4Ra, optionally wherein the signaling is IL-4R signaling.

35. The method of claim 1, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-21R, optionally wherein the signaling is IL-21R signaling.

36. The method of claim 1, wherein the intracellular region or the intracellular signaling domain comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 105-115 and 533-540.

37. The method of claim 1, wherein the intracellular region or the intracellular signaling domain comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 105-115 and 533-540.

38. The method of claim 1, wherein the engineered immune signaling construct consists essentially of the extracellular region, the transmembrane region, and the intracellular region.

39. The method of claim 1, wherein the engineered immune signaling construct consists of the extracellular region, the transmembrane region, and the intracellular region.

40. The method of claim 1, wherein the extracellular region lacks a ligand-binding domain of a cytokine receptor.

41. The method of claim 1, wherein the extracellular region lacks a ligand-binding domain of an immune inhibitory receptor.

42. The method of claim 1, wherein the method increases signaling of the common gamma chain family cytokine receptor in the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct.

43. The method of claim 1, wherein the method increases signaling of the common gamma chain family cytokine receptor in a bystander cell that lacks the engineered immune signaling construct by at least 5%, as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signaling construct.

44. The method of claim 42, wherein the increase in signaling is as determined by a STAT5 phosphorylation assay.

45. The method of claim 1, wherein the method enhances survival or persistence of the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct.

46. The method of claim 1, wherein the method enhances survival or persistence of a bystander cell that lacks the engineered immune signaling construct by at least 5% as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signaling construct.

47. The method of claim 45, wherein the survival is as determined by an IL-2 starvation assay.

48. The method of claim 1, wherein the method enhances proliferation of the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct.

49. The method of claim 1, wherein the method enhances proliferation of a bystander cell that lacks the engineered immune signaling construct by at least 5% as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signaling construct.

50. The method of claim 48, wherein the proliferation is as determined by a lymphoproliferation assay.

51. An engineered immune signaling construct comprising:(a) an extracellular region comprising a common gamma chain-binding domain, wherein the extracellular region lacks a soluble ligand-binding domain of a receptor;(b) a transmembrane region; and(c) an intracellular region comprising an intracellular signaling domain of a cytokine receptor subunit, wherein the cytokine receptor subunit is a common gamma chain family cytokine receptor subunit and is heterologous to the common gamma chain-binding domain.

52. The engineered immune signaling construct of claim 51, wherein the extracellular region consists essentially of (i) the common gamma chain-binding domain, and (ii) optionally a hinge, linker, and / or spacer.

53. The engineered immune signaling construct of claim 51, wherein the extracellular region consists of (i) the common gamma chain-binding domain, and (ii) optionally a hinge, linker, and / or spacer.

54. The engineered immune signaling construct of claim 51, wherein the extracellular region or the gamma chain-binding domain comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 1-91.

55. The engineered immune signaling construct of claim 51, wherein the extracellular region or the gamma chain-binding domain comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 1-91.

56. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises a cytokine or a gamma chain-binding domain thereof.

57. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises a wild type cytokine or a gamma chain-binding domain thereof.

58. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises a mutein cytokine or a gamma chain-binding domain thereof.

59. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises IL-7 or a gamma chain-binding domain thereof.

60. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises IL-2 or a gamma chain-binding domain thereof.

61. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises IL-4 or a gamma chain-binding domain thereof.

62. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises IL-9 or a gamma chain-binding domain thereof.

63. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises IL-15 or a gamma chain-binding domain thereof.

64. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises IL-21 or a gamma chain-binding domain thereof.

65. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises TSLP or a TSLP receptor (TSLPR) chain-binding domain thereof.

66. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises an antibody or antigen-binding fragment thereof.

67. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises a single chain variable fragment (scFv).

68. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises a fragment antigen-binding (Fab).

69. The engineered immune signaling construct of claim 51, wherein the common gamma chain-binding domain comprises a single domain antibody.

70. The engineered immune signaling construct of claim 51, wherein the transmembrane region comprises a transmembrane domain of an immune receptor.

71. The engineered immune signaling construct of claim 51, wherein the transmembrane region comprises a transmembrane domain of a cytokine receptor.

72. The engineered immune signaling construct of claim 51, wherein the transmembrane region comprises a transmembrane domain of a common gamma chain family cytokine receptor.

73. The engineered immune signaling construct of claim 51, wherein the transmembrane region comprises a transmembrane domain of IL-2Ra, IL-2Rb, IL-4Ra, IL-7Ra, IL-9Ra, IL-15Ra, or IL-21R.

74. The engineered immune signaling construct of claim 51, wherein the transmembrane region comprises an IL-7Ra transmembrane domain.

75. The engineered immune signaling construct of claim 51, wherein the transmembrane region comprises a CD8a transmembrane domain.

76. The engineered immune signaling construct of claim 51, wherein the transmembrane region comprises a CD28 transmembrane domain.

77. The engineered immune signaling construct of claim 51, wherein the transmembrane region comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 92-104.

78. The engineered immune signaling construct of claim 51, wherein the transmembrane region comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 92-104.

79. The engineered immune signaling construct of claim 51, wherein the intracellular signaling domain comprises a JAK1 binding domain.

80. The engineered immune signaling construct of claim 51, wherein the intracellular signaling domain comprises a BOX1 motif.

81. The engineered immune signaling construct of claim 51 , wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-2Rb, optionally wherein the signaling is IL-2R signaling.

