Regulatable boosters for engineered receptor immune cells

Abstract

Provided herein are immune cells configured to be activated by an antigen or independently by a small molecule, as well as related compositions and methods, such as methods of use in treating cancer.

Description

[0001] REGULATABLE BOOSTERS FOR ENGINEERED RECEPTOR IMMUNE CELLS

[0002] RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 651,619 filed on May 24, 2024; U.S. Provisional Application No. 63 / 651,622 filed on May 24, 2024; and U.S. Provisional Application No. 63 / 683,241 filed on August 14, 2024, the contents of which are incorporated herein by reference in their entireties.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 061250-561001WO, created on April 23, 2025, and is 313 kilobytes in size. The information in electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] T cells are regulated in complex ways, but the three most important groups of stimuli that generate and maintain an antigen-specific response in T cells are: (i) antigen, otherwise called signal 1; (ii) costimulation, otherwise called signal 2; and (iii) cytokine, otherwise called signal 3. These three major stimuli interact with each other, as well as additional sources of stimulation such as innate immune signaling molecules (e.g., toll-like receptors), checkpoint receptors (e.g., PD-1, CTLA-4), and homing receptors (e.g., CCL19). Signal 1 (antigen) is arguably the most important stimulus because it not only initiates the adaptive immune response but also maintains it and provides the means of recalling the response upon subsequent exposure to antigen. Antigen is the essence of specificity in the adaptive immune response and the basis for vaccination. Thus, signal 1 is not only the key mediator via TCRs of activation in naive and memory T cells but also stokes the fire of the immune response, once ignited.

[0008] One of the most active areas in CAR-T cell engineering involves boosting the performance of immune cells. Some approaches utilize exogenous agents, such as cytokines, lympho-depleting chemotherapeutics, STING agonists, and vaccines to enhance function, including persistence of immune cells in the body.

[0009] Onboard engineering of boosters is another approach. Onboard boosters are especially desirable because they mitigate issues of systemic toxicity, biodistribution, and excretion of certain exogenous agents. A variety of approaches have been explored preclinically and in the clinic, including (i) addition of genes that encode signaling molecules that provide signal 2 (costimulation); (ii) engineering genes that “armor” CAR-Ts via expression of cytokines of various types and in various configurations (e.g., IL-15; Lands et al., 2021 PMID: 33156338 ); (iii) addition of elements of the innate immune response that are regulated by antigen or other means (e.g., MyD88; Collinson-Pautz et al., 2019 PMID: 30816327); (iv) expression of inhibitors of apoptosis (e.g., RNAi directed at FAS, etc.; Santoro et al. J ImmunoTher of Cancer 2021;9:doi: 10.1136 / jitc-2021-SITC2021.21); (v) expression of inhibitors of checkpoints (e.g., PD-1 dominant-negative constructs; Liu et al., 2016 PMID: 26979791); and (vi) inhibitors of the TGFP pathway (e.g., dominant-negative TGFBR2; Kloss et al., 2018 PMID: 29807781).

[0010] In addition, some efforts have focused on increasing sensitivity to antigen by improvement in ligand-binding domain affinity / sensitivity or structure (Xu et al., 2020 PMID: 32768859; Calderon et al., 2022 PMID: 31707680).

[0011] However, despite the limited antigens available to T cells infused in the blood to treat solid tumors, few efforts have explored boosting signal 1 directly beyond engineering of the scFvs and intracellular domains of CARs. Although some have appreciated the possible benefits of tonic (ligand-independent) signaling in T cells (Chen et al., 2023 PMID: 36882513), generally researchers have viewed tonic signaling as a problematic feature of CARs that must be overcome (Gennert et al., 2021 PMID: 34285077; Calderon et al., 2022 PMID: 31707680). At best, tonic signaling has been viewed as a double-edged sword.

[0012] Some efforts to boost performance of CAR-Ts have centered on downstream elements of TCR signaling (e.g, LAT, SLP76, LCK, etc.; Mock et al., 2022 PMID: 34039676; Tousley et al., 2023 PMID: 36890224). These have in some cases produced new designs for CARs that extend beyond the classical ITAMs of TCRs.

[0013] Finally, drug discovery has also considered methods to control T cell response via exogenous molecules that regulate the activation process in cells engineered with proteins that bind such agents (e.g., rapamycin and its analogs; Leung et al., 2019 PMID: 31039141; Foster et al., 2017 PMID: 28697888; Jaijour et al., US patent 10,457,731; Brogdon et al., US patent 10,287,354).

[0014] Antigen (signal 1) is the ignition and fuel for T cell responses. Chimeric antigen receptors co-opt TCR signaling domains (e.g., ITAM elements) to redirect T cell response to specific antigens. Beyond efforts to improve sensitivity of the CAR ligand-binding domains and enhance the TCR ITAMs through added intracellular domains from other signaling proteins (e.g., CD28 and 4-1BB), little effort has been expended to further manipulate signal 1, other than vaccination (e.g., CLDN6; Mackensen et al., 2023 PMID: 37872225). Most cell therapy engineering is designed to preserve antigen dependence, while boosting sensitivity, persistence, etc.

[0015] Though substantial energy has been applied to boost antigen sensitivity and T cell response, few efforts have been directed at regulating the tonic component or mimicking the antigen stimulus. The invention provides compositions and methods to, for example, boost function of engineered immune cells by manipulating antigen signaling in a tunable fashion.

[0016] SUMMARY

[0017] The present inventors have recognized that solid tumors pose unique challenges compared to blood cancers; for example: (i) access to tumor tissues that express target antigen is highly restricted by the blood vessel walls; and (ii) with limiting antigen, it is unclear how antigen-dependent boosters can be brought into action. The invention described here addresses this problem.

[0018] Many signals enhance the response to antigen once an antigen-stimulus is encountered. Although initiation of the adaptive immune responds depends on exposure to antigen, the amplification of the response is equally important for proper immune function. A variety of molecules are involved in amplifying immune responses. Many of these molecules are typically grouped as signal 2 (co-stimulatory) or signal 3 (cytokine) proteins and they stimulate intracellular signaling pathways that have both common and distinctive elements. When effector cells, e.g., T cells, experience stimulation by all three signaling agents, i.e., 1) antigen;

[0019] 2) co-stimulation; and 3), an effective immune response may be maximized. Each of these stimuli activates downstream signaling cascades within the effector cells that are partly independent and partly overlapping. For example, signal 1 is biased toward signaling through the NFAT transcription factor and signal 2 favors activation via NF-KB. Signal 3 primarily signals through the JAK / STAT pathways. Despite differences in the details of the signaling pathways stimulated by these three key immune signals, they often ultimately converge on a set of downstream effector mechanisms that control entry into the cell cycle, proliferation, survival, secretion of additional stimulatory factors, adhesion, migration, and other aspects of immune function. Signal 3, often a cytokine, is perhaps the most significant amplifier of the response initiated by signal 1. A normal immune effector cell such as a T cell, responds to cytokines such as IL-2, by maintaining a state of balance between proliferation and apoptosis. In vitro, at least, T cells exposed to cytokines without antigen typically survive but do not proliferate. However, in the presence of antigen, activation occurs and the T cells begin to divide and express other characteristics of activated effector cells. Once activated by signal 1, T cells become hyper- responsive to signal 3, including IL-2, in part by expression of higher levels of IL-2R (CD25 protein) on their surface. Under certain conditions, signal 3 ligands can maintain the ongoing immune response, including proliferation, even in the absence of continuing exposure to antigen. The process of engineering cell therapies with signal 2 or 3 enhancers is sometimes referred to as “armoring.” In all cases, these “armored CARs” are designed to maintain antigen-dependence. Thus, to come into play, the cells must either be activated in advance of patient-infusion or must encounter antigen in the patient’s body.

[0020] Accordingly, provided herein is, in one aspect, an immune cell configured to be activated by an antigen or independently by a small molecule (e.g., a switch molecule for a switchable receptor or an inducer molecule for an inducible receptor).

[0021] In embodiments, the immune cell comprises an artificial receptor configured to specifically activate in response to an antigen and independently to a small molecule.

[0022] In embodiments, the artificial receptor is a multipartite chimeric antigen receptor (CAR).

[0023] In embodiments, the artificial receptor is a multipartite chimeric antigen receptor (CAR).

[0024] In embodiments, the multipartite CAR comprises: a first polypeptide chain comprising optionally a hinge domain, a transmembrane domain, at least one first switch domain, and a scaffolding intracellular domain (e.g., a LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3(^, 4-1BB, ZAP70, or GADs domain); and a second polypeptide chain comprising an antigen-binding domain (e.g., an scFv), a transmembrane domain, one or more costimulatory intracellular domains (e.g., a CD28 domain or a 41BB domain), and a signaling intracellular domain (e.g., a CD3zeta domain), optionally further comprising at least one second switch domain, wherein the at least one first switch domain and the at least one second switch domain when present are configured to selectively dimerize in the presence of a switch molecule (e.g., rapamycin or a rapalog or an estrogen receptor modulator, e.g., tamoxifen).

[0025] In embodiments, the artificial receptor is a multipartite T cell receptor (TCR).

[0026] In embodiments, the multipartite TCR comprises: a first polypeptide chain comprising optionally a hinge domain, a transmembrane domain, at least one first switch domain, and a scaffolding intracellular domain (e.g., a LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4-1BB, ZAP70, or GADs domain); and a second polypeptide chain comprising a transmembrane domain, an signaling intracellular domain (e.g., a CD3zeta domain), and optionally further comprising at least one second switch domain; and a third polypeptide chain comprising an antigen-binding domain (e.g., a TCR extracellular domain) and a transmembrane domain, wherein the at least one first switch domain and the at least one second switch domain when present are configured to selectively dimerize in the presence of a switch molecule (e.g., rapamycin or a rapalog or an estrogen receptor modulator, e.g., tamoxifen).

[0027] In embodiments, the first and second switch domains are selected from the pairs disclosed in Table 1 and the small molecule is the corresponding switch molecule in Table 1, or a functional analogue thereof.

[0028] In embodiments, the artificial receptor is an inducible multipartite chimeric antigen receptor (CAR).

[0029] In embodiments, such that the immune cell is configured to express the inducible CAR, the immune cell comprises: a first polynucleotide operatively linked to an inducible promoter, the first polynucleotide encoding a first polypeptide chain comprising, at least one first switch domain, and a scaffolding intracellular domain (e.g., aLAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4-1BB, ZAP70, or GADs domain), and optionally a hinge domain, and / or a transmembrane domain wherein the inducible promoter is configured to induce expression of the first polypeptide chain in response to an inducer molecule; and a second polynucleotide encoding a second polypeptide chain comprising an antigen-binding domain (e.g., an scFv), a transmembrane domain, one or more costimulatory intracellular domains (e.g., a CD28 domain or a 41BB domain), and an signaling intracellular domain (e.g., a CD3zeta domain), optionally further comprising at least one second switch domain.

[0030] In embodiments, the artificial receptor is an inducible multipartite T cell receptor (TCR).

[0031] In embodiments, such that the immune cell is configured to express the inducible TCR, the immune cell comprises: a first polynucleotide operatively linked to an inducible promoter, the first polynucleotide encoding a first polypeptide chain comprising at least one switch domain, and a intracellular domain (e.g., a LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3(^, 4-1BB, ZAP70, or GADs domain), and optionally a hinge domain, and / or a transmembrane domain wherein the inducible promoter is configured to induce expression of the first polypeptide chain in response to an inducer molecule; and a second polynucleotide encoding a second polypeptide chain comprising a transmembrane domain, an signaling intracellular domain (e.g., a CD3zeta domain), optionally further comprising at least one second switch domain; and a third polynucleotide encoding a third polypeptide chain comprising an antigen-binding domain (e.g., a TCR extracellular domain) and a transmembrane domain.

[0032] In embodiments, the first polypeptide comprises at least two first switch domains selected from Table 1 and / or the second polypeptide comprises at least two second switch domains selected from Table 1.

[0033] In embodiments, the at least two first switch domains comprise a pair of switch domains disclosed in a row of Table 1 and / or the at least two second switch domains comprise a pair of switch domains disclosed in a row of Table 1.

[0034] In embodiments, the inducible promoter is selected from chemically / biochemically- regulated and physically-regulated promoters such as alcohol -regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline responsive promoter systems, which include a tetracycline repressor protein (TetR, or TetRKRAB), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA), and a tetracycline operator sequence (tetO) and a reverse tetracycline transactivator fusion protein (rtTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid 25 receptor superfamily), metal -regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadi azole (BTH)), temperature / heat- inducible promoters (e.g., heat shock promoters), pH-regulated promoters, and light-regulated promoters.

[0035] In embodiments, the scaffolding intracellular domain is selected from LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4-1BB, ZAP70, or GADs or a functional variant thereof.

[0036] In embodiments, the one or more costimulatory intracellular domains is selected from the group consisting of IL-2RP, Fc Receptor gamma (FcRy), Fc Receptor beta (FcRP), CD3g molecule gamma (CD3y), CD35, CD3s, CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen- 1 (LFA-1), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domains, or functional variants thereof.

[0037] In embodiments, the immune cell further comprises an inhibitory receptor.

[0038] In embodiments, the inhibitory receptor is specific to a target disclosed in Table 5.

[0039] In embodiments, the inhibitory receptor comprises an intracellular domain of a LILRB 1 and optionally the hinge and / or transmembrane domains of LILRB 1.

[0040] In embodiments, the second polypeptide comprises at least one second switch domain.

[0041] In embodiments, the second polypeptide does not comprise at least one second switch domain.

[0042] In embodiments, the immune cell is a T cell.

[0043] In embodiments, the immune cell is a natural killer (NK) cell.

[0044] The present disclosure provides a pharmaceutical composition comprising a population of immune cells disclosed herein.

[0045] The present disclosure provides a method of expanding an immune cell population, the method comprising providing a population of immune cells disclosed herein and contacting the immune cells with a small molecule from Table 1 to boost activation of the immune cells, thereby expanding the immune cell population.

[0046] The present disclosure provides a method of treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein and further administering a small molecule from Table 1 to boost activation of the immune cells.

[0047] In embodiments, the cancer is a solid tumor.

[0048] The present disclosure provides a polynucleotide or a plurality of polynucleotides encoding an artificial receptor, a multipartite chimeric antigen receptor (CAR), or a multipartite T cell receptor (TCR) as disclosed herein or one or more of the polypeptide chains thereof.

[0049] The present disclosure provides a vector comprising the polynucleotide or the plurality of polynucleotides described herein.

[0050] The present disclosure provides a host cell comprising the polynucleotide or the plurality of polynucleotides described herein.

[0051] Any cell, pharmaceutical composition, method, polynucleotide, or vector disclosed herein is applicable to any herein-disclosed cell, pharmaceutical composition, method, polynucleotide, or vector In other words, any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG. 1 is a schematic of a platform to induce activation of T cell Signal 1, independent of antigen engagement. TCR signaling molecule, such as LAT, is induced to dimerize with CAR via FKBP switch domains and small molecule Rimiducid (Rim), which triggers CAR signaling.

[0053] FIG. 2A is a graph depicting tonic immune cell signaling in Jurkat cells expressing the indicated signal 1 booster construct after 6 hours of treatment with or without rimiducid (Rim).

[0054] FIG. 2B is a graph depicting tonic immune cell signaling in Jurkat cells expressing the indicated signal 1 booster construct and MSLN CAR-2xFKBP after 6 hours of treatment with or without rimiducid (Rim).

[0055] FIG. 3A is a graph depicting tonic immune cell signaling in Jurkat cells expressing the LAT or the LAT mutant (G160D) signal 1 booster transfected with or without the MSLN C AR- 2xFKBP. Jurkat cells were incubated with increasing doses of Rim for 6 hours prior to measuring tonic signaling.

[0056] FIG. 3B is a graph depicting tonic immune cell signaling in Jurkat cells expressing the LAT or the LAT mutant (G160D) signal 1 booster transfected with or without the MSLN C AR- Ix FKBP. Jurkat cells were incubated with increasing doses of Rim for 6 hours prior to measuring tonic signaling.

[0057] FIG. 4 is a graph providing results from and a cartoon depicting tonic immune cell signaling in Jurkat cells expressing the LAT signal 1 booster transfected with or without the CAR. Immune cell signaling was also measured in Jurkat cells expressing the LAT signal 1 booster transfected with or without the CAR co-cultured with Hela target cells.

[0058] FIG. 5A is a graph depicting antigen dependent immune cell activation in Jurkat cells transfected with MSLN CAR-2xFKBP and indicated signal 1 booster construct. MSLN KO HeLa cells were transfected with increasing levels of MSLN mRNA to generate a titration of antigen level on target cells. Transfected Jurkat cells were cocultured with target cells for 6 hours without Rim.

[0059] FIG. 5B is a graph depicting antigen dependent immune cell activation and sensitivity in Jurkat cells transfected with MSLN CAR-2xFKBP and indicated signal 1 booster construct. MSLN KO HeLa cells were transfected with increasing levels of MSLN mRNA to generate a titration of antigen level on target cells. Transfected Jurkat cells were cocultured with target cells for 6 hours with 50nM Rim. FIG. 6A is a graph depicting tonic immune cell signaling in Jurkat cells expressing the MSLN CAR-2xFKBP or MSLN CAR with and without co-expression of the indicated LAT signal 1 booster . Jurkat cells were treated with or without 50nM Rim.

[0060] FIG. 6B is a graph depicting immune cell activation in Jurkat cells expressing the MSLN CAR-2xFKBP or MSLN CAR with and without co-expression of the indicated LAT signal 1 booster after co-culture with HeLa cells expressing MSLN antigen. Jurkat cells were treated with or without 50nM Rim.

[0061] FIG. 7A is a graph depicting antigen dependent immune cell activation and sensitivity in Jurkat cells transfected with MSLN activator CAR-2xFKBP, HLA-A*02 blocker CAR and a LAT signal 1 booster . MSLN KO HeLa cells were transfected with increasing levels of MSLN mRNA to generate a titration of antigen level on target cells. Transfected Jurkat cells were co-cultured with target cells for 6 hours without 50nM Rim.

[0062] FIG. 7B is a graph depicting antigen dependent immune cell activation and sensitivity in Jurkat cells transfected with a MSLN activator CAR, a HLA-A*02 blocker CAR and a LAT signal 1 booster . MSLN KO HeLa cells were transfected with increasing levels of MSLN mRNA to generate a titration of antigen level on target cells. Transfected Jurkat cells were cocultured with target cells for 6 hours with 50nM Rim.

[0063] FIG. 8A is a graph depicting antigen dependent immune cell blocking in Jurkat cells transfected with a MSLN activator CAR, a HLA-A*02 blocker CAR and a LAT signal 1 booster construct. MSLN(+) HeLa cells were transfected with increasing levels of HLA-A*02 mRNA to generate a titration of blocker antigen level on target cells. Transfected Jurkat cells were co-cultured with target cells for 6 hours without 50nM Rim.

[0064] FIG. 8B is a graph depicting antigen dependent immune cell blocking in Jurkat cells transfected with a MSLN activator CAR, a HLA-A*02 blocker CAR and a LAT signal booster . MSLN(+) HeLa cells were transfected with increasing levels of HLA-A*02 mRNA to generate a titration of blocker antigen level on target cells. Transfected Jurkat cells were co- cultured with target cells for 6 hours with 50nM Rim.

[0065] FIG. 9 is a graph depicting immune cell activation in Jurkat cells expressing the indicated switchable artificial receptor constructs after treatment with increasing doses of rimiducid (Rim).

[0066] FIG. 10 is a schematic of a platform to induce activation of T cell Signal 1, independent of antigen engagement. TCR signaling molecule, such as LAT, is induced to dimerize with a second LAT molecule via FKBP switch domains and small molecule Rimiducid (Rim), which when expressed with CAR (without switch domains) increases tonic signaling. FIG. 11 is a graph quantifying the secretion of IFN-y by JNL cells expressing CAR alone, CAR + LAT-lxFKBP, or Lat-2xFKBP treated with Rimiducid (Rim).

[0067] FIG. 12 is a grid depicting immune cell activation in Jurkat cells expressing the indicated Signal 1 booster constructs and / or activator receptors after treatment with rimiducid (Rim). The induction ratio is relative to boosted JNL cells not treated with Rim. Also included are Signal 1 booster constructs that comprise an Estrogen Receptor (ER) switch domain and are responsive to, at least, tamoxifen; see bottom row and second to right column. The induction ratio there is relative to boosted JNL cells not treated with tamoxifen.

[0068] FIG. 13 is a graph depicting immune cell activation in Jurkat cells expressing the indicated Signal 1 booster constructs and / or activator receptors after treatment with increasing doses of rimiducid (Rim). The induction ratio is relative to boosted JNL cells not treated with Rim .

[0069] FIG. 14A is a set of graphs showing the use of small molecules (Rimiducid or tamoxifen) to induce dimerization of booster constructs in immune cells. Rimiducid and Tamoxifen can be used to drive the dimerization of booster constructs containing the appropriate switch domain, i.e., FKBP and ER, respectively.

[0070] FIG. 14B is a set of graphs showing the use of small molecules (Rimiducid, Rapalog- AP21967 or tamoxifen) to induce dimerization of booster constructs in immune cells. Rimiducid, Rapamycin, and Tamoxifen can be used to drive the dimerization of booster constructs containing the appropriate switch domain.

[0071] FIG. 15 is a cartoon depicting the combination of the dual receptor system with the small molecule Signal 1 booster and the transcription-driven signal 3 booster.

[0072] FIG. 16 is a grid depicting immune cell activation in Jurkat cells expressing the indicated Signal 1 booster constructs and / or activator receptors after treatment with rapalog. The induction ratio is relative to boosted JNL cells not treated with rapalog . Also included are induction ratios of Jurkat cells expressing the indicated activator receptors co-cultured with target cells.

[0073] FIG. 17 is a set of graphs showing quantification of CAR expression, CD25 expression and secretion of IFN-y by primary T cells expressing the indicated Signal 1 booster constructs and / or activator receptors after treatment with rapalog (lOnm or lOOOnm) or TransAct.

[0074] FIG. 18 is a graph showing the percentage of specific killing of target cells by primary T cells expressing the indicated activator receptors with and without switch domains.

[0075] FIG. 19 is a grid depicting immune cell activation in Jurkat cells expressing the indicated Signal 1 booster constructs and / or activator receptors after treatment with tamoxifen. The induction ratio is relative to boosted JNL cells not treated with tamoxifen. Also included are induction ratios of Jurkat cells expressing the indicated Signal 1 booster constructs and / or activator receptors co-cultured with target cells.

[0076] FIG. 20 Is a grid depicting immune cell activation in Jurkat cells expressing the indicated Signal 1 booster constructs and / or activator receptors after treatment with Rim. The induction ratio is relative to boosted JNL cells not treated with Rim.

[0077] FIG. 21A is a flow cytometry plot showing expression of the activator receptor (CAR) and indicated Signal 1 boosters in primary T cells.

[0078] FIG. 21B is a graph showing quantification of secreted IFN-y from primary T cells expressing the indicated Signal 1 booster constructs and / or activator receptors after treatment with Rim.

[0079] DETAILED DESCRIPTION

[0080] The invention disclosed herein relates, in part, to synthetic circuits engineered in immune cells to bring the major signaling pathways that underlie the adaptive immune response under the control of a small molecule that, depending on dose, mimics the behavior of antigen binding and activates the signaling pathways downstream of signal 1. Further, the activation triggered by the small molecule induces expression of, or otherwise activates, an onboard signal 3 (and / or 2) stimulus; e.g., a cytokine. Thus, one aspect of the invention comprises a complete circuit for activation, amplification, and maintenance of response of the engineered immune cell, when the cell is exposed to the small molecule inducer. In the absence of the small molecule, the engineered immune cell may return to a state of quiescence. Critically, the system described herein bypasses the requirement for antigen-exposure in the patient’s blood, a key constraint and limitation of many therapies that target solid tumors, i.e., where the targeting-antigen is largely sequestered outside of the patient’s blood vessels. The approach described herein increases the timeframe of activation and cell numbers to improve the odds that the engineered immune cells can access the tumor environment and react directly to the native antigen expressed on tumor cells.

[0081] In embodiments, the engineered receptor may be an artificial multipartite chimeric antigen receptor (CAR) or T-cell receptor (TCR) that may include one or more switch domains configured to dimerize in the presence of a small molecule. Embodiments of the invention include a polynucleotide that encodes a multipartite receptor composed of a first polypeptide that comprises a conventional CAR or TCR modified to comprise a switch domain, and a second polypeptide that comprises a complementary switch domain and a domain from the TCR signaling pathway.

[0082] An illustrative embodiment is provided in FIG. 1, which depicts a switchable multipartite chimeric antigen receptor (CAR) composed of two polypeptide chains. The first polypeptide chain (on right) comprises a transmembrane domain, a first switch domain comprising two “FK506 binding protein” (FKBP) domains, and a scaffolding intracellular domain which in the depicted embodiment is a “linker for activation of T cells” (LAT) domain. The second polypeptide (on left) comprises an antigen-binding domain which is a single-chain variable fragment (scFv), a transmembrane domain, a second switch domain likewise comprising two FKBP domains, two costimulatory intracellular domains which are a CD28 domain and a 4 IBB domain, and a signaling intracellular domain which is a CD3zeta domain. The small molecule API 903 (a lipid-permeable tacrolimus analogue known as rimiducid, labeled “Rim”) is depicted as selectively dimerizing the switch domain. In a variation of this embodiment, the second polypeptide lacks the costimulatory intracellular domains (as in a so- called second-generation CAR, rather than the third generation CAR depicted). In another variation of this embodiment, termed a switchable multipartite T cell receptor (TCR), the second polypeptide chain is TCR comprising TCRalpha and TCRbeta extracellular domains (or a single-chain TCR), a transmembrane domain, and a switch domain. In other variations, the switch domain comprises only one FKBP domain or comprises three or more FKBP domains. In further variations the first polypeptide lacks a transmembrane domain, and is termed a cytosolic or soluble first polypeptide. Further variations employing alternative transmembrane switch domains (see Table 1), and intracellular domains, are described below.

[0083] Further embodiments of the invention include an immune cell that expresses such a multipartite receptor. Further embodiments involve administering such immune cells to a subject and then administering a small molecule to trigger assembly of the multipartite CAR or TCR, and thereby, in embodiments and without being bound by theory, boost a tonic response. In further embodiments, the immune cells may selectively express a component of the multipartite receptor in response to a small molecule, and thereby enhance tonic signaling.

[0084] Embodiments of the invention may be employed medically to improve treatment of cancer, such as solid tumors. Without being bound by theory, the present invention relates in part to the recognition by the present inventors that, beyond efforts to improve sensitivity of the CAR ligand-binding domain and enhance the TCR ITAMs through added intracellular costimulatory domains (ICDS), little effort by has been expended in the past to further manipulate signal 1, other than vaccination (e.g., CLDN6 CARVac). Most other cellular engineering is designed to preserve antigen dependence, while boosting sensitivity, persistence, etc. Accordingly, it is counterintuitive to (as done in embodiments herein) generate a receptor that retains its ability to be selectively activated by an antigen but in addition may be independently activated by a small molecule.

[0085] Further, without being bound by theory, solid tumors pose unique challenges compared to blood cancers. Access to tumor tissues that express target antigen is severely restricted by the blood vessel walls. With limiting antigen, it is unclear how antigen-dependent boosters can be brought into action. In at least some embodiments described herein, the present inventors address this problem by mimicking antigen with a small molecule that triggers signaling by the receptor (e.g., CAR or TCR). Accordingly, provided herein is, in embodiments, a signal 1 mimetic, a type of booster, that allows tuned stimulation to improve the quality and performance of the T cell product in patients. In embodiments, such a booster may bypass the need for antigen exposure in the patient’s blood, a key difference between blood and solid tumor therapy. In embodiments, the invention further provides for use of such a signal 1 booster complemented by a signal 3 booster (e.g., a membrane bound interleukin, also known as an “armored T cell”).

[0086] In one aspect, the disclosure provides, an immune cell configured to be activated by an antigen or independently by a small molecule (e.g., a switch molecule for a switchable receptor or an inducer molecule for an inducible receptor). The immune cell may include an artificial receptor configured to specifically activate in response to an antigen and independently to a small molecule. The term “receptor” is used herein to refer to surface-expressed macromolecule or macromolecular complex that responds an antigen. While conventional chimeric antigen receptors generally consist of a single polypeptide chain with an antigenbinding domain, sometimes a hinge, a transmembrane domain, and intracellular domains, embodiments described herein include so-called “multipartite” receptors that include several (two, three, or more) polypeptide chains.

[0087] In embodiments, the disclosure provides receptor that is a switchable multipartite chimeric antigen receptor (CAR). The term “switchable” is used to refer to a receptor that may be regulated by use of a switch molecule, such as the small molecule rapamycin, rapalogs (analogs of rapamycin), an estrogen receptor modulator, e.g., tamoxifen, or any of the various switch molecules known in the art, including but not limited to those described in U.S. Patent No. 10,287,735, U.S. Patent No. 10,457,731, U.S. Patent No. 11,478,510, U.S. Patent No. 10,287,354 and Kolos et al. Front Pharmacol 9: 1425 (2018) (PMC6290070), the entire contents of which are incorporated by reference herein. Switch domains

[0088] In embodiments, switchable receptors may include a first switch domain on a first polypeptide chain and a second switch domain on the second polypeptide chain, where the two switch domains are selected to homo- or heterodimerize in response to a cognate switch molecule. Illustrative switch domain pairs and cognate switch molecules are provided in Table 1. Where the switch domains form heterodimers, it should be understood that the first and second switch domains may be interchanged between the two polypeptide chains. While representative sequences are provided, it is possible to identify functional variants of the provided sequences by varying the amino acid sequence and testing for selective dimerization in the presence of the switch molecule. Accordingly, functional variants having at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity may be used. In embodiments, tandem switch domains are employed, meaning that each polypeptide chain included two (or more) switch domains in series.

[0089] A “switch domain,” as used herein, refers to a polypeptide molecule that, in the presence of a switch molecule, associates with another switch domain. The association of the polypeptides and small molecule results in a functional coupling of a first polypeptide molecule that comprises a switch domain to a second polypeptide molecule that comprises a switch domain. In embodiments the first and second switch domain are the same as one another, z.e., they are polypeptides having the same (or similar) amino acid sequence, and referred to collectively as a homodimerization switch. In embodiments, the first and second switch domains are different from one another, z.e., they include polypeptide sequences having different amino acid sequence, and are referred to collectively as a heterodimerization switch. In an embodiment, the switch domain is intracellular. In embodiments, the switch domain is extracellular.

[0090] Table 1: Switch domains and switch molecules In embodiments, the switch domain is a polypeptide comprising FKBP and a second polypeptide comprising FKBP, and the switch molecule is a small molecule, e.g., Rim. In embodiments, the switch is made up of a switch domain comprising FKBP and a second switch domain comprising FRB and the switch molecule is a small molecule, e.g., a rapalog. In embodiments, the first switch domain comprises FKBP and the second switch domain comprises calcineurin and the switch molecule is a small molecule, e.g., a rapalog. In embodiments, the first switch domain comprises FKBP and the second switch domain comprises cyclophilin and the switch molecule is a small molecule, e.g., a rapalog. In embodiments, the first switch domain comprises FKBP and the second switch domain comprises bacterial dihydrofolate reductase (DHFR) and the switch molecule is a small molecule, e.g., a rapalog or methotrexate. In embodiments, the first switch domain comprises calcineurin and the second switch domain comprises cyclophilin and the switch molecule is a small molecule, e.g., cyclosporin A or rapalog. In embodiments, the first switch domain comprises PYRl-like 1 (PYL1) and the second switch domain comprises of abscisic acid insensitive 1 (ABI1) and the switch molecule is a small molecule, e.g., abscisic acid. In embodiments, the first switch domain comprises GIB1 and the second switch domain comprises GAI and the switch molecule is a small molecule, e.g. gibberellin. In some embodiments, the first switch domain comprises an estrogen receptor and the second switch domain comprises an estrogen receptor and the switch molecule is a small molecule, e.g. tamoxifen or 4-hydroxytamoxifen.

[0091] FKBP12 (FKBP, or FK506 binding protein) is an abundant cytoplasmic protein that serves as the initial intracellular target for rapamycin. Rapamycin binds to FKBP and to the large P13K homolog FRAP (RAFT, mTOR), thereby acting to dimerize molecules. In embodiments, the FKBP switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 23. In embodiments, the FKBP switch domain comprises or consists essentially of SEQ ID NO: 23. In embodiments, the FKBP switch domain comprises a F36V mutation.

[0092] In embodiments, an FKBP / FRAP based switch, also referred to herein as an FKBP / FRB, based switch can use a heterodimerization molecule, e.g., rapamycin or a rapamycin analog. FRB is a 93 amino acid portion of FRAP, that is sufficient for binding the FKBP-rapamycin complex (Chen, J., Zheng, X. F., Brown, E. J. & Schreiber, S. L. (1995). Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP 12- rapamycin-associated protein and characterization of a critical serine residue has been reported in Proc Natl Acad Sci USA 92: 4947-51). The FRB and FKBP protein can be mutated at certain residues to increase or decrease their affinity for rapamycin or rapalogs. In embodiments, the FKBP is mutated at F36V. FKBP with F36V mutations has been reported to have lOOOx higher affinity for Rim than the native FKBP ( Clackson, et al. "Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity." PNAS 95.18 (1998): 10437- 10442) The F36V mutations may also allow the FKBP interaction with Rim to be a reversible reaction (Rollins, et al. "A ligand-reversible dimerization system for controlling proteinprotein interactions." PNAS 97.13 (2000): 7096-7101). The F36V mutation is underlined table Mutations that can increase or decrease binding affinity are described in U.S. Patent App. No. US20230026049A1 which is incorporated here in its entirety. In embodiments, the switch domain comprises an FRB binding fragment or analog of FKBP and an FKBP binding fragment or analog of FRB, and the FKBP binding fragment or analog of FRB comprises one or more mutations which enhances the formation of a complex between FKBP or FRB and the switch molecule. The FKBP binding fragment or analog of FRB may comprise: an E2032 mutation, e.g., an E2032I mutation or E2032L mutation; a T2098 mutation, e.g., a T2098L mutation; or an E2032 and a T2098 mutation, e.g., an E2032I and a T2098L or an E2032L and a T2098L mutation.

[0093] In embodiments, the FRB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 24. In embodiments, the FRB switch domain comprises or consists essentially of SEQ ID NO: 24.

[0094] In embodiments, calcineurin catalytic subunit A (also known as PPP3CA; CALN; CALNA; CALNA1; CCN1; CNA1; PPP2B; CAM-PRP catalytic subunit; calcineurin A alpha; calmodulin-dependent calcineurin A subunit alpha isoform; protein phosphatase 2B, catalytic subunit, alpha isoform; etc.) is the switch domain. Calcineurin (protein phosphatase 2B) is a Ca(2+) and calmodulin-dependent protein phosphatase with diverse functions. Calcineurin interacts with ryanodine receptor (RyR) to regulate Ca+ release channels. The association between calcineurin and RyR involves FKB12, an accessory unit of RyR. In embodiments, the calcineurin switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 25. In embodiments, the calcineurin switch domain comprises or consists essentially of SEQ ID NO: 25.

[0095] In embodiments, the switch domain is a domain of a cyclophilin (also known as cyclophilin A, PPIA, CYPA, CYPH, PPIase A, etc.) capable of dimerizing. Cyclophilins are part of a group of proteins known as immunophilins that have peptidyl-prolyl cis-trans isomerase activity. Mammalian cyclophilin A is the major cellular target for, and thus mediates the actions of, the immunosuppressive drug cyclosporin A. The cyclophilins and FKBP naturally bind to cyclosporin and rapamycin to mediate their immunosuppressive and toxic effects. In embodiments, the cyclophilin switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 26. In embodiments, the cyclophilin switch domain comprises or consists essentially of SEQ ID NO: 26.

[0096] In embodiments, the switch domain is a domain of a DHFR (also known as dihydrofolate reductase, DHFRP1, and DYR) capable of dimerizing in response to a small molecule. In embodiments, the DHFR switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 27. In embodiments, the cyclophilin switch domain comprises or consists essentially of SEQ ID NO: 27.

[0097] In embodiments, the switch domain is a domain of a PYL protein ( also known as abscisic acid receptor and as RCAR) capable of dimerizing in response to a small molecule. For example, a switch domain can be derived from proteins such as those of Arabidopsis thaliana: PYR1, RCAR1(PYL9), PYL1, PYL2, PYL3, PYL4, PYL5, PYL6, PYL7, PYL8 (RCAR3), PYL10, PYL11, PYL12, PYL13. In embodiments, the PYL1 switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 28. In embodiments, the PYL1 switch domain comprises or consists essentially of SEQ ID NO: 28.