82. The engineered immune signaling construct of claim 51, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-7Ra, optionally wherein the signaling is IL-7R signaling.

83. The engineered immune signaling construct of claim 51, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-9Ra, optionally wherein the signaling is IL-9R signaling.

84. The engineered immune signaling construct of claim 51, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-4Ra, optionally wherein the signaling is IL-4R signaling.

85. The engineered immune signaling construct of claim 51, wherein the intracellular signaling domain comprises an intracellular signaling domain of IL-21R, optionally wherein the signaling is IL-21R signaling.

86. The engineered immune signaling construct of claim 51, wherein the intracellular region or the intracellular signaling domain comprises, consists essentially of, or consists of an amino acid sequence with at least 90% sequence identity to any one of SEQ ID NOs: 105- 115 and 533-540.

87. The engineered immune signaling construct of claim 51, wherein the intracellular region or the intracellular signaling domain comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 105-115 and 533-540.

88. The engineered immune signaling construct of claim 51, wherein the engineered immune signaling construct consists essentially of the extracellular region, the transmembrane region, and the intracellular region.

89. The engineered immune signaling construct of claim 51, wherein the engineered immune signaling construct consists of the extracellular region, the transmembrane region, and the intracellular region.

90. The engineered immune signaling construct of claim 51, wherein the extracellular region lacks a soluble ligand-binding domain.

91. A polynucleotide encoding the engineered immune signaling construct of claim 51.

92. A vector comprising the polynucleotide of claim 91.

93. The vector of claim 92, wherein the vector is a viral vector.

94. The vector of claim 92, wherein the vector is a non-viral vector.

95. A method of making an engineered cell, the method comprising contacting a cell with the vector of claim 92.

96. The method of claim 95, wherein the contacting is in vitro or ex vivo.

97. The method of claim 95, wherein the contacting is in vivo.

98. A cell expressing the engineered immune signaling construct of claim 51.

99. The cell of claim 98, wherein the cell is an immune cell.

100. The cell of claim 98, wherein the cell is a lymphocyte.

101. The cell of claim 98, wherein the cell is a T cell.

102. The cell of claim 98, wherein the cell is an NK cell.

103. The cell of claim 98, wherein the cell expresses an exogenous antigen-recognition receptor.

104. The cell of claim 103, wherein the exogenous antigen-recognition receptor is a chimeric antigen receptor.

105. The cell of claim 103, wherein the exogenous antigen-recognition receptor is a T cell receptor.

106. The cell of claim 104, wherein if the cell expresses common gamma chain, the engineered immune signaling construct constitutively induces signaling of the intracellular signaling domain in the cell.

107. The cell of claim 104, wherein the cell expresses common gamma chain.

108. The cell of claim 107, wherein the engineered immune signaling construct constitutively binds to the common gamma chain.

109. The cell of claim 104, wherein the cell expresses common gamma chain, and the engineered immune signaling construct constitutively induces signaling of the intracellular signaling domain in the cell.

110. The cell of claim 104, wherein the engineered immune signaling construct increases signaling of a common gamma chain family cytokine receptor comprising the cytokine receptor subunit and common gamma chain in the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct.

111. The cell of claim 104, wherein the engineered immune signaling construct increases signaling of a gamma chain family cytokine receptor in a bystander cell that lacks the engineered immune signaling construct by at least 5%, as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signaling construct.

112. The cell of claim 110, wherein the increase in signaling is as determined by a STAT5 phosphorylation assay.

113. The cell of claim 104, wherein the engineered immune signaling construct enhances survival or persistence of the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct.

114. The cell of claim 104, wherein the engineered immune signaling construct enhances survival or persistence of a bystander cell that lacks the engineered immune signaling construct by at least 5% as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signaling construct.

115. The cell of claim 113, wherein the survival is as determined by an IL-2 starvation assay.

116. The cell of claim 104, wherein the engineered immune signaling construct enhances proliferation of the cell by at least 5% as compared to a corresponding control cell that lacks the engineered immune signaling construct.

117. The cell of claim 104, wherein the engineered immune signaling construct enhances proliferation of a bystander cell that lacks the engineered immune signaling construct by at least 5% as compared to a corresponding control condition in which both the cell and the bystander cell lack the engineered immune signaling construct.

118. The cell of claim 116, wherein the proliferation is as determined by a lymphoproliferation assay.

119. A pharmaceutical composition comprising the polynucleotide of claim 91 and a pharmaceutically-acceptable excipient, vehicle, carrier, or diluent.

120. A pharmaceutical composition comprising the cell of claim 98 and a pharmaceutically- acceptable excipient, vehicle, carrier, or diluent.

121. A method of treating a condition in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of claim 120.

122. The method of claim 121, wherein the condition comprises or is cancer.

123. The method of claim 121, wherein the condition comprises or is an infectious disease.

124. The method of claim 121, wherein the condition comprises or is an autoimmune disease.

125. A method of enhancing the efficacy of a therapeutic regimen that comprises an engineered cell, the method comprising expressing the engineered immune signaling construct of claim 51 in the engineered cell, thereby enhancing the efficacy of the therapeutic regimen.

126. A method of enhancing survival, proliferation, differentiation, persistence, or effector function of an engineered cell, the method comprising expressing the engineered immune signaling construct of claim 51 in the engineered cell, thereby enhancing the survival, proliferation, differentiation, persistence, or effector function of the engineered cell.

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