[0098] In embodiments, the switch domain is a domain of a ABI protein (also known as Abscisic Acid-Insensitive) capable of dimerizing in response to a small molecule. For example, a switch domain can be derived from proteins such as those of I: ABH (Also known as ABSCISIC ACID-INSENSITIVE 1, Protein phosphatase 2C56, AtPP2C56, P2C56, and PP2C ABI1) and / or ABI2 (also known as P2C77, Protein phosphatase 2C77, AtPP2C77, ABSCISIC ACID-INSENSITIVE 2, Protein phosphatase 2C AB 12, and PP2C ABI2). In embodiments, the ABI switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 29. In embodiments, the ABI switch domain comprises or consists essentially of SEQ ID NO: 29. In alternative embodiments, the switch domain can comprise a contiguous stretch of from about 100 amino acids to about 110 amino acids (aa), from about 110 aa to about 115 aa, from about 115 aa to about 120 aa, from about 120 aa to about 130 aa, from about 130 aa to about 140 aa, from about 140 aa to about 150 aa, from about 150 aa to about 160 aa, from about 160 aa to about 170 aa, from about 170 aa to about 180 aa, from about 180 aa to about 190 aa, or from about 190 aa to about 200 aa of any of the amino acid in SEQ ID NO: 29.

[0099] In embodiments, the switch domain is a domain of a GID1 Arabidopsis thaliana protein (also known as Gibberellin receptor GID1) capable of dimerizing in response to a small molecule. In embodiments, the GID1 switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 30. In embodiments, the GID1 switch domain comprises or consists essentially of SEQ ID NO: 30.

[0100] In embodiments, the switch domain is a domain of a GAI I protein (also known as Gibberellic Acid Insensitive, and DELLA protein GAI) capable of dimerizing in response to a small molecule. In embodiments, the GAI switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 31. In embodiments, the GAI switch domain comprises or consists essentially of SEQ ID NO: 31.

[0101] In embodiments, the switch domain is a domain of GyrB (also known as DNA gyrase subunit B) capable of dimerizing in response to a small molecule. In embodiments, the GyrB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 32. In embodiments, the GyrB switch domain comprises or consists essentially of SEQ ID NO: 32.

[0102] In embodiments, the switch domain is a domain of DmrB (i.e. DmrB homodimerization domain) capable of dimerizing in response to a small molecule. In embodiments, the DmrB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 33. In embodiments, the DmrB switch domain comprises or consists essentially of SEQ ID NO: 33.

[0103] In embodiments, the switch domain is a domain of the estrogen receptor capable of dimerizing in response to a small molecule, e.g., tamoxifen. In embodiments, the estrogen switch domain comprises the estrogen receptor. In embodiments, the estrogen switch domain comprises a portion of the estrogen receptor. In embodiments, the estrogen switch domain comprises estrogen receptor alpha. In embodiments, the estrogen switch domain comprises estrogen receptor beta. In embodiments, the estrogen switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity to SEQ ID NO: 121, or a fragment thereof and which is capable of binding the small molecule, e.g., tamoxifen. In embodiments, the estrogen switch domain comprises or consists essentially of SEQ ID NO: 121. In embodiments, the switch domains dimerize upon treatment with a small molecule or “switch molecule” selected from rapamycin or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)- synthetic ligand for FKBP (SLF) or a derivative thereof, tamoxifen or a derivative thereof (e.g. 4-hydroxytamoxifen) or any combination thereof.

[0104] 'Rapalog" may refer to an analog or derivative of rapamycin, examples include everolimus (RAD001), temsirolimus (CCL-779), ridaforolimus, dactolisib (NVP-BEZ235), GSK2126458, XL 765, AZD8055, INK128 / MLN0128, OS1027, AP1903 (Rimiducid) and rapalinks. Additional examples of rapalogs are known in the art and are described in Abdel- Magid et al., 2019 PMID: 31223435.

[0105] “Tamoxifen” may refer to an analog or derivative of tamoxifen and is capable of acting as a selective estrogen receptor modulator.

[0106] The switch domains listed in the present specification are meant to be examples not limiting. The switch domains can be combined in any combination and can be truncated or mutated to increase or decrease affinity for a specific small molecule.

[0107] In embodiments, the first polypeptide of the multipartite receptor comprises a switch domain. In embodiments, the switch domain forms a homodimer upon binding of the small molecule. In embodiments, the switch domain forms a heterodimer upon binding of the small molecule. In embodiments, the first polypeptide of the multipartite receptor comprises a single switch domain. In embodiments, the first polypeptide of the multipartite receptor comprises more than one switch domain. In embodiments, the first polypeptide of the multipartite receptor comprises two switch domains. In embodiments, the first polypeptide of the multipartite receptor comprises three switch domains.

[0108] In embodiments, the switch domain of the first polypeptide is a FKBP switch domain. In embodiments, the FKBP switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 23. In embodiments, the FKBP switch domain comprises or consists essentially of SEQ ID NO 23.

[0109] In embodiments, the switch domain of the first polypeptide is a FRB switch domain. In embodiments, the FRB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 23. In embodiments, the FRB switch domain comprises or consists essentially of SEQ ID NO 24.

[0110] In embodiments, the switch domain of the first polypeptide is a calcineurin switch domain. In embodiments, the calcineurin switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 25. In embodiments, the calcineurin switch domain comprises or consists essentially of SEQ ID NO 25.

[0111] In embodiments, the switch domain of the first polypeptide is cyclophilin switch domain. In embodiments, the cyclophilin switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 26. In embodiments, the cyclophilin switch domain comprises or consists essentially of SEQ ID NO 26.

[0112] In embodiments, the switch domain of the first polypeptide is a bacterial DHFR switch domain. In embodiments, the bacterial DHFR switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 27. In embodiments, the bacterial DHFR switch domain comprises or consists essentially of SEQ ID NO 27.

[0113] In embodiments, the switch domain of the first polypeptide is a PYLI switch domain. In embodiments, the PYLI switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 28. In embodiments, the PYLI switch domain comprises or consists essentially of SEQ ID NO 28.

[0114] In embodiments, the switch domain of the first polypeptide is a ABI1 switch domain. In embodiments, the ABI1 switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 29. In embodiments, the bacterial ABH switch domain comprises or consists essentially of SEQ ID NO 29.

[0115] In embodiments, the switch domain of the first polypeptide is a GIB IB switch domain. In embodiments, the GIB IB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 30. In embodiments, the GIB IB switch domain comprises or consists essentially of SEQ ID NO 30.

[0116] In embodiments, the switch domain of the first polypeptide is a GAI switch domain. In embodiments, the GAI switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 31. In embodiments, the GAI switch domain comprises or consists essentially of SEQ ID NO 31.

[0117] In embodiments, the switch domain of the first polypeptide is a GyrB switch domain. In embodiments, the GyrB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 32. In embodiments, the GyrB switch domain comprises or consists essentially of SEQ ID NO 32.

[0118] In embodiments, the switch domain of the first polypeptide is a DmrB switch domain. In embodiments, the DmrB switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 33. In embodiments, the DmrB switch domain comprises or consists essentially of SEQ ID NO 33.

[0119] In embodiments, the switch domain of the first polypeptide is an estrogen receptor switch domain. In embodiments, the estrogen switch domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to SEQ ID NO: 121, or a fragment thereof and which is capable of binding the small molecule, e.g., tamoxifen. In embodiments, the estrogen switch domain comprises or consists essentially of SEQ ID NO: 121.

[0120] The first polypeptide chains described herein are termed a “booster ” or “booster module” in the sense that the chain is intended to increase the activity of the multipartite receptor compared to the activity of a conventional receptor. However, for clarity, the first polypeptide chains described herein to do, at least in most embodiments, function with conventional receptors because conventional receptors lack the counterpart switch domain or dimerization domain, but still retain sensitivity to the booster .

[0121] It is contemplated that the first polypeptide chain of a multipartite receptor (termed a “booster ” or “booster module”) may include a transmembrane domain and therefore may be membrane bound when expressed. Alternatively, the first polypeptide chain may lack a transmembrane domain and therefore may be considered to be cytoplasmic (“cyto”), except when presence of the switch molecule causes the booster to localize to the membrane along with other components of its receptor. Choice of a membrane-bound or cytoplasmic booster may be used to tune the activation of the multipartite receptor. Illustrative transmembrane domains include: In embodiments, the first polypeptide comprises a transmembrane domain having at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 97% identity or at least 99% identity to a sequence shown in Table 7 below:

[0122] Table 7 Illustrative transmembrane domains

[0123] Scaffolding Intracellular domain

[0124] In embodiments, the first polypeptide chain includes a scaffolding intracellular domain.

[0125] The term “scaffolding intracellular domain” as used herein refers to an intracellular polypeptide molecule that promotes immune effector function of the switchable receptor. Illustrative scaffolding intracellular domain include, but are not limited to LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4-1BB, ZAP70, or GADs and any functional variants or any N terminal or C terminal truncations thereof (see Table A).

[0126] Table A: Illustrative Examples of Scaffolding Intracellular Domains In embodiments, the truncations considered in this disclosure retain the activity of the full-length scaffolding intracellular domain, e.g., as a Signal 1 booster. In some cases, the scaffolding intracellular domain is a LAT comprising a 129 amino acid N-terminal deletion and in other cases, the scaffolding intracellular domain is a LAT comprising a 30 amino acid C- terminal deletion. See FIG. 12.

[0127] As illustrated in FIG. 12, numerous combinations of scaffolding intracellular domains may be expressed by a cell. As shown, the cell of the fourth column from left comprises a first construct comprising a CD3(^, scaffolding intracellular domain and a second construct comprising a LAT scaffolding intracellular domain. In the fifth column, the cell comprises a first construct comprising a SLP76, scaffolding intracellular domain and a second construct comprising a LAT scaffolding intracellular domain. Similarly, In the sixth column, the cell comprises a first construct comprising a ZAP70, scaffolding intracellular domain and a second construct comprising a LAT scaffolding intracellular domain. Any combinations of scaffolding intracellular domains, as disclosed herein, are considered in the present disclosure. Further, as shown, some of the constructs shown a the top of the grid are membrane tethered and other constructs are soluble intracellularly, i.e., they are untethered and lack the membrane tether. Any combination of tethered constructs with tethered constructs, tethered constructs with untethered constructs, and untethered constructs with untethered constructs is considered. Also, the second from right column shows a construct that comprise an Estrogen Receptor (ER) switch domain which is responsive to, at least, tamoxifen ER, whereas the remaining constructs include a FKBP switch domain which is response to the small molecule Rimiducid (Rim). Any combination of FKBP switch domain and ER switch domain constructs are considered, e.g., two FKBP switch domain constructs, one FKBP switch domain construct with an ER switch domain construct, or two ER switch domain constructs; also, these switch domain variants may be combined with any of the scaffolding intracellular domains disclosed herein. Finally, to the left of the grid are shown illustrative switch domain that may be included in a CAR or a TCR. Any CAR or TCR of the present disclosure may have one or both of the FKBP switch domain and / or the ER switch domain; alternately, a CAR or TCR may lack either or both of the FKBP switch domain and / or the ER switch domain.

[0128] Conventional CARs do not include scaffolding intracellular domains. However, the present disclosure demonstrates that, in embodiments, a switchable or inducible multipartite receptor may be activated by a small molecule or antigen.

[0129] In embodiments, the first polypeptide of the multipartite receptor comprises a scaffolding intracellular domain. In embodiments, the scaffolding intracellular domain is a TCR signaling domain. In embodiments, the scaffolding intracellular domain is linker for activation of T cells (LAT). In embodiments, the scaffolding intracellular domain is a variant of the LAT domain other than the native LAT domain. LAT is an important adaptor molecule which recruits multiple key enzymes and signaling molecules to the TCR complex to mediate downstream signaling after TCR ligation. Inclusion of LAT in scaffolding intracellular domain can enhance T cell activation. The LAT scaffolding intracellular domain may be at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identical to the native LAT domain provided here: MEEAILVPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVA PWPPAYPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGI RGAQAGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPGYLVVLPDSTPATST AAPSAPALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAA KTEPAALSSQEAEEVEEEGAPDYENLQELN (SEQ ID NO: 23).

[0130] In embodiments, the LAT domain includes the amino acid substitution G160D. LAT G160D may enhance activation of PLC-gamma (PLCgl). Recruitment and activation of PLCgl can be a key step in the T-cell activation process triggered by the TCR. The activated enzyme may hydrolyze phosphatidylinositol-4,5-bisphosphate to inositol -1,4, 5 -trisphosphate (IP3), which can stimulate the release of Ca2+ from intracellular stores, and diacylglycerol, which activates protein kinase C and RasGRP-dependent signaling pathways. The increase in intracellular free Ca2+ concentration triggered by IP3 may play a crucial role in the induction of numerous T-cell activation-associated responses. The LAT G160D scaffolding intracellular domain may be at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identical to: GSYDSTSSDSLYPRGIQFKRPHTVAPWPPAYPPVTSYPPLSQPDLLPIPRSPQPLGGSHR TPSSRRDSDGANSVASYENEGASGIRGAQAGWGVWGPSWTRLTPVSLPPEPACEDAD EDEDDYHNPDYLVVLPDSTPATSTAAPSAPALSTPGIRDSAFSMESIDDYVNVPESGES AEASLDGSREYVNVSQELHPGAAKTEPAALSSQEAEEVEEEGAPDYENLQELN (SEQ ID NO: 36).

[0131] In embodiments, the scaffolding intracellular domain is LAT-ZAP70 fusion. ZAP70 is activated by Lek (lymphocyte-specific protein tyrosine kinase) while bound to the ITAMs in the TCR complex and phosphorylates tyrosine residues of two important adaptor molecules, LAT and SLP76. Whereas ZAP70 is thought to be a key mediator of TCR activation and downstream signaling, inclusion of ZAP70 in scaffolding intracellular domain can boost amplification of TCR signaling leading to more potent T cell activation. In embodiments, the Zap70 domain comprises the Zap70 kinase domain, the Zap70 interdomain, or a combination thereof. The LAT-ZAP70 fusion scaffolding intracellular domain may be at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identical to: GSYDSTSSDSLYPRGIQFKRPHTVAPWPPAYPPVTSYPPLSQPDLLPIPRSPQPLGGSHR TPSSRRDSDGANSVASYENEGASGIRGAQAGWGVWGPSWTRLTPVSLPPEPACEDAD EDEDDYHNPGYLVVLPDSTPATSTAAPSAPALSTPGIRDSAFSMESIDDYVNVPESGES AEASLDGSREYVNVSQELHPGAAKTEPAALSSQEAEEVEEEGAPDYENLQELNGGG GSGGGGSPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPR PMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKK QIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAG GGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHY AKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMW EALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDF LTVEQRMRACYYSL (SEQ ID NO: 38).

[0132] In embodiments, the scaffolding intracellular domain is LAT-SLP76 fusion protein. SLP76 (also known as lymphocyte cytosolic protein 2 or LCP2). SLP76 is recruited to the LAT complex and activates key enzymes and signaling molecules involved in downstream signaling and cell adhesion. Inclusion of SLP76 in the scaffolding intracellular domain can enhance T cell activation. In embodiments, the scaffolding intracellular domain is a fusion of a variant of the LAT domain with another protein. The LAT intracellular protein may be fused with SLP76. The SLP76 scaffolding intracellular domain may be at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identical to: ALRNVPFRSEVLGWDPDSLADYFI<I<LNYI<DCEI<AVI<I<YHIDGARFLNLTENDIQI<FP KLRVPILSKLSQEINKNEERRSIFTRKPQVPRFPEETESHEEDNGGWSSFEEDDYESPN DDQDGEDDGDYESPNEEEEAPVEDDADYEPPPSNDEEALQNSILPAKPFPNSNSMYID RPPSGKTPQQPPVPPQRPMAALPPPPAGRNHSPLPPPQTNHEEPSRSRNHKTAKLPAPS IDRSTKPPLDRSLAPFDREPFTLGKKPPFSDKPSIPAGRSLGEHLPKIQKPPLPPTTERHE RSSPLPGKKPPVPKHGWGPDRRENDEDDVHQRPLPQPALLPMSSNTFPSRSTKPSPM NPLPSSHMPGAFSESNSSFPQSASLPPYFSQGPSNRPPIRAEGRNFPLPLPNKPRPPSPAE EENSLNEEWYVSYITRPEAEAALRKINQDGTFLVRDSSKKTTTNPYVLMVLYKDKVY NIQIRYQKESQVYLLGTGLRGKEDFLSVSDIIDYFRKMPLLLIDGKNRGSRYQCTLTH AAGYPGNS (SEQ ID NO: 39).

[0133] In embodiments, the scaffolding intracellular domain is membrane tethered SLP76. SLP76 (also known as lymphocyte cytosolic protein 2 or LCP2). SLP76 is a signal transducing adapter protein expressed in immune cells that may be important in T cell receptor signaling. SLP76 is a component of the ITAM signaling pathway. In embodiments, the SLP76 is tethered to the membrane by a transmembrane polypeptide. In embodiments, the SLP76 is cytosolic and not bound to the membrane.

[0134] In embodiments, the scaffolding intracellular domain is FYN (also known as Protooncogene tyrosine-protein kinase Fyn (p59-FYN, Slk, Syn, MGC45350). FYN is a member of the Src family of kinases that may regulate T cell and neuronal signaling. The FYN scaffolding intracellular domain may be at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identical to:

[0135] GCVQCKDKEATKLTEERDGSLNQSSGYRYGTDPTPQHYPSFGVTSIPNYNNFHAAGG QGLTVFGGVNS S SHTGTLRTRGGTGVTLF VALYDYEARTEDDLSFHKGEKFQILNS SE GDWWEARSLTTGETGYIPSNYVAPVDSIQAEEWYFGKLGRKDAERQLLSFGNPRGTF LIRESETTKGAYSLSIRDWDDMKGDHVKHYKIRKLDNGGYYITTRAQFETLQQLVQH YSERAAGLCCRLVVPCHKGMPRLTDLSVKTKDVWEIPRESLQLIKRLGNGQFGEVW MGTWNGNTKVAIKTLKPGTMSPESFLEEAQIMKKLKHDKLVQLYAVVSEEPIYIVTEY MNKGSLLDFLKDGEGRALKLPNLVDMAAQVAAGMAYIERMNYIHRDLRSANILVGN GLICKIADFGLARLIEDNEYTARQGAKFPIKWTAPEAALYGRFTIKSDVWSFGILLTEL VTKGRVPYPGMNNREVLEQVERGYRMPCPQDCPISLHELMIHCWKKDPEERPTFEY LQSFLEDYFTATEPQYQPGENL (SEQ ID NO: 41).

[0136] In embodiments, the scaffolding intracellular domain is cytoplasmic PLCgl (also known as Phospholipase C gamma 1, PLCgammal). PLCgl can be a critical enzyme regulating nuclear factor-KB (NF-KB), extracellular signal-related kinase, mitogen-activated protein kinase, and nuclear factor of activated T cells signaling pathway. In embodiments, the PLCgl is tethered to the membrane by a transmembrane polypeptide. In embodiments, the PLCgl is cytosolic and not bound to the membrane. The PLCgl scaffolding intracellular domain may be at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identical to: AGAASPCANGCGPGAPSDAEVLHLCRSLEVGTVMTLFYSKKSQRPERKTFQVKLET RQITWSRGADKIEGAIDIREIKEIRPGKTSRDFDRYQEDPAFRPDQSHCFVILYGMEFRL KTLSLQATSEDEVNMWIKGLTWLMEDTLQAPTPLQIERWLRKQFYSVDRNREDRISA KDLKNMLSQVNYRVPNMRFLRERLTDLEQRSGDITYGQFAQLYRSLMYSAQKTMDL PFLEASTLRAGERPELCRVSLPEFQQFLLDYQGELWAVDRLQVQEFMLSFLRDPLREI EEP YFFLDEF VTFLF SKENS VWNSQLD AVCPDTMNNPL SHYWIS S SHNT YLTGDQF S S ESSLEAYARCLRMGCRCIELDCWDGPDGMPVIYHGHTLTTKIKFSDVLHTIKEHAFVA SEYPVILSIEDHCSIAQQRNMAQYFKKVLGDTLLTKPVEISADGLPSPNQLKRKILIKH KKLAEGSAYEEVPTSMMYSENDISNSIKNGILYLEDPVNHEWYPHYFVLTSSKIYYSE ETS SDQGNEDEEEPKEVS S STELHSNEKWFHGKLGAGRDGRHIAERLLTEYCIETGAP DGSFLVRESETFVGDYTLSFWRNGKVQHCRIHSRQDAGTPKFFLTDNLVFDSLYDLIT HYQQVPLRCNEFEMRLSEPVPQTNAHESKEWYHASLTRAQAEHMLMRVPRDGAFL VRKRNEPNSYAISFRAEGKIKHCRVQQEGQTVMLGNSEFDSLVDLISYYEKHPLYRK MI<LRYPINEEALEI<IGTAEPDYGALYEGRNPGFYVEANPMPTFI<CAVI<ALFDYI<AQR EDELTFIKSAIIQNVEKQEGGWWRGDYGGKKQLWFPSNYVEEMVNPVALEPEREHL DENSPLGDLLRGVLDVPACQIAIRPEGKNNRLFVFSISMASVAHWSLDVAADSQEELQ DWVKKIREVAQTADARLTEGKIMERRKKIALELSELVVYCRPVPFDEEKIGTERACYR DMSSFPETKAEKYVNKAKGKKFLQYNRLQLSRIYPKGQRLDSSNYDPLPMWICGSQ LVALNFQTPDKPMQMNQALFMTGRHCGYVLQPSTMRDEAFDPFDKSSLRGLEPCAIS IEVLGARHLPKNGRGIVCPFVEIEVAGAEYDSTKQKTEFVVDNGLNPVWPAKPFHFQI SNPEFAFLRFVVYEEDMFSDQNFLAQATFPVKGLKTGYRAVPLKNNYSEDLELASLLI KIDIFPAKENGDLSPFSGTSLRERGSDASGQLFHGRAREGSFESRYQQPFEDFRISQEH LADHFDSRERRAPRRTRVNGDNRLGSGATNFSLLKQAGDVEENPGPMGAGATGRAM DGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPC LDSVTFSDVVSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCE ACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRE CTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGVVTTVMGS SQPVVTRGTTDNLIPVYCSILAAVVVGLVAYIAFKR (SEQ ID NO: 43).

[0137] In embodiments, the scaffolding intracellular domain is Grb2-related adaptor protein (GADs). Gads functions to couple the activated TCR to distal signaling events through its interactions with the leukocyte-specific signaling proteins SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) and LAT (linker for activated T cells). The PLCgl scaffolding intracellular domain may be at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identical to: MEAVAKFDFTASGEDELSFHTGDVLKILSNQEEWFKAELGSQEGYVPKNFIDIQFPKW FHEGLSRHQAENLLMGKEVGFFIIRASQSSPGDFSISVRHEDDVQHFKVMRDNKGNY FLWTEKFPSLNKLVDYYRTNSISRQKQIFLRDRTREDQGHRGNSLDRRSQGGPHLSGA VGEEIRPSMNRKLSDHPPTLPLQQHQHQPQPPQYAPAPQQLQQPPQQRYLQHHHFHQ ERRGGSLDINDGHCGTGLGSEMNAALMHRRHTDPVQLQAAGRVRWARALYDFEAL EDDELGFHSGEVVEVLDSSNPSWWTGRLHNKLGLFPANYVAPMTR (SEQ ID NO: 53).

[0138] In embodiments, the scaffolding intracellular domain is T cell surface glycoprotein CD3 zeta chain (CD3 . CD3^ together with T cell receptor alpha / beta and gamma / delta heterodimers and CD3 gamma, delta and epsilon forms the T cell receptor-CD3 complex. The CD3(^ can be involved in coupling antigen recognition to several intracellular signaltransduction pathways. The CD3^ scaffolding intracellular domain may be at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identical to SEQ ID NO: 122 yet retains the activity of the full-length CD3(^ scaffolding intracellular domain.

[0139] QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDE DHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIV DICITGGLLLLVYYWSKNRKAKAKPV (SEQ ID NO: 122).

[0140] Chimeric Antigen Receptor

[0141] In embodiments, the multipartite receptor described herein is a multipartite Chimeric Antigen Receptor (CAR). One component of the CAR disclosed herein may be an antigenbinding domain. The antigen-binding domain may be specific for a tumor antigen. The tumor antigen may be TNF receptor superfamily member 9 (4-1BB, CD137), 5'-nucleotidase, trophoblast glycoprotein (5T4), activin receptor-like kinase 1, alpha-fetoprotein, angiopoietin 2, TNF superfamily member 13b (BAFF), TNF receptor superfamily member 17 (BCMA). mucin 16, cell surface associated (CA-125), C-C motif chemokine receptor 4 (CCR4), interleukin 3 receptor subunit alpha (CD123), TNF receptor superfamily member 4 (CD134), cytotoxic T-lymphocyte associated protein 4 (CD 152), CD 19 molecule (CD 19), membrane spanning 4-domains Al (CD20), CD200 molecule (CD200), CD22 molecule (CD22), Fc fragment of IgE receptor II (CD23, IgE receptor), interleukin 2 receptor subunit alpha (CD25), CD27 molecule (CD27), CD276 molecule (CD276), TNF receptor superfamily member 8 (CD30, TNFRSF8), CD33 molecule (CD33), CD37 molecule (CD37), CD38 molecule (CD38), CD44 molecule v6 (CD44 v6), integrin subunit alpha V (CD51), neural cell adhesion molecule 1 (CD56), CD6 molecule (CD6), CD70 molecule (CD70), CD74 molecule (CD74), CD79B molecule (CD79B), CD80 molecule (CD80), CEA cell adhesion molecule 5 (CEA), Claudin 18 Isoform 2, Colony stimulating factor 1 (CSF1), colony stimulating factor 1 receptor (CSF1R), Colony stimulating factor 2 (CSF2), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), CXCR4 (CD 184), dendritic cell-associated lectin 2, delta like canonical Notch ligand 3 (DLL3), delta like canonical Notch ligand 4 (DLL4), TNF receptor superfamily member 10b (DR5), EGF like domain multiple 7 (EGFL7), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM),EPH receptor A3 (EPHA3), erb-b2 receptor tyrosine kinase 3 (ERBB3, HER3), fibroblast growth factor receptor (FGFR2), Frizzled receptor, GD2 ganglioside, GD3 ganglioside, GD3 ganglioside, glypican 3, glycoprotein nmb (GPNMB), epidermal growth factor receptor (HER1), erb-b2 receptor tyrosine kinase 2 (HER2), hepatocyte growth factor (HGF), MET proto-oncogene, receptor tyrosine kinase (HGFR), IGF-1 receptor (CD221), Interleukin 3 receptor, Interleukin 1 alpha (ILIA), Interleukin 2 (IL2), integrin a5pi, integrin avP3, lymphocyte activating 3 (LAG3), C- C motif chemokine ligand 2 (MCP-1), Mesothelin, Mucin 1, NGNA ganglioside, Notch 1, Notch receptor, neuropilin 1 (NRP1), programmed cell death 1 (PD-1), CD274 molecule (PD- Ll), receptor tyrosine kinase like orphan receptor 1 (R0R1), tenascin C, transforming growth factor beta 1 (TGF- ), VEGF-A, VEGFR-1, VEGFR2

[0142] Various antigen-binding domain are known in the art, and include, without limitation those described in U.S. Patent No. 10,882,922, US Patent No. 11,602,544, U.S. patent No. 11,254,726, US Patent No. 11,602,543, U.S. Patent No. 11,433,100, and US Patent No. 11,730,764, all which are incorporated here in the entirety. The disclosure contemplates both antibody -based antigen-binding domains for switchable or inducible chimeric antigen receptor (CAR) receptors and TCR-based antigen-binding domains for switchable or inducible TCR receptors. In embodiments, the second polypeptide of the multipartite CAR comprises a switch domain. In embodiments, the second polypeptide of the multipartite CAR comprises two switch domains. In embodiments, the second polypeptide of the multipartite CAR does not comprise a switch domain.

[0143] In embodiments, the second polypeptide of the multipartite CAR receptor comprises a hinge domain. In embodiments, the CARs of the present disclosure comprise an extracellular hinge region. Incorporation of a hinge region can affect cytokine production from CAR-T cells and improve expansion of CAR-T cells in vivo. Exemplary hinges can be isolated or derived from IgD and CD8 domains, for example IgGl. In embodiments, the hinge is isolated or derived from CD8a or CD28.

[0144] In embodiments, the hinge is isolated or derived from CD8a or CD28. In embodiments, the CD8a hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 71). In embodiments, the CD8a hinge comprises SEQ ID NO: 71. In embodiments, the CD8a hinge consists essentially of SEQ ID NO: 71. In embodiments, the CD8a hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAG CCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCAC ACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 72). In embodiments, the CD8a hinge is encoded by SEQ ID NO: 72.

[0145] In embodiments, the CD28 hinge comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of CTIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 73). In embodiments, the CD28 hinge comprises or consists essentially of SEQ ID NO: 73. In embodiments, the CD28 hinge is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

[0146] TGTACCATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATG GAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGG ACCTTCTAAGCCC (SEQ ID NO: 74). In embodiments, the CD28 hinge is encoded by SEQ ID NO: 74.

[0147] In embodiments, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. For example, a CAR comprising a CD28 co-stimulatory domain might also use a CD28 transmembrane domain. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0148] The transmembrane domain can be derived either from a natural or from a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions can be isolated or derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or from an immunoglobulin such as IgG4. Alternatively, the transmembrane domain can be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length can form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker. In embodiments of the CARs of the disclosure, the CARs comprise a CD28

[0149] 980 transmembrane domain. In embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 75). In embodiments, the CD28 transmembrane domain comprises or consists essentially of SEQ ID NO: 75. In embodiments,

[0150] 985 the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of TTCTGGGTGCTGGTCGTTGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGA CAGTGGCCTTCATCATCTTTTGGGTG (SEQ ID NO: 76). In embodiments, the CD28

[0151] 990 transmembrane domain is encoded by SEQ ID NO: 76. In embodiments, the CD28 transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 157. In embodiments, the CD28 transmembrane domain is encoded by SEQ ID NO: 157.

[0152] 995 In embodiments of the CARs of the disclosure, the CARs comprise an IL-2Rbeta transmembrane domain. In embodiments, the IL-2Rbeta transmembrane domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of IPWLGHLLVGLSGAFGFIILVYLLI (SEQ ID NO: 77). In embodiments, the IL-2Rbeta transmembrane domain comprises or

[0153] 1000 consists essentially of SEQ ID NO: 77. In embodiments, the IL-2Rbeta transmembrane domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

[0154] ATTCCGTGGCTCGGCCACCTCCTCGTGGGCCTCAGCGGGGCTTTTGGCTTCATCA TCTTAGTGTACTTGCTGATC (SEQ ID NO: 78). In embodiments, the IL-2Rbeta

[0155] 1005 transmembrane domain is encoded by SEQ ID NO: 78.

[0156] Signaling Intracellular Domain

[0157] Another component of receptors disclosed herein may be a costimulatory domain. The cytoplasmic domain or otherwise the intracellular signaling domain of the CARs of the instant disclosure is responsible for activation of at least one of the normal effector functions of the

[0158] 1010 immune cell in which the CAR has been placed. The term “effector function” refers to a specialized function of a cell. Thus, the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion

[0159] 1015 of the intracellular signaling domain is used, such truncated portion can be used in place of the intact chain as long as it transduces the effector function signal. In some cases, multiple intracellular domains can be combined to achieve the desired functions of the CAR-T cells of the instant disclosure. The term intracellular signaling domain is thus meant to include any truncated portion of one or more intracellular signaling domains sufficient to transduce the

[0160] 1020 effector function signal.

[0161] Examples of intracellular signaling domains for use in the CARs of the instant disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same

[0162] 1025 functional capability.

[0163] Accordingly, the intracellular domain of CARs of the instant disclosure comprises at least one cytoplasmic activation domain. In embodiments, the intracellular activation domain ensures that there is T-cell receptor (TCR) signaling necessary to activate the effector functions of the CAR T-cell. In embodiments, the at least one cytoplasmic activation is a CD247

[0164] 1030 molecule (CD3Q activation domain, a stimulatory killer immunoglobulin-like receptor (KIR) KIR2DS2 activation domain, or a DNAX-activating protein of 12 kDa (DAP12) activation domain.

[0165] In embodiments, the CD3(^ activation domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is

[0166] 1035 identical to a sequence of RVI<FSRSADAPAYI<QGQNQLYNELNLGRREEYDVLDI<RRGRDPEMGGI<PRRI<NPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 79).

[0167] In embodiments, the CD3(^ activation domain comprises or consists essentially of SEQ

[0168] 1040 ID NO: 79. In embodiments, the CD3(^ activation domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGA 1045 CAAGCGTAGAGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTT TACCAGGGACTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGC (SEQ ID NO: 80). In embodiments, the CD3(^ activation domain is

[0169] 1050 encoded by SEQ ID NO: 80. In embodiments, the CD3(^ activation domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 163. In embodiments, the CD3(^ activation domain is encoded by SEQ ID NO: 163.

[0170] It is known that signals generated through the TCR alone are often insufficient for full

[0171] 1055 activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0172] 1060 Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner can contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. In embodiments, the ITAM contains a tyrosine separated from a leucine or an isoleucine by any two other amino acids

[0173] 1065 (YxxL / I (SEQ ID NO: 983). In embodiments, the cytoplasmic domain contains 1, 2, 3, 4 or 5 ITAMs. An exemplary ITAM containing cytoplasmic domain is the CD3(^ activation domain. Further examples of ITAM containing primary cytoplasmic signaling sequences that can be used in the CARs of the instant disclosure include those derived from TCR^, FcRy, FcRP, CD3y, CD35, CD3s, CD3i CD5, CD22, CD79a, CD79b, and CD66d.

[0174] 1070 In embodiments, the CD3(^ activation domain comprising a single ITAM comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 81). In embodiments, the CD3(^ activation domain comprises SEQ ID NO: 81. In embodiments, the

[0175] 1075 CD3(^ activation domain comprising a single ITAM consists essentially of an amino acid sequence of RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLHMQALPPR (SEQ ID NO: 81 ) . In embodiments, the CD3(^ activation domain comprising a single ITAM is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

[0176] 1080 AGAGTGAAGT TCAGCAGGAG CGCAGACGCC CCCGCGTACC AGCAGGGCCA GAACCAGCTC TATAACGAGC TCAATCTAGG ACGAAGAGAG GAGTACGATG TTTTGCACAT GCAGGCCCTG CCCCCTCGC (SEQ ID NO: 82). In embodiments, the CD3(^ activation domain is encoded by SEQ ID NO: 82.

[0177] In embodiments, the cytoplasmic domain of the receptor can be designed to comprise

[0178] 1085 the CD3(^ signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the receptor of the instant disclosure. For example, the cytoplasmic domain of the CAR can comprise a CD3(^ chain portion and a co-stimulatory domain.

[0179] Costimulatory Intracellular Domain

[0180] The co-stimulatory domain refers to a portion of the CAR comprising the intracellular

[0181] 1090 domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include the co-stimulatory domain is selected from the group consisting of IL-2RP, Fc Receptor gamma (FcRy), Fc Receptor beta (FcRP), CD3g molecule gamma (CD3y), CD35, CD3s, CD5 molecule (CD5), CD22 molecule (CD22),

[0182] 1095 CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen- 1 (LFA-1),

[0183] 1100 CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domains, or functional variants thereof. In embodiments, the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain. In embodiments, the co-stimulatory domain is isolated or derived from CD28.

[0184] 1105 In embodiments, the intracellular domains of CARs of the instant disclosure comprise at least one co-stimulatory domain. In embodiments, the co-stimulatory domain is isolated or derived from CD28. In embodiments, the CD28 co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of 1110 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 83). In embodiments, the CD28 co-stimulatory domain comprises or consists essentially of SEQ ID NO: 83). In embodiments, the CD28 co-stimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is identical to a sequence of

[0185] 1115 AGGAGCAAGCGGAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCCCGG AGGCCTGGCCCCACCCGGAAGCACTACCAGCCCTACGCCCCTCCCAGGGATTTCG CCGCCTACCGGAGC (SEQ ID NO: 84). In embodiments, the CD28 co-stimulatory domain is encoded by SEQ ID NO: 84. In embodiments, the CD28 co-stimulatory domain is encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95%

[0186] 1120 identity, at least 99% identity or is identical to a sequence of SEQ ID NO: 160. In embodiments, the CD28 co-stimulatory domain is encoded by SEQ ID NO: 160.

[0187] In embodiments, the co-stimulatory domain is isolated or derived from 4-1BB. In embodiments, the 4-1BB co-stimulatory domain comprises an amino acid sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity or is

[0188] 1125 identical to a sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 161). In embodiments, the 4-1BB co-stimulatory domain comprises or consists essentially of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 161). In embodiments, the 4-1BB co-stimulatory domain s encoded by a nucleotide sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99%

[0189] 1130 identity or is identical to a sequence of AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGGCCA GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAA GAAGGAGGATGTGAACTG (SEQ ID NO: 162).

[0190] In embodiments, the intracellular domain of the CAR comprises a CD28 co-stimulatory

[0191] 1135 domain, a 4-1BB costimulatory domain, and a CD3(^ activation domain. In embodiments, the intracellular domain of the CAR comprises a sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPF MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRR EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK

[0192] 1140 GHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 158), or a sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity thereto. In embodiments, the intracellular domain of the CAR is encoded by SEQ ID NO: 159, or a sequence having at least 80% identity, at least 90% identity, at least 95% identity, at least 99% identity thereto. In embodiments, the intracellular domain of the CAR is encoded by SEQ ID

[0193] 1145 NO: 159.

[0194] The cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure can be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example between 2 and 10 amino acids in length can form the linkage. A glycine-serine doublet provides an example of a suitable linker. An exemplary linker

[0195] 1150 comprises a sequence of GGGGSGGGGSGGGGSGG.

[0196] The cytoplasmic domains within the cytoplasmic signaling portion of the CARs of the instant disclosure can be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example between 2 and 10 amino acids in length can form the linkage. A glycine-serine doublet provides an example of a suitable linker.

[0197] 1155 In embodiments, the costimulatory domains are included on the same polypeptide chain as the antigen-binding domain and the signaling domain. An advantage of this configuration may be that the receptor retains an ability to signal in response to binding of antigen in the absence of the switch molecule - that is, when the booster module is not associated to the remainder of the receptor.

[0198] 1160 T-Cell Receptor (TCR)

[0199] In embodiments, the receptor is a multipartite T cell receptor (TCR). In embodiments, the switchable TCR comprises a first polypeptide chain comprising optionally a hinge domain, a transmembrane domain, a first switch domain, and a scaffolding intracellular domain (e.g., a LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4-1BB,

[0200] 1165 ZAP70, or GADs domain); and a second polypeptide chain comprising a transmembrane domain, an signaling intracellular domain (e.g., a CD3zeta domain), optionally a second switch domain; and a third polypeptide chain comprising an antigen-binding domain (e.g., a TCR extracellular domain) and a transmembrane domain, wherein the switch domains are configured to selectively dimerize in the presence of a switch molecule (e.g., rapamycin or a

[0201] 1170 rapalog or an estrogen receptor modulator, e.g., tamoxifen).

[0202] In embodiments, the first polypeptide of the multipartite TCR comprises the same components as the first polypeptide of the multipartite CAR described above. In embodiments, the same options for hinge, transmembrane switch domain and scaffolding intracellular domain are contemplated with the multipartite TCR as they are in the multipartite CAR receptor

[0203] 1175 described above. In embodiments, the second polypeptide of the multipartite TCR comprises a transmembrane domain, a second switch domain, and an signaling intracellular domain (e.g., a CD3zeta domain). . In embodiments, the second polypeptide of the multipartite TCR comprises a switch domain. In embodiments, the second polypeptide of the multipartite TCR comprises

[0204] 1180 two switch domains. In embodiments, the second polypeptide of the multipartite TCR does not comprise a switch domain. In embodiments, the transmembrane of the second polypeptide can be any transmembrane described herein or a portion of any transmembrane sequence described herein.

[0205] In embodiments, the signaling intracellular domain can be any naturally occurring

[0206] 1185 intracellular signaling domain. The intracellular signaling domains described in the multipartite CAR receptor section can be incorporated into the second polypeptide of the multipartite TCR receptor.

[0207] In embodiments, the third polypeptide of the multipartite TCR comprises an antigenbinding domain (e.g., a TCR extracellular domain) and a transmembrane domain. Exemplary

[0208] 1190 TCRs comprising intracellular domains for use in the instant disclosure are described in PCT / US2020 / 045250 filed on September 6, 2020, the contents of which are incorporated herein by reference.

[0209] As used herein, a “TCR”, sometimes also called a “TCR complex” or “TCR / CD3 complex” refers to a protein complex comprising a TCR alpha chain, a TCR beta chain, and

[0210] 1195 one or more of the invariant CD3 chains (zeta, gamma, delta and epsilon), sometimes referred to as subunits. The TCR alpha and beta chains can be disulfide-linked to function as a heterodimer to bind to peptide-MHC complexes. Once the TCR alpha / beta heterodimer engages peptide-MHC, conformational changes in the TCR complex in the associated invariant CD3 subunits are induced, which leads to their phosphorylation and association with

[0211] 1200 downstream proteins, thereby transducing a primary stimulatory signal. In an exemplary TCR complex, the TCR alpha and TCR beta polypeptides form a heterodimer, CD3 epsilon and CD3 delta form a heterodimer, CD3 epsilon and CD3 gamma for a heterodimer, and two CD3 zeta form a homodimer.

[0212] Any suitable ligand binding domain can be fused to an extracellular domain, hinge

[0213] 1205 domain or transmembrane of the TCRs described herein. For example, the ligand binding domain can be an antigen binding domain of an antibody or TCR, or comprise an antibody fragment, a VP only domain, a linear antibody, a single-chain variable fragment (scFv), or a single domain antibody (sdAb). In embodiments, the ligand binding domain is fused to one or more extracellular

[0214] 1210 domains or transmembrane domains of one or more TCR subunits. The TCR subunit can be TCR alpha, TCR beta, CD3 delta, CD3 epsilon, CD3 gamma or CD3 zeta. For example, the ligand binding domain can be fused to TCR alpha, or TCR beta, or portions of the ligand binding can be fused to two subunits, for example portions of the ligand binding domain can be fused to both TCR alpha and TCR beta.

[0215] 1215 TCR subunits include TCR alpha, TCR beta, CD3 zeta, CD3 delta, CD3 gamma and CD3 epsilon. Any one or more of TCR alpha, TCR beta chain, CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta, or fragments or derivative thereof, can be fused to one or more domains capable of providing a stimulatory signal of the disclosure, thereby enhancing TCR function and activity.

[0216] 1220 TCR transmembrane domains isolated or derived from any source are envisaged as within the scope of the disclosure. The transmembrane domain can be derived either from a natural or from a recombinant source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

[0217] In embodiments, the transmembrane domain is capable of signaling to the intracellular

[0218] 1225 domain(s) whenever the TCR complex has bound to a target. A transmembrane domain of particular use can include at least the transmembrane region(s) of e.g, the alpha, beta or zeta chain of the TCR, CD3 delta, CD3 epsilon or CD3 gamma, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.

[0219] In embodiments, the transmembrane domain can be attached to the extracellular region

[0220] 1230 of a polypeptide of the TCR, e.g, the antigen binding domain of the TCR alpha or beta chain, via a hinge, e.g., a hinge from a human protein. For example, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge. In embodiments, the hinge is isolated or derived from CD8a or CD28.

[0221] In embodiments, the extracellular ligand binding domain is attached to one or more

[0222] 1235 transmembrane domains of the TCR. In embodiments, the transmembrane domain comprises a TCR alpha transmembrane domain, a TCR beta transmembrane domain, or both. In embodiments, the transmembrane comprises a CD3 zeta transmembrane domain.

[0223] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region

[0224] 1240 of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the extracellular region) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or up to 15 amino acids of the intracellular region).

[0225] In embodiments, the transmembrane domain can be selected or modified by amino acid

[0226] 1245 substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.

[0227] When present, the transmembrane domain can be a natural TCR transmembrane domain, a natural transmembrane domain from a heterologous membrane protein, or an

[0228] 1250 artificial transmembrane domain. The transmembrane domain can be a membrane anchor domain. Without limitation, a natural or artificial transmembrane domain can comprise a hydrophobic a-helix of about 20 amino acids, often with positive charges flanking the transmembrane segment. The transmembrane domain can have one transmembrane segment or more than one transmembrane segment. Prediction of transmembrane domains / segments can

[0229] 1255 be made using publicly available prediction tools (e.g. TMHMM, Krogh et al. Journal of Molecular Biology 2001; 305(3):567-580; or TMpred, Hofmann & Stoffel Biol. Chem. Hoppe- Seyler 1993; 347: 166). Non-limiting examples of membrane anchor systems include platelet derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchor (added post- translationally to a signal sequence)

[0230] 1260 and the like.

[0231] In embodiments, the transmembrane domain comprises a TCR alpha transmembrane domain. In embodiments, the TCR alpha transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a

[0232] 1265 sequence of: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 85). In embodiments, the TCR alpha transmembrane domain comprises, or consists essentially of, SEQ ID NO: 85. In embodiments, the TCR alpha transmembrane domain is encoded by a sequence of GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGA CGCTGCGGCTGTGG (SEQ ID NO: 86).

[0233] 1270 In embodiments, the transmembrane domain comprises a TCR beta transmembrane domain. In embodiments, the TCR beta transmembrane domain comprises an amino acid sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity or is identical to a sequence of: TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 87). In embodiments, the TCR 1275 beta transmembrane domain comprises, or consists essentially of, SEQ ID NO: 87. In embodiments, the TCR beta transmembrane domain is encoded by a sequence of ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCA GTGCCCTCGTGCTG (SEQ ID NO: 88).

[0234] Inhibitory Receptor

[0235] 1280 In embodiments, the immune cell includes a second receptor. The second receptor may be an inhibitory receptor. Illustrative inhibitory receptors are listed in Table 5. Additional illustrative inhibitory receptors are described in US patent No. 11,254,725, PCT / US2023 / 065860, US Patent No. 11,602,544, U.S. patent No. 11,254,726, US Patent No. 11,602,543, U.S. Patent No. 11,433,100, and US Patent No. 11,730,764, U.S. Patent App. No.

[0236] 1285 US20200261499A1, and U.S. Patent App. No. US20220380434A1, the entire contents of which are incorporated by reference herein. In embodiments, the inhibitory receptor comprises the hinge, transmembrane, and / or intracellular domains of LILRB1 in combination with a heterologous antigen-binding domain.

[0237] Table 5 Inhibitory Receptors

[0238] 1290

[0239] Inducible Receptor

[0240] In embodiments, the receptor is an inducible receptor. The term “inducible” receptor, CAR, or TCR is used herein to refer to a receptor where at least one polypeptide chain of the receptor is expressed under the control of an inducible promoter. Rather than using a switch 1295 molecule to control assembly of the receptor, one may use an inducer molecule to control expression of a component of the receptor, such as the first polypeptide (referred to as the booster or booster module). Advantage of such embodiments may include more selective control of receptor, because the booster is not present without the inducer molecule.

[0241] An “inducible promoter” is one that is characterized by initiating or enhancing

[0242] 1300 transcriptional activity when in the presence of, influenced by, or contacted by an inducing agent. An inducing agent may be endogenous or a normally exogenous condition, compound, agent, or protein that contacts an engineered nucleic acid in such a way as to be active in inducing transcriptional activity from the inducible promoter. In certain embodiments, an inducing agent is a tetracycline-sensitive protein (e.g., tTA or rtTA, TetR family regulators).

[0243] 1305 Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically / biochemically-regulated and physically-regulated promoters such as alcohol -regulated promoters, tetracycline-regulated promoters (e.g, anhydrotetracycline (aTc)-responsive promoters and other tetracycline

[0244] 1310 responsive promoter systems, which include a tetracycline repressor protein (TetR, or TetRKRAB), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA), and a tetracycline operator sequence (tetO) and a reverse tetracycline transactivator fusion protein (rtTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters

[0245] 1315 from the steroid / retinoid / thyroid 25 receptor superfamily), metal-regulated promoters (e.g, promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g, induced by salicylic acid, ethylene or benzothiadi azole (BTH)), temperature / heat- inducible promoters (e.g., heat shock promoters), pH-regulated promoters, and light-regulated promoters. A non-limiting

[0246] 1320 example of an inducible system that uses a light-regulated promoter is provided in Wang et al, Nat. Methods. 2012 Feb 12;9(3):266-9.

[0247] Additional non-limiting examples of inducible promoters include mifepristone- responsive promoters (e.g., GAL4-Elb promoter) and coumermycin-responsive promoters. See, e.g., Zhao et al., Hum Gene Then 2003 Nov 20; 14(17): 1619-29.

[0248] 1325 Furthermore, there are a variety of inducer molecules that may be used, including but not limited to tetracycline or a derivative thereof, cumate or a derivative thereof, rapamycin or a derivative thereof, FKCsa or a derivative thereof, abscisic acid or a derivative thereof, tamoxifen or a derivative thereof, and gibberellin or a derivative thereof. The inducible receptors described herein may include a dimerization domain. The

[0249] 1330 dimerization domain may be a constitutive dimerization domain.

[0250] Signal 1 + Signal 3 Booster

[0251] Though adoptive transfer of immune cells has achieved remarkable results in many blood cancers, efficacy in solid tumors has been challenging. One explanation for the mixed performance of solid tumor cell therapy is the result of limited access to tumor-associated

[0252] 1335 antigens outside the blood. Thus, it may be advantageous to mimic the effects of antigen with SIB under the control of a small molecule. Further, it may be advantageous in some instances to include a means to amplify the response to SIB in the immune cell product by bringing a second or third stimulus under the control of the small molecule. This can be accomplished by introducing an additional construct that encodes a signal 3 protein or variant, e.g., a cytokine,

[0253] 1340 under the control of the SIB signaling pathway of the signal 1 booster; for instance, by designing the signal 3 booster such that it is induced by NFAT or NF-KB activation. This design, composed of 3 different parts (antigen receptor, SIB, and inducible signal 3 booster) brings the complete circuit of immune cell activation under the control of a small molecule. Thus, in a clinical setting, this discovery will allow physicians to tune the immune response to

[0254] 1345 maximize efficacy, while maintaining a therapeutic window. In the event of idiosyncratic toxicity, the activation of the engineered immune cells can be shut down by cessation of smallmolecule dosing. Thus, the system includes a tuning mechanism to ensure maximum efficacy with reasonable safety, including the means to switch off the response.

[0255] FIG. 15 illustrates the incorporation of signal 1 and signal 3 boosters into an engineered

[0256] 1350 immune cell. The engineered immune cell shown comprises 3 parts: 1) one or more signaling receptor, either engineered (e.g., CARs or TCRs) or endogenous (e.g., TCRs) - here shown by a pair of CARs, 2) Signal 1 booster construct that is controlled by a small molecule inducer that mimics the effect of antigen and activates signaling through the CAR or TCR - here shown by the “SM-inducible signal 1 booster and which is responsive to the small molecule “SM”;

[0257] 1355 and 3) a signal- 1 -responsive booster that mimics the effect of signal 2 and / or signal 3 (e.g., an NFAT-responsive cytokine construct or an NF-KB responsive cytokine construct) - here shown as the NFAT-regulated signal 3 booster. In some cases, the signal 1 booster is activated by the small molecule, which increases activity of transcription factors of the Nuclear Factor of Activated T-cells (NFAT) family. Activated NFAT promotes expression of NFAT-responsive

[0258] 1360 genes thereby resulting in the production of downstream proteins that are relevant to an effective immune response, e.g., cytokines. Alternatively, the signal 1 booster is activated by the small molecule, which increases activity of the transcription factor Nuclear Factor kappa light chain enhancer of activated B cells (NF-KB). Activated NF-KB promotes expression of NF-KB responsive genes thereby resulting in the production of downstream proteins that are

[0259] 1365 relevant to an effective immune response, e.g. cytokines. The engineered immune cell maintains the specificity and selectivity of the dual receptor system while also being responsive to a small molecule that can induce the signal 1 booster to activate tonic signaling and drive transcription of signal 3 booster. The small molecule allows for tunable activation of immune cells.

[0260] 1370 Polynucleotides / Vectors

[0261] The disclosure provides polynucleotides encoding sequence(s) of multipartite receptors. The disclosure further provides T cells comprising the polynucleotides and vectors described herein.

[0262] In embodiments, the sequence encoding the first polypeptide and / or the second and / or

[0263] 1375 the third is operably linked to a promoter. In embodiments, the sequence encoding the first polypeptide is operably linked to a first promoter, the sequence encoding the second polypeptide is operably linked to a second promoter, and the sequence encoding the third polypeptide is operably linked to a third promoter.

[0264] The disclosure provides vectors comprising the polynucleotides described herein.

[0265] 1380 In embodiments, the first, second and optionally third polynucleotide are encoded by a single vector. Methods of encoding multiple polypeptides using a single vector will be known to persons of ordinary skill in the art, and include, inter alia, encoding multiple polypeptides under control of different promoters, or, if a single promoter is used to control transcription of multiple polypeptides, use of sequences encoding internal ribosome entry sites (IRES) and / or

[0266] 1385 self-cleaving peptides. Exemplary self-cleaving peptides include T2A, P2A, E2A and F2A selfcleaving peptides. In embodiments, the T2A self-cleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 489). In embodiments, the P2A self-cleaving peptide comprises a sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 186). In embodiments, the E2A self-cleaving peptide comprises a sequence of

[0267] 1390 QCTNYALLKLAGDVESNPGP (SEQ ID NO: 490). In embodiments, the F2A self-cleaving peptide comprises a sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 491). In embodiments, the T2A self-cleaving peptide comprises a sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 489). Any of the foregoing can also include an N terminal GSG linker. For example, a T2A self-cleaving peptide can also comprise a sequence 1395 of GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 181), which can be encoded by a sequence of

[0268] GGATCCGGAGAGGGCAGAGGCAGCCTGCTGACATGTGGCGACGTGGAAGAGAA CCCTGGCCCC (SEQ ID NO: 492).

[0269] In embodiments, the first polypeptide is operably linked to a promoter. In embodiments,

[0270] 1400 the first polynucleotide encoding a first polypeptide chain comprises, at least one first switch domain, and a scaffolding intracellular domain (e.g., aLAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4-1BB, ZAP70, or GADs domain). In embodiments, the first the first polypeptide chain further comprises a hinge domain. In-I-n embodiments, the first the first polypeptide chain further comprises a transmembrane domain.

[0271] 1405 In embodiments, the second polynucleotide encodes a second polypeptide comprising an antigen binding domain a transmembrane domain, one or more costimulatory intracellular domains (e.g., a CD28 domain or a 41BB domain), and a signaling intracellular domain (e.g., a CD3zeta domain). In embodiments, the second polypeptide further comprises at least one second switch domain. In embodiments, the second polypeptide does not comprise a second

[0272] 1410 switch domain.

[0273] In embodiments, the third polynucleotide encodes a third polypeptide chain comprising an antigen-binding domain (e.g., a TCR extracellular domain) and a transmembrane domain.

[0274] In embodiments, the vector is an expression vector, i.e. for the expression of the first, second and optionally third polypeptide in a suitable cell.

[0275] 1415 Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce nonproliferating cells, such as hepatocytes. They also have the added advantage of low

[0276] 1420 immunogenicity.

[0277] In embodiments, the vector is a lentiviral vector.

[0278] Immune Cell Manufacturing

[0279] Another application of embodiments is in the manufacture of immune cells.

[0280] An advantages of some embodiments may be observed improvements in immune cell

[0281] 1425 expansion in a subject when antigen levels are low or availability in the bloodstream is limited. Accordingly, the invention may be applied in treatment of solid tumors having low antigen levels or low bloodstream availability of antigen. Another advantage of some embodiments may be an ability to tune the tonic signal in a patient over time (e.g., by pulsing) or in degree. In manufacture of immune cells, embodiments may provide, in some cases, improved the

[0282] 1430 quality of the cell product during manufacturing (e.g., its proliferation rate, purity, etc.).

[0283] In embodiments, a method of expanding immune cells is described. In embodiments, the method is used to activate immune cells in vitro. In embodiments, the method is used to activate and expand immune cells in vitro. In embodiments, the immune cell is a T cell. In embodiments, the immune cell is an NK cell. In embodiments, the immune cells are contacted

[0284] 1435 with a small molecule to boost activation of the immune cells. In embodiments, the multipartite receptors described herein can be used in immune cell manufacturing.

[0285] In variations of any of the foregoing embodiments, the second polypeptide does not comprise a second switch domain. An illustrative embodiment is provided in FIG. 9, which depicts a multipartite chimeric antigen receptor (CAR) composed of two polypeptide chains.

[0286] 1440 The signal booster described herein surprisingly works in the absence of a switch domain on the second polypeptide of the multipartite CAR or multipartite TCR as illustrated in Example 4 and FIG. 8. The first polypeptide chain (on right) comprises a transmembrane domain, a switch domain comprising two “FK506 binding protein” (FKBP) domains, and a scaffolding intracellular domain which in the depicted embodiment is a “linker for activation of T cells”

[0287] 1445 (LAT) domain. The second polypeptide (on left) comprises an antigen-binding domain which is a single-chain variable fragment (scFv), a transmembrane domain, two costimulatory intracellular domains which are a CD28 domain and a 4 IBB domain, and a signaling intracellular domain which is a CD3zeta domain. The small molecule AP1903 (a lipid- permeable tacrolimus analogue known as rimiducid, labeled “Rim”) is depicted as selectively

[0288] 1450 dimerizing the switch domains present on the first polypeptide. In a variation of this embodiment, the second polypeptide lacks the costimulatory intracellular domains (as in a so- called second-generation CAR, rather than the third generation CAR depicted). In another variation of this embodiment, termed a switchable multipartite T cell receptor (TCR), the second polypeptide chain is TCR comprising TCRalpha and TCRbeta extracellular domains

[0289] 1455 (or a single-chain TCR), and a transmembrane domain. In other variations, the switch domain on the first polypeptide comprises only one FKBP domain or comprises three or more FKBP domains. In further variations the first polypeptide lacks a transmembrane domain and is termed a cytosolic or soluble first polypeptide. Further variations employing alternative transmembrane switch domains (see Table 1), and intracellular domains, are described above.

[0290] 1460 In embodiments, an immune cell comprises an artificial receptor configured to specifically activate in response to an antigen and independently to a small molecule. In embodiments, the artificial receptor is a multipartite chimeric antigen receptor (CAR). In embodiments, the multipartite CAR comprises a first polypeptide chain. In embodiments, the first polypeptide comprises a transmembrane domain. In embodiments, the first polypeptide

[0291] 1465 comprises at least one first switch domain. In embodiments, the first polypeptide further comprises a scaffolding intracellular domain (e.g., a LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4-1BB, ZAP70, or GADs domain). In embodiments, the first polypeptide does not comprise a transmembrane domain. In embodiments, the first polypeptide further comprises a hinge domain. In embodiments, the multipartite CAR

[0292] 1470 comprises a second polypeptide chain. In embodiments, the second polypeptide comprises an antigen-binding domain (e.g., an scFv), a transmembrane domain, one or more costimulatory intracellular domains (e.g., a CD28 domain or a 41BB domain), and a signaling intracellular domain (e.g., a CD3zeta domain). In embodiments, the second polypeptide does not contain at least one switch domain. In embodiments, the switch domains on the first polypeptide are

[0293] 1475 configured to selectively dimerize in the presence of a switch molecule (e.g., rapamycin or a rapalog or an estrogen receptor modulator, e.g., tamoxifen).

[0294] In embodiments, the artificial receptor is a multipartite T cell receptor (TCR). In embodiments, the multipartite TCR comprises a first polypeptide chain. In embodiments, the first polypeptide comprises a transmembrane domain. In embodiments, the first polypeptide

[0295] 1480 comprises at least one switch domain. In embodiments, the multipartite TCR further comprises a second polypeptide. In embodiments, the second polypeptide comprises a transmembrane domain. In embodiments, the second polypeptide further comprises an intracellular domain (e.g., a CD3zeta domain). In embodiments, the second polypeptide does not comprise at least one switch domain. In embodiments, the multipartite TCR further comprises a third

[0296] 1485 polypeptide. In embodiments, the third polypeptide comprises an antigen-binding domain (e.g., a TCR extracellular domain). In embodiments, the third polypeptide comprises a transmembrane domain. In embodiments, the switch domains on the first polypeptide are configured to selectively dimerize in the presence of a switch molecule (e.g., rapamycin or a rapalog or an estrogen receptor modulator, e.g., tamoxifen).

[0297] 1490 In embodiments, the first polypeptide of the multipartite CAR is selected from the pairs disclosed in Table 1 and the small molecule is the corresponding switch molecule in Table 1, or a functional analogue thereof. In embodiments, the first polypeptide of the multipartite TCR is selected from the pairs disclosed in Table 1 and the small molecule is the corresponding switch molecule in Table 1, or a functional analogue thereof. 1495 In embodiments, the artificial receptor is an inducible multipartite chimeric antigen receptor (CAR). In embodiments, the inducible multipartite CAR comprises a first polypeptide operatively linked to an inducible promoter. In embodiments, the first polypeptide comprises a transmembrane domain. In embodiments, the first polypeptide comprises at least one switch domain. In embodiments, the first polypeptide further comprises a scaffolding intracellular

[0298] 1500 domain (e.g., a LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3(^, 4-1BB, ZAP70, or GADs domain). In embodiments, the first polypeptide does not comprise a transmembrane domain. In embodiments, the first polypeptide further comprises a hinge domain. In embodiments, the inducible multipartite CAR comprises a second polypeptide chain. In embodiments, the second polypeptide comprises an antigen-binding

[0299] 1505 domain (e.g., an scFv), a transmembrane domain, one or more costimulatory intracellular domains (e.g., a CD28 domain or a 41BB domain), and a signaling intracellular domain (e.g., a CD3zeta domain). In embodiments, the second polypeptide does not contain at least one switch domain. In embodiments, the switch domains on the first polypeptide are configured to selectively dimerize in the presence of a switch molecule (e.g., rapamycin or a rapalog or an

[0300] 1510 estrogen receptor modulator, e.g., tamoxifen).

[0301] In embodiments, the artificial receptor is an inducible multipartite T cell receptor (TCR). In embodiments, the inducible multipartite TCR comprises a first polypeptide chain. In embodiments, the first polypeptide comprises a transmembrane domain. In embodiments, the first polypeptide comprises at least one switch domain. In embodiments, the multipartite TCR

[0302] 1515 further comprises a second polypeptide. In embodiments, the second polypeptide comprises a transmembrane domain. In embodiments, the second polypeptide further comprises an intracellular domain (e.g., a CD3zeta domain). In embodiments, the second polypeptide does not comprise at least one switch domain. In embodiments, the multipartite TCR further comprises a third polypeptide. In embodiments, the third polypeptide comprises an antigen¬

[0303] 1520 binding domain (e.g., a TCR extracellular domain). In embodiments, the third polypeptide comprises a transmembrane domain. In embodiments, the switch domains on the first polypeptide are configured to selectively dimerize in the presence of a switch molecule (e.g., rapamycin or a rapalog or an estrogen receptor modulator, e.g., tamoxifen).

[0304] In embodiments, the first polypeptide of the inducible multipartite CAR is selected from

[0305] 1525 the pairs disclosed in Table 1 and the small molecule is the corresponding switch molecule in Table 1, or a functional analogue thereof. In embodiments, the first polypeptide of the inducible multipartite TCR is selected from the pairs disclosed in Table 1 and the small molecule is the corresponding switch molecule in Table 1, or a functional analogue thereof. In embodiments, the first polypeptide comprises at least two first switch domains

[0306] 1530 selected from Table 1. In embodiments, the second polypeptide does not comprise a switch domain. In embodiments, the first polypeptide comprises a pair of switch domains disclosed in a row of Table 1.

[0307] In embodiments, the inducible promoter of the inducible multipartite CAR or inducible multipartite TCR is selected from chemically / biochemically-regulated and physically-

[0308] 1535 regulated promoters such as alcohol -regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline responsive promoter systems, which include a tetracycline repressor protein (TetR, or TetRKRAB), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA), and a tetracycline operator sequence (tetO) and a reverse tetracycline transactivator fusion protein

[0309] 1540 (rtTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid 25 receptor superfamily), metal -regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene

[0310] 1545 or benzothiadi azole (BTH)), temperature / heat- inducible promoters (e.g., heat shock promoters), pH-regulated promoters, and light-regulated promoters.

[0311] In embodiments, the first polypeptide comprises a scaffolding intracellular domain selected from LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4- IBB, ZAP70, or GADs or a functional variant thereof. In embodiments, the multipartite

[0312] 1550 CAR is expressed with an inhibitory receptor. In embodiments, the multipartite TCR is expressed with an inhibitory receptor. In embodiments, the inhibitory receptor is specific to a target disclosed in Table 5. In embodiments, the inhibitory receptor comprises an intracellular domain of a LILRB1 and optionally the hinge and / or transmembrane domains of LILRB1.

[0313] In variations of any of the previous embodiments, the first polypeptide comprising a

[0314] 1555 transmembrane domain, at least one switch domain, and a scaffolding intracellular domain (e g., a LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4- 1BB, ZAP70, or GADs domain) is able to dimerize in response to a small molecule and increase tonic signaling in immune cells expressing the multipartite CAR independent of antigen. In variations of any of the previous embodiments, the first polypeptide comprising a

[0315] 1560 transmembrane domain, at least one switch domain, and a scaffolding intracellular domain (e g., a LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4- 1BB, ZAP70, or GADs domain) is able to dimerize in response to a small molecule and increase tonic signaling in immune cells expressing the multipartite TCR independent of antigen. In variations of any of the previous embodiments, the first polypeptide comprising a

[0316] 1565 transmembrane domain, at least one switch domain, and a scaffolding intracellular domain (e g., a LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4- 1BB, ZAP70, or GADs domain) is able to dimerize in response to a small molecule and increase tonic signaling in immune cells expressing the inducible multipartite CAR independent of antigen. In variations of any of the previous embodiments, the first polypeptide

[0317] 1570 comprising a transmembrane domain, at least one switch domain, and a scaffolding intracellular domain (e.g., a LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3(^, 4-1BB, ZAP70, or GADs domain) is able to dimerize in response to a small molecule and increase tonic signaling in immune cells expressing the inducible multipartite TCR independent of antigen.

[0318] 1575 EXAMPLES

[0319] The following Examples are intended for illustration only and do not limit the scope of the invention.

[0320] Example 1. Selective activation of switchable CAR by switch molecule

[0321] This Example describes design and testing of switchable multipartite chimeric antigen

[0322] 1580 receptors (individually, a “switchable CAR”). The Example demonstrates that tonic signaling from the switchable CAR can be triggered by the switch molecule in the absence of cognate antigen for the receptor. The LAT and LAT G160D constructs are shown to selectively activate in the presence of the switch molecule.

[0323] In each case the tested immune cells were engineered to express the switchable CAR

[0324] 1585 by introducing into the cells a polynucleotide (1) encoding a first polypeptide chain (referred to as a “booster construct”) that included two tandem FKBP-based switch domains followed by a scaffolding intracellular domain, or a pair of scaffolding intracellular domains fused to each other (see Table A); and (2) a second polypeptide chain (SEQ ID NO: 19) that includes as an extracellular domain a single-chain variable fragment (scFv) specific to mesothelin

[0325] 1590 (MSLN), a transmembrane domain, two tandem FKBP-based switch domains followed by the intracellular domains of a conventional third-generation chimeric antigen receptor (CD28, 4- 1BB, and CD3zeta). Some first polypeptide chains also included a transmembrane domain. The intracellular scaffolding domains tested were LAT, LAT with a G160D amino acid substitution (LAT G160D), SLP76, FYN, or PLCGL Also tested was a fusion of LAT and ZAP70. The 1595 following sequences were tested in this experiment, SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 13. A representative switchable CAR is depicted in FIG 1 and sequences of the tested first polypeptide chains are provided in Table A.

[0326] Seven scaffolding intracellular domains were designed and tested for their ability to increase tonic signaling in immune cells when incorporated into a switchable multipartite CAR.

[0327] 1600 The scaffolding intracellular domains were designed to induce immune cell activation independent of antigen binding. Wildtype LAT, LAT G160D, LAT-ZAP70 fusion, membrane tethered- SLP76, FYN, cytoplasmic SLP76 and cytoplasmic-PLCGl were expressed alone or in combination with an activator chimeric antigen receptor specific for mesothelin (“MSLN CAR”). The multipartite switchable CARs were designed to have two tandem FKBP switch

[0328] 1605 domains (one on each polypeptide), such that upon treatment with rimiducid (Rim) or another rapalog, the multipartite switchable CAR assembles by dimerization of the scaffolding intracellular domains and the antigen-binding second polypeptide chain.

[0329] Tonic signaling was measured in Jurkat cells expressing the booster alone or in combination with MSLN CAR using the NF AT luciferase assay. Jurkat cells were transfected

[0330] 1610 with a booster construct comprising two tandem FKBP-based switch domains followed by a scaffolding intracellular domain alone or in combination with MSLN CAR. Jurkat cells were treated with 50nM Rim for 6 hours and then their activation was quantified by luminescence. Jurkat cells transfected with scaffolding intracellular domain alone showed little to no increase in tonic signaling expect for LAT-ZAP70 (FIG. 2A). LAT-ZAP70 constitutively increased tonic

[0331] 1615 signaling (with and without the addition of Rim). Co-expression of booster construct and the MSLN CAR resulted in increased tonic signaling upon the addition of 50nM Rim. Cells expressing the LAT and LAT (G160D) scaffolding intracellular domain from the first polypeptide with the switchable multipartite CAR showed the highest Rim-dependent induction of tonic signaling (FIG. 2B). Membrane-tethered SLP76 (mem-SLP76) and

[0332] 1620 cytosolic SLP76 scaffolding intracellular domains also showed some Rim-dependent tonic increase when co-expressed with MSLN CAR. LAT-ZAP70 constitutively increased tonic signaling when co-expressed with MSLN CAR (with and without the addition of Rim).

[0333] This example shows that co-expression of scaffolding intracellular domain polypeptide comprising two FKBP switch domains co-expressed with MSLN CAR comprising two FKBP

[0334] 1625 switch domains results in increased tonic signaling upon treatment with Rim. This shows that the booster constructs described herein are able to activate immune cells independent of antigen engagement. Example 2. Independent activation of switchable CAR by antigen or switch molecule

[0335] This Example demonstrates that switchable CARs using LAT or LAT G160D may be

[0336] 1630 activated by antigen (cells expressing MLSN) or independently by the switch molecule (Rim).

[0337] The switchable CARs using LAT and LAT (G160D) constructs from Example 1 were further characterized (SEQ ID NOs: 1,3). First, tonic signaling was measured in Jurkat cells transfected with the LAT or LAT (G160D) booster and the MSLN CAR (SEQ ID NO: 19). Both constructs contain 2 FKBP switch domains. Jurkat cells were treated with increasing

[0338] 1635 doses of Rim for 6 hours and then activation was measured. Jurkat cells co-expressing the CAR and either booster showed a Rim dose-dependent increase in tonic signaling (FIG. 3A). Expression of the MSLN CAR alone did not increase tonic signaling. The same experiment was repeated with LAT (SEQ ID NO: 157). and LAT G160D boosters and MSLN constructs that contain only 1 FKBP switch domain Similarly, Jurkat cells co-expressing the MSLN-

[0339] 1640 IxFKBP CAR (SEQ ID NO: 21) and the booster showed a Rim dose-dependent increase in tonic signaling (FIG. 3B). A similar trend was when activation with increasing doses of Rim compared to co-culture with target cells (FIG. 4). The following constructs were tested in FIG. 4: SEQ ID NOs: 21 and 157. Expression of the MSLN CAR or either booster alone did not increase tonic signaling in either experiment.

[0340] 1645 Next, the ability of immune cells expressing a switchable multipartite CAR to respond to antigens present on the surface of cells was tested. For this experiment, Jurkat cells transfected with the CAR alone, the CAR with FKPB switch domains or the CAR with FKPB switch domains and the scaffolding intracellular domain with FKBP switch domains. Jurkat cells were co-cultured with MSLN knockout (KO) HeLa cells that were transfected with

[0341] 1650 increasing concentrations of MSLN mRNA. The Jurkat cells and the MLSN KO HeLa cells were co-cultured for six hours and then their activation was measured via NF AT reporter assay. The following sequences were tested in this experiment: SEQ ID NOs: 1, 3, 7, 19, and 183. Jurkat cells expressing either booster construct and a CAR with FKBP switch domains showed antigen-dependent increase in activation without Rim (FIG. 5A). Jurkat cells expressing either

[0342] 1655 booster and a CAR with FKBP switch domains treated with Rim showed increased tonic signaling and Emin (FIG. 5B). Addition of the FKBP domains to the CAR did not increase tonic signaling unless co-expressed with a booster . Expression of a signal booster alone did not increase tonic signaling. This was further confirmed when the same Jurkat cells were grown in the absence of target cells and treated with Rim. The addition of Rim increased tonic 1660 signaling when the CAR and the booster (both containing FKBP switch domains) were coexpressed in Jurkat cells (FIG. 4).

[0343] Example 3. Switchable CAR in combination in inhibitory receptor

[0344] This Example shows that a dual receptor (activator / blocker) immune cell can be selectively activated by the switch molecule in the presence of antigen recognized by the

[0345] 1665 blocker receptor.

[0346] Antigen dependent activation and sensitivity were quantified next in Jurkat cells expressing a dual CAR system comprising an MSLN activator CAR with FKBP switch domain, an HLA-A*02 blocker CAR in combination with a LAT booster construct. MSLN KO HeLa cells were transfected with increasing concentrations of MSLN mRNA to generate a titration

[0347] 1670 of antigen levels on target cells. Jurkat cells expressing the indicated constructs were cocultured with target cells with or without Rim. The following sequences were tested in this experiment: SEQ ID NOs: 1, 3, 19, 21, and 182 Jurkat cells expressing the dual CAR system and the LAT booster construct showed increased tonic signaling and Emin after the addition of Rim compared to Jurkat cells only expressing the dual CAR system(FIGs. 6A-6B).

[0348] 1675 Antigen dependent activation was measured next in Jurkat cells expressing the same dual CAR system as above and a LAT booster construct. HeLa cells were transfected with increasing amount of MSLN mRNAto generate a titration of activator antigen on target cells. Jurkat cells expressing dual CAR system and LAT booster showed increased tonic signaling and Emax with the addition of Rim compared to Jurkat cells only expressing the dual CAR

[0349] 1680 system. (FIGs. 7A-7B). The following sequences were tested in this experiment: SEQ ID NOs: 1, 21, 182, and 183.

[0350] Antigen dependent blocking was measured next in Jurkat cells expressing the same dual CAR system as above and a LAT booster construct. MSLN positive HeLa cells were transfected with increasing amount of HLA-A*02 mRNA to generate a titration of blocker

[0351] 1685 antigen on target cells. Jurkat cells expressing dual CAR system and LAT booster showed increased tonic signaling and Emax with the addition of Rim compared to Jurkat cells only expressing the dual CAR system. (FIGs. 8A-8B). The following sequences were tested in this experiment: SEQ ID NOs: 1, 21, 182, and 183. Importantly, the HLA-A*02 blocker CAR was sufficient to reduce activation of immune cells co-cultured with target expressing HLA-A*02

[0352] 1690 and MSLN.

[0353] Taken together, these examples show that immune cells expressing an activator CAR with FKBP switch domains in combination with a signal booster with FKBP switch domain have increased tonic signaling in the presence of Rim compared to immune cells expressing a CAR alone. Additionally, co-expression of the activator CAR with FKBP switch domains and

[0354] 1695 signal booster with FKBP switch domains results in increased antigen-dependent activation. The expression of a blocker CAR in combination with an activator CAR and booster was sufficient to reduce the killing of cells expressing the blocker antigen. This shows that the booster can be expressed with an activator CAR and blocker CAR to increase tonic signaling in immune cells while maintaining a degree of selectivity for target cells.

[0355] 1700 Example 4. CAR-independent booster activity

[0356] This Example shows that the booster constructs described in previous examples is capable of increasing tonic signaling in immune cells independent of a CAR comprising a switch domain.

[0357] Antigen independent activation was quantified in Jurkat cells expressing different

[0358] 1705 combinations of LAT booster construct with and without FKBP switch domains, MSLN activator CAR with and without FKBP switch domains and an HLA-A*02 blocker CAR. The following sequences were tested in this experiment: SEQ ID NOs: 21, 34, 91 andl82. Expression of HLA-A*02 blocker CAR alone or in combination with a LAT booster construct with or without FKBP switch domains did not increase tonic signaling in Jurkat cells in

[0359] 1710 response to increasing doses of Rim. Expression of MSLN activator CAR with or without FKBP switch domains and an HLA-A*02 blocker CAR did not increase tonic signaling in Jurkat cells in response to increasing doses of Rim. Expression of MSLN activator CAR without FKBP switch domains , HLA-A*02 blocker CAR and LAT booster construct without FKBP switch domains increased tonic signaling in Jurkat cells slightly but it did not increase

[0360] 1715 in response to increasing doses of Rim. As shown in the previous examples, expression of the LAT booster construct with FKBP switch domains, MSLN activator CAR with FKBP switch domains and HLA-A*02 blocker CAR increased tonic signaling in Jurkat cells in response to increasing doses of Rim. Surprisingly, expression of LAT booster construct with FKBP switch domains, MSLN activator CAR without FKBP switch domains and HLA-A*02 blocker in

[0361] 1720 Jurkat cells increased tonic signaling in response to increasing doses of Rim (FIG. 9). The following sequences were tested in this experiment: SEQ ID NOs: 1, 21, 34, 182, and 183.

[0362] This example shows that co-expression of scaffolding intracellular domain polypeptide comprising two FKBP switch domains co-expressed with MSLN CAR without switch domains results in increased tonic signaling upon treatment with Rim. This shows that the booster 1725 constructs described herein are able to activate immune cells independent of antigen engagement.

[0363] Example 5. Expression of Signal 1 booster increases IFN-y

[0364] This Example demonstrates that immune cells expressing a CAR and a switchable LAT- Ix FKBP booster have increased gamma interferoni (IFN-y) production when treated with

[0365] 1730 Rimiducid.

[0366] JNL cells were transfected with CAR alone, CAR+ LAT-lx FKBP or LAT-2xFKBP and expression of each was confirmed via flow cytometry (FIG. 11). The following sequences were tested in this experiment: SEQ ID NOs: 1, 19 and 88. Next the cells were treated with increasing doses of Rimiducid for 24 hours and then IFN-y production was measured in cell media.

[0367] 1735 Untransduced (UTD) JNL did not show any increase in IFN-y production upon treatment with Rimiducid. Expression of CAR alone or LAT-2xFKBP resulted in a basal increase in IFN-y production but treatment with Rimiducid did not increase IFN-y production. Expression of CAR+ LAT-lx FKBP increased the basal production of IFN-y. Treatment with increasing doses of Rimiducid further increased IFN-y production.

[0368] 1740 This example shows that expression of CAR with LAT-lx FKBP results in a basal increase in IFN-y in JNL cells. Additionally, treatment with Rimiducid (switch molecule) results in a dose dependent increase in IFN-y production. Thus, the CAR + LAT-lx FKBP system allows for tunable increase in tonic signaling and in production of cytokines (Signal 3).

[0369] Example 6. Characterization of additional Signal 1 boosters

[0370] 1745 This example demonstrates that switchable signal 1 boosters can activate tonic signaling in immune cells expressing CARs with and without switch domain when treated with Rimiducid (switch molecule).

[0371] For the first set of experiments JNL cells were transfected with DNA encoding a MSLN CAR and with DNA encoding one of the following SIBs : LAT-FKBP , CD3z-FKBP+LAT-

[0372] 1750 FKBP , SLP76-FKBP+LAT-FKBP , memSLP76-FKBP+LAT-FKBP , ZAP70-FKBP+LAT- FKBP , or PLCgl-FKBP LAT-FKBP . The following sequences were tested in this experiment: SEQ ID NOS: 11, 12, 21, 74, 88, 100, 109, 110, 111, 143, 167, 195 and 198. After transfection, the JNL cells were treated with Rimiducid and activation was measured based on luminescence (FIG. 12 top row). FIG. 12 shows the induction ratio for immune cell activation of transfected

[0373] 1755 JNL cells treated with Rimiducid compared to transfected JNL cells not treated with Rimiducid. This data demonstrates that expression of a signal 1 booster construct with a CAR (without switch domains) results in increased tonic signaling upon treatment with Rimiducid (switch molecule).

[0374] Next, JNL cells were transfected with DNA encoding MSLN-lxFKBP CAR and with

[0375] 1760 DNA encoding one of the following SIBs: LAT-FKBP , 1 / 2LAT [129aa N-term del.]-FKBP (SEQ ID NO: 99), LAT [30aa C-term del.]-FKBP , SLP76-FKBP LAT-FKBP , memSLP76- FKBP+LAT-FKBP , or ZAP70-FKBP+LAT-FKBP . After transfection, the JNL cells were treated with Rimiducid and activation was measured based on luminescence (FIG. 12 second row). This data demonstrates that expression of a signal 1 booster construct with a CAR-

[0376] 1765 IxFKBP results in increased tonic signaling upon treatment with Rimiducid (switch molecule). FIG. 13 shows a dose response curve for immune cell activation upon treatment with increasing doses of Rimiducid (switch molecule). The same constructs described in the second row of FIG. 12 are tested in FIG. 13.

[0377] Next, JNL cells were transfected with DNA encoding a T cell receptor (TCR) (native,

[0378] 1770 unmodified) and with DNA encoding one of the following SIBs: LAT-FKBP , CD3z- FKBP+LAT-FKBP , SLP76-FKBP LAT-FKBP , memSLP76-FKBP+LAT-FKBP , ZAP70- FKBP+LAT-FKBP , or PLCgl-FKBP LAT-FKBP . After transfection, the JNL cells were treated with Rimiducid and activation was measured based on luminescence (FIG. 12 third row). Surprisingly, expression of a TCR (native, unmodified) with SLP76-FKBP LAT-FKBP

[0379] 1775 resulted in higher immune cell activation than exposure to target cells (FIG. 12 last column). This data demonstrates that expression of a signal 1 booster construct with a TCR (native, unmodified) results in increased tonic signaling upon treatment with Rimiducid (switch molecule).

[0380] Next, JNL cells were transfected with DNA encoding TCR-FKBP (native, CD3e-

[0381] 1780 FKBP) and with DNA encoding one of the following SIBs: LAT-FKBP , SLP76-FKBP LAT- FKBP or memSLP76-FKBP+LAT-FKBP . After transfection, the JNL cells were treated with Rimiducid and activation was measured based on luminescence (FIG. 12 fourth row). Surprisingly, expression of TCR-FKBP with SLP76-FKBP LAT-FKBP resulted in higher immune cell activation than exposure to target cells (FIG. 12 last column). This data

[0382] 1785 demonstrates that expression of a signal 1 booster construct with a TCR-FKBP results in increased tonic signaling upon treatment with Rimiducid (switch molecule).

[0383] Next, the switch molecule tamoxifen was tested for its ability to induce immune cell activation in immune cells expressing a LAT-ER (LAT-estrogen receptor) with a CAR-ER (CAR-estrogen receptor). For this experiment, JNL cells were transfected with DNA encoding

[0384] 1790 CAR-ER and with DNA encoding LAT-ER. After transfection, the JNL cells were treated with doses of Tamoxifin and activation was measured based on luminescence (FIG. 12 last row). Expression of CAR-ER and LAT-ER with tamoxifen treatment increases tonic signaling. This data demonstrates that co-expression of a signal 1 booster construct with lx ER switch domain with a CAR construct with a lx ER switch domain results in increased tonic signaling upon

[0385] 1795 treatment with tamoxifen (switch molecule).

[0386] FIG. 14A shows that Rim and tamoxifen can act as switch molecules to increase tonic signaling in immune cells expressing a booster construct with a switch domain. For this experiment JNL cells were engineered to express MSLN CAR alone, MSLN CAR+ LAT- IxFKBP, CAR-lxFKBP or CAR-lxFKBP+LAT-lxFKBP (Seq ID NOs: 55 and 88). The same

[0387] 1800 set of booster constructs were expressed with the lx FKBP switch domain changed to an ESRI (ER) switch domain (SEQ ID NOs: 183 and 195). Analysis of immune cell activation shows that JNL cells expressing the CAR alone or the CAR-lxFKBP do not increase immune cell activation as Rim concentration increases. JNL cells expressing MSLN CAR+ LAT-lxFKBP or CAR-lxFKBP+LAT-lxFKBP did show a dose dependent increase in immune cell activation

[0388] 1805 as Rim concentration increased. A similar trend was seen with ESRI switch domains and titrated Tamoxifen.

[0389] Next, a similar set of experiments were run but rapalog AP21967was tested as a switch molecule. For this experiment, JNL cells were engineered to express one of the following activator receptors: CAR IxFKBP, CAR 2x FRB, or CAR 2x ESR1. JNL cells were further

[0390] 1810 engineered to express one of the following signal booster constructs: LAT lx FKBP, LAT lx FKBP+SLP76 2x FKBP, LAT IxESRl or Lat lx ESR1+ SLP76 lx ESR1. The FKBP containing signal booster constructs were tested with both the CAR lx FKBP and the CAR 2x FRB. Expression of any of the activator receptors alone did not increase immune cell activation even as the switch molecule concentration increased (FIG. 14B). Expression of the CAR

[0391] 1815 IxFKBP with LAT lx FKBP or LAT lx FKBP+SLP76 2x FKBP resulted in dose dependent increase in immune cell activation as rapalog levels increased. Expression of the CAR 2x FRB with LAT lx FKBP or LAT lx FKBP+SLP76 2x FKBP resulted in dose dependent increase in immune cell activation as rapalog levels increased. Expression of the CAR 2x ESRI with LAT IxESRl or Lat lx ESR1+ SLP76 lx ESRI resulted in dose dependent increase in immune cell

[0392] 1820 activation as tamoxifen levels increased (FIG. 14B) The addition of SLP76 signal booster to the CAR+LAT system results in greater immune cell activation, equivalent or exceeding on target activation. In each system tested (FKBP+RIM, FKBP / FRB+ Rapamycin or ESRl+tamxofen), the addition of the SLP76 signal booster increased immune cell activation. This approach can also be extended to the FRB / FKBP switch domains with switch molecule

[0393] 1825 AP21967 (rapalog).

[0394] As a whole this example demonstrates one can use a switch molecule to control the tonic signaling and activation of immune cells expressing a signal 1 booster with a switch domain. Surprisingly, the signal 1 booster increased immune cell signaling independent of antigen engagement and dimerization with CAR. Addition of a SLP76 signal booster with a

[0395] 1830 CAR and LAT signal booster results in greater immune cell activation, equivalent or exceeding on target activation.

[0396] Example 7. Development of Signal 1 and Signal 3 Expressing Immune cells

[0397] FIG. 15 illustrates the incorporation of signal 1 and signal 3 boosters into an engineered immune cell to generate a cell therapy system with tumor selectivity whose activity is tunable

[0398] 1835 based on the presence of a switch molecule. The engineered immune cell shown comprises 3 parts: 1) one or more signaling receptor, either engineered (e.g., CARs or TCRs) or endogenous (e.g., TCRs) - here shown by a pair of CARs, 2) Signal 1 booster construct that is controlled by a small molecule inducer that mimics the effect of antigen and activates signaling through the CAR or TCR - here shonw by the “SM-inducible signal 1 booster and which is responsive

[0399] 1840 to the small molecule “SM”; and 3) a signal- 1 -responsive booster that mimics the effect of signal 2 and / or signal 3 (e.g., an NFAT-responsive cytokine construct or NF-KB-responsive cytokine construct) - here shown as the NFAT-regulated signal 3 booster. In some cases, the signal 1 booster is activated by the small molecule, which increases activity of transcription factors of the Nuclear Factor of Activated T-cells (NF AT) family. Activated NF AT promotes

[0400] 1845 expression of NFAT-responsive genes thereby resulting in the production of downstream proteins that are relevant to an effective immune response, e.g., cytokines. Alternatively, the signal 1 booster is activated by the small molecule, which increases activity of the transcription factor Nuclear Factor kappa light chain enhancer of activated B cells (NF-KB). Activated NF- KB promotes expression of NF-KB responsive genes thereby resulting in the production of

[0401] 1850 downstream proteins that are relevant to an effective immune response, e.g. cytokines. The engineered immune cell maintains the specificity and selectivity of the dual receptor system while also being responsive to a small molecule that can induce the signal 1 booster to activate tonic signaling and drive transcription of signal 3 booster. The small molecule allows for tunable activation of immune cells. 1855 Example 7. Characterization of switchable CAR by FRB / Rapamycin system

[0402] This Example describes design and testing of switchable multipartite chimeric antigen receptors (individually, a “switchable CAR”). The Example demonstrates that tonic signaling from the switchable CAR can be triggered by the switch molecule in the absence of cognate antigen for the receptor.

[0403] 1860 In each case the tested immune cells were engineered to express the switchable CAR by introducing into the cells at least two polynucleotide sequences. The first polynucleotide sequence encodes a first polypeptide chain comprising an extracellular domain of a singlechain variable fragment (scFv) specific to mesothelin (MSLN), a transmembrane domain and the intracellular domains of a conventional second-generation chimeric antigen receptor

[0404] 1865 (CD28 and CD3zeta) or third-generation chimeric antigen receptor (CD28, 4-1BB, and CD3zeta). The first polypeptide may comprise one or two FRB-based switch domains and the intracellular domains of a conventional second-generation chimeric antigen receptor (CD28 and CD3zeta) or third-generation chimeric antigen receptor (CD28, 4- IBB, and CD3zeta) in any order. The one or two FRB-based switch domains can be followed by the intracellular

[0405] 1870 domains of a conventional second-generation chimeric antigen receptor (CD28 and CD3zeta) or third-generation chimeric antigen receptor (CD28, 4-1BB, and CD3zeta). The intracellular domains of a conventional second-generation chimeric antigen receptor (CD28 and CD3zeta) or third-generation chimeric antigen receptor (CD28, 4- IBB, and CD3zeta) can be followed by one or two FRB-based switch domains. The first polypeptide can comprise any

[0406] 1875 combination of the intracellular domains of a second-generation chimeric antigen receptor (CD28 and CD3zeta) or conventional third-generation chimeric antigen receptor (CD28, 4- 1BB, and CD3zeta). In FIG. 16 the abbreviation 28BBz means the construct comprises CD28, 4- IBB, and CD3zeta. In FIG. 16 the abbreviation 28z means the construct comprises CD28, and CD3zeta. The second polynucleotide (referred to as a “booster construct”)

[0407] 1880 encodes a second polypeptide that comprises one or two FRB-based switch domains and followed by a scaffolding intracellular domain, or a pair of scaffolding intracellular domains fused to each other. The following intracellular domains were tested in this experiment: lx FKBP-LAT, 2x FKBP-LAT, LAT-lxFKBP (c-term), lx FKBP-SLP76, LAT-lxFKBP IxFRB- SLP76, LAT-lxFKBP+2xFRB-SLP76, LAT-2xFKBP+lxFRB-SLP76 and LAT-

[0408] 1885 2xFKBP+2xFRB-SLP76 (SEQ ID NOs: 1, 12, 88, 89, 91, 192, and 193). The following activator receptor constructs were tested in this experiment: SEQ ID NOs: 183-191. In the first set of experiments, after transfection, the JNL cells were treated with rapalog and luminescence was measured after 24 hours to measure immune cell activation (FIG. 16). The values in FIG.16 represent induction ratios of JNL treated with rapalog compared to those

[0409] 1890 not treated with rapalog. Analysis of activation shows that immune cells expressing the combination of CAR-lxFRB-28BBz with IxFKBP-LAT and treated with rapalog resulted in a 16.9 fold induction in activation compared to cells not treated with rapalog. Analysis of activation shows that immune cells expressing the combination of CAR-2xFRB-28BBz with IxFKBP-LAT and treated with rapalog resulted in a 18.3 fold induction in activation

[0410] 1895 compared to cells not treated with rapalog. Additionally, immune cells expressing the combination of CAR-lxFRB-28z with IxFKBP-LAT and treated with rapalog resulted in a 19.2 fold induction in activation compared to cells not treated with rapalog. This shows that the combination of a second or third generation CAR comprising 1 or 2 FRB domains expressed with IxFKBP-LAT can increase tonic signaling in immune cells with a small

[0411] 1900 molecule, independent of antigen binding.

[0412] Next the same set of constructs were tested in a co-culture assay with MSLN+ HeLa cells. In this experiment, after transfection, the JNL cells were co-cultured with MSLN+ HeLa cells. Luminescence was measured after 24 hours to measure immune cell activation (FIG.16 bottom row). Analysis of activation shows that immune cells expressing the

[0413] 1905 combination of CAR-lxFRB-28BBz with IxFKBP-LAT co-cultured with MSLN+ HeLa cells resulted in a 19.8 fold induction in activation compared to immune cells not co-cultured with target cells. Analysis of activation shows that immune cells expressing the combination of CAR-2xFRB-28BBz with IxFKBP-LAT , co-cultured with MSLN+ HeLa cells resulted in a 18.3 fold induction in activation. Additionally, immune cells expressing the combination of

[0414] 1910 CAR-lxFRB-28z with IxFKBP-LAT, co-cultured with MSLN+ HeLa cells resulted in a 19.2 fold induction in activation. This shows that the combination of a second or third generation CAR comprising 1 or 2 FRB domains expressed with IxFKBP-LAT are activated when co- cultured with tumor cells. The CAR retained the ability to activate immune cells even when engineered with switch module.

[0415] 1915 Next, a subset of these constructs were tested in primary T cells. For these experiments, primary T cells were engineered to express a first polypeptide encoding one of the following activator receptor constructs: CAR lx FRB 28BBZ, CAR 28z lx FRB (c-term) or CAR 28BBZ (SEQ ID NOs: 184, 190 and 183). The immune cells were engineered to express the first polypeptide alone or in combination with a booster construct. In some groups

[0416] 1920 the primary T cells were further engineered to express one of the following booster constructs: lx FKBP LAT, 2x FKBP LAT, LAT IxFKBP (c-term) 9Seq ID NOs,: 88, 89 and 91). Each of the booster constructs was tested in combination with each of the CAR constructs. After transfection, primary T cells were seeded in a 48-well plate and treated with 100 lU / ml of IL-2 and one of the following drugs: DMSO, lOnM Rapalog, lOOnM rapalog or

[0417] 1925 1 : 100 TransAct. TransAct is T cell activation reagent that activates immune cells via CD3 and CD28. TransAct was used as a control in these experiments. After six days of culture T cells were harvested and analyzed via Flow cytometry for CAR expression and CD25 expression. The conditioned media was analyzed for secreted IFN-y.

[0418] Analysis of CAR expression shows that primary T cells expressing the CAR 28z lx

[0419] 1930 FRB (c-term) alone or in combination with the booster constructs and treated with lOOOnM rapalog had the highest expression of CAR (FIG. 17). The CAR expression in the CAR 28z lx FRB (c-term) alone or in combination with the booster constructs and treated with lOOOnM rapalog was higher than TransAct stimulated primary T cells. The CAR expression was induced 2-3 fold compared to the same cells treated with DMSO. Primary T cells

[0420] 1935 expressing the CARlxFRB 28BBZ alone or in combination with the booster constructs and treated with lOOOnM rapalog was similar to TransAct stimulated primary T cells. The CAR expression was induced ~2 compared to untreated controls.

[0421] Analysis of CD25 (IL-2 receptor) expression shows increased expression in primary T cells expressing the CAR 28z lx FRB (c-term) or CAR IxFRB 28BBz alone or in

[0422] 1940 combination with the booster constructs and treated with lOOOnM rapalog (FIG.17). Expression of CD25 is increased in activated immune cells. The CAR expression in the CAR 28z lx FRB (c-term) alone or in combination with the booster constructs and treated with lOOOnM rapalog was similar to TransAct stimulated primary T cells.

[0423] Analysis of secreted IFN-y in conditioned media shows that primary T cells

[0424] 1945 expressing CAR lx FRB 28BBZ or CAR 28z lx FRB (c-term) alone or in combination with booster construct and treated with rapalog increase IFN-y secretion 2-3 fold over cells treated with DMSO (FIG. 17). The level of IFN-y secretion is similar or higher to primary T cells activated with TransAct.

[0425] Next primary T cells expressing activator receptors were tested in a cell kill assay. For

[0426] 1950 this experiment, primary T cells engineered to express one of the following activator receptors were tested: CAR 28BBZ, CAR IxFRB 28BBZ or CAR 28z IxFRB. After transduction, primary T cells were co-cultured with MSLN+ HeLa cells. 48 hours after coculture the number of target cells was quantified. All three activator constructs showed a dose dependent increase in specific killing of target cells as the E:T (effecter cell: tumor cell) ratio 1955 increased (FIG 18). This shows that activator receptors that incorporate switch domains still function to activate primary T cells to kill tumor cells.

[0427] Example 8. Characterization of switchable CAR by ESRl / Tamoxifen system

[0428] This Example describes design and testing of switchable multipartite chimeric antigen receptors (individually, a “switchable CAR”). The Example demonstrates that tonic signaling

[0429] 1960 from the switchable CAR can be triggered by the switch molecule in the absence of cognate antigen for the receptor.

[0430] In each case the tested immune cells were engineered to express the switchable CAR by introducing into the cells at least two polynucleotide sequences. The first polynucleotide sequence encodes a first polypeptide chain comprising an extracellular domain of a single¬

[0431] 1965 chain variable fragment (scFv) specific to mesothelin (MSLN), a transmembrane domain and the intracellular domains of a conventional third-generation chimeric antigen receptor (CD28, 4- IBB, and CD3zeta). The first polypeptide may comprise one ESRI -based switch domains and the intracellular domains of a conventional second generation chimeric antigen receptor (CD28 and CD3zeta). The one ESRI based switch domains can be followed by the

[0432] 1970 intracellular domains of a conventional second generation CAR. The one ESRI based switch domains can be preceded by the intracellular domains of a conventional second generation CAR. The second polynucleotide (referred to as a “booster construct”) encodes a second polypeptide that comprises one ESRI -based switch domains and followed by a scaffolding intracellular domain, or a pair of scaffolding intracellular domains fused to each other. The

[0433] 1975 following activator receptor constructs were tested: SEQ ID NOs: 72, 73 and 183. The following intracellular domains were tested in this experiment: ESR1-LAT, LAT-ESR1, ESR1-SLP76, SLP76-ESR1, ESR1-LAT and ESR1-SLP76, LAT-ESR1 and ESR1-SLP76, ESR1-LAT and SLP76-ESR1 or LAT-ESR1 and SLP76 ESRI (SEQ ID NOs: 194-197). In the first set of experiments, after transfection, the JNL cells were treated with 500nM

[0434] 1980 tamoxifen and luminescence was measured after 24 hours to measure immune cell activation (FIG.19). Analysis of immune cell activation shows that JNL cells expressing the ESR1-LAT signal booster construct combined with any of the CAR constructs and treated with tamoxifen results in at least a 3-fold increase in immune cell activation compared to immune cells not treated with tamoxifen. Expression of the CAR 28z-ESRl construct with the ESR1-LAT

[0435] 1985 booster construct resulted in a 12.1 fold induction of immune cell activation. Analysis of immune cell activation shows that JNL cells expressing the ESR1-LAT and ESR1-SLP76 signal booster construct combined with any of the CAR constructs and treated with tamoxifen results in at least a 3-fold increase in immune cell activation compared to immune cells not treated with tamoxifen. Expression of the CAR 28z-ESRl construct with the ESR1-LAT and

[0436] 1990 ESR1-SLP76 booster construct resulted in a 8.9 fold induction of immune cell activation.

[0437] This shows that the combination of a second CAR comprising 1 ESRI switch domains expressed with signal boosters can increase tonic signaling in immune cells with a small molecule, independent of antigen binding. Additionally expression of the ESR1-LAT and ESR1-SLP76 or ESR1-LAT and SLP76-ESR1 alone and treated with tamoxifen increase

[0438] 1995 immune cell activation by 4.3 fold and 6.2 fold. This shows these booster constructs can be used to stimulate immune cell activation independent of antigen binding.

[0439] Next the same set of constructs were tested in a co-culture assay with MSLN+ MS751 cells. In this experiment, after transfection, the JNL cells were co-cultured with MSLN+ MS751 cells. Luminescence was measured after 24 hours to measure immune cell activation

[0440] 2000 (FIG. 19 right side). Analysis of activation shows that immune cells expressing the combination of CAR-ESRl-28z with ESR1-LAT signal booster, co-cultured with MSLN+ MS751 cells resulted in a 20.3 fold induction in activation compared to immune cells not co- cultured with target cells. The 20.3 fold induction in this group was more than JNL cells expressing only the third generation MSLN CAR.

[0441] 2005 Example 9. Characterization of switchable CAR by FKBP / Rim system

[0442] This example demonstrates that switchable signal 1 boosters can activate tonic signaling in immune cells expressing CARs with and without switch domain when treated with Rim (switch molecule).

[0443] For the first set of experiments JNL cells were transfected with DNA encoding one of

[0444] 2010 the following activator receptors: CAR-KFBP, CAR, CAR ' / 2LAT-FKBP or TCR-FKBP (SEQ ID NOs, 57, 153 and 183). JNL cells were further transfected with DNA encoding one of the booster constructs: LAT-FKBP, LAT(129AA M-Term del.)-FKBP, LAT [30AA C-term del.]- FKBP, MyD88-FKBP, CD3z-FKBP+LAT-FKBP , SLP76-FKBP+LAT-FKBP, memSLP76- FKBP+LAT-FKBP , ZAP70-FKBP+LAT-FKBP , or PLCgl-FKBP LAT-FKBP (SEQ ID

[0445] 2015 NOs: l, 12, 55, 84, 88, 100,109, 110, 111, 116, 167, and 198). JNL cells were engineered to express an activator receptor alone or in combination with signal booster. After transfection, the JNL cells were treated with Rim and activation was measured based on luminescence (FIG. 20) FIG. 20 shows the induction ratio for immune cell activation of transfected JNL cells treated with Rim compared to transfected JNL cells not treated with Rim. This data 2020 demonstrates that expression of a signal 1 booster construct with a CAR (without switch domains) results in increased tonic signaling upon treatment with Rim (switch molecule).

[0446] Next an additional LAT-lxFKBP+SLP76-lxFKBP (SEQ ID NOs: 88 and 198) signal booster construct was tested in primary T cells. For this experiment, primary human T cells were engineered to express a MSLN CAR alone or in combination with the LAT-

[0447] 2025 lxFKBP+SLP76-lxFKBP signal booster construct. Flow cytometry analysis confirmed expression of both the MSLN CAR and the LAT-lxFKBP+SLP76-lxFKBP signal booster construct (FIG. 21A). Next the same primary human T cells were treated with increasing doses of Rim and secreted IFN-y was measured in the conditioned media. Analysis of secreted IFN-y shows that primary T cells only expressing the MSLN CAR do not increase

[0448] 2030 IFN-y secretion regardless of the Rim dose. Conversely, primary T cells expressing the MSLN CAR and the LAT-lxFKBP+SLP76-lxFKBP signal booster showed a dose dependent increase in IFN-y secretion as the Rim dose increased (FIG. 21B) . This shows that the LAT- lxFKBP+SLP76-lxFKBP signal booster construct can increase IFN-y secretion independent of antigen binding.

[0449] 2035

Claims

1. CLAIMSWHAT IS CLAIMED IS:

1. An immune cell configured to be activated by an antigen or independently by a small2040 molecule (e.g., a switch molecule for a switchable receptor or an inducer molecule for an inducible receptor).

2. The immune cell of claim 1, wherein the immune cell comprises an artificial receptor configured to specifically activate in response to an antigen and independently to a small molecule. 45 3. The immune cell of claim 2, wherein the artificial receptor is a multipartite chimeric antigen receptor (CAR).

4. The immune cell of claim 3, wherein the multipartite CAR comprises: a first polypeptide chain comprising optionally a hinge domain, a transmembrane domain, at least one first switch domain, and a scaffolding intracellular domain (e.g., a LAT, 50 LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4-1BB, ZAP70, or GADs domain); and a second polypeptide chain comprising an antigen-binding domain (e.g., an scFv), a transmembrane domain, one or more costimulatory intracellular domains (e.g., a CD28 domain or a 41BB domain), and a signaling intracellular domain (e.g., a CD3zeta domain), optionally 55 further comprising at least one second switch domain, wherein the at least one first switch domain and the at least one second switch domain when present are configured to selectively dimerize in the presence of a switch molecule (e.g., rapamycin or a rapalog or an estrogen receptor modulator, e.g., tamoxifen).

5. The immune cell of claim 2, wherein the artificial receptor is a multipartite T cell 60 receptor (TCR).

6. The immune cell of claim 5, wherein the multipartite TCR comprises: a first polypeptide chain comprising optionally a hinge domain, a transmembrane domain, at least one first switch domain, and a scaffolding intracellular domain (e.g., a LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3< 4-1BB, ZAP70, or 65 GADs domain); anda second polypeptide chain comprising a transmembrane domain, an signaling intracellular domain (e.g., a CD3zeta domain), and optionally further comprising at least one second switch domain; and a third polypeptide chain comprising an antigen-binding domain (e.g., a TCR2070 extracellular domain) and a transmembrane domain, wherein the at least one first switch domain and the at least one second switch domain when present are configured to selectively dimerize in the presence of a switch molecule (e.g., rapamycin or a rapalog or an estrogen receptor modulator, e.g., tamoxifen).

7. The immune cell of any one of claims 1-6, wherein the first and second switch domains 75 are selected from the pairs disclosed in Table 1 and the small molecule is the corresponding switch molecule in Table 1, or a functional analogue thereof.

8. The immune cell of claim 2, wherein the artificial receptor is an inducible multipartite chimeric antigen receptor (CAR).

9. The immune cell of claim 8, wherein, such that the immune cell is configured to express 80 the inducible CAR, the immune cell comprises: a first polynucleotide operatively linked to an inducible promoter, the first polynucleotide encoding a first polypeptide chain comprising, at least one first switch domain, and a scaffolding intracellular domain (e.g., aLAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3(^, 4-1BB, ZAP70, or GADs domain), and optionally a hinge 85 domain, and / or a transmembrane domain wherein the inducible promoter is configured to induce expression of the first polypeptide chain in response to an inducer molecule; and a second polynucleotide encoding a second polypeptide chain comprising an antigenbinding domain (e.g., an scFv), a transmembrane domain, one or more costimulatory intracellular domains (e.g., a CD28 domain or a 4 IBB domain), and an signaling intracellular 90 domain (e.g., a CD3zeta domain), optionally further comprising at least one second switch domain.

10. The immune cell of claim 2, wherein the artificial receptor is an inducible multipartite T cell receptor (TCR).

11. The immune cell of claim 10, wherein, such that the immune cell is configured to 95 express the inducible TCR, the immune cell comprises: a first polynucleotide operatively linked to an inducible promoter, the first polynucleotide encoding a first polypeptide chain comprising at least one switch domain, anda intracellular domain (e.g., a LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3(^, 4-1BB, ZAP70, or GADs domain), and optionally a hinge domain, and / or a2100 transmembrane domain wherein the inducible promoter is configured to induce expression of the first polypeptide chain in response to an inducer molecule; and a second polynucleotide encoding a second polypeptide chain comprising a transmembrane domain, an signaling intracellular domain (e.g., a CD3zeta domain), optionally further comprising at least one second switch domain; and 05 a third polynucleotide encoding a third polypeptide chain comprising an antigenbinding domain (e.g., a TCR extracellular domain) and a transmembrane domain.

12. The immune cell of any one of claims 1-11, wherein the first polypeptide comprises at least two first switch domains selected from Table 1 and / or the second polypeptide comprises at least two second switch domains selected from Table 1. 10 13. The immune cell of claim 12, wherein the at least two first switch domains comprise a pair of switch domains disclosed in a row of Table 1 and / or the at least two second switch domains comprise a pair of switch domains disclosed in a row of Table 1.

14. The immune cell of claim 9 or claim 11, wherein the inducible promoter is selected from chemically / biochemically-regulated and physically-regulated promoters such as alcohol- 15 regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)- responsive promoters and other tetracycline responsive promoter systems, which include a tetracycline repressor protein (TetR, or TetRKRAB), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA), and a tetracycline operator sequence (tetO) and a reverse tetracycline transactivator fusion protein (rtTA)), steroid-regulated 20 promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadi azole 25 (BTH)), temperature / heat- inducible promoters e.g., heat shock promoters), pH-regulated promoters, and light-regulated promoters.

15. The immune cell of any one of claims 4, 6-7, 9, or 11-14, wherein the scaffolding intracellular domain is selected from LAT, LAT (G160D), SLP76, LCK, FYN, PLCgl, CD28, CD4, CD8alpha, CD3(^, 4-1BB, ZAP70, or GADs or a functional variant thereof.2130 16. The immune cell of claim 15, wherein the scaffolding intracellular domain comprises any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,17, 88-114, 157-181, 192-198 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

17. The immune cell of any one of claims 4, 6, 7, 9, or 11-16, wherein the one or more2135 costimulatory intracellular domains is selected from the group consisting of IL-2RP, Fc Receptor gamma (FcRy), Fc Receptor beta (FcRP), CD3g molecule gamma (CD3y), CD35, CD3s, CD5 molecule (CD5), CD22 molecule (CD22), CD79a molecule (CD79a), CD79b molecule (CD79b), carcinoembryonic antigen related cell adhesion molecule 3 (CD66d), CD27 molecule (CD27), CD28 molecule (CD28), TNF receptor superfamily member 9 (4-1BB), TNF 40 receptor superfamily member 4 (0X40), TNF receptor superfamily member 8 (CD30), CD40 molecule (CD40), programmed cell death 1 (PD-1), inducible T cell costimulatory (ICOS), lymphocyte function-associated antigen- 1 (LFA-1), CD2 molecule (CD2), CD7 molecule (CD7), TNF superfamily member 14 (LIGHT), killer cell lectin like receptor C2 (NKG2C) and CD276 molecule (B7-H3) c-stimulatory domains, or functional variants thereof. 45 18. The immune cells of any one of claim 1-17, further comprising an inhibitory receptor.

19. The immune cell of claim 18, wherein the inhibitory receptor is specific to a target disclosed in Table 5.

20. The immune cell of claim 18 or claim 19, wherein the inhibitory receptor comprises an intracellular domain of a LILRB1 and optionally the hinge and / or transmembrane domains of 50 LILRB1.

21. The immune cell of claim 20, wherein the inhibitory receptor comprises SEQ ID NO 182, or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.

22. The immune cell of any one of claims 4, 6, 7, 9, or 11-21, wherein the second 55 polypeptide comprises at least one second switch domain.

23. The immune cell of claim 22, wherein the second polypeptide comprises any one of SEQ ID NOs: 19, 21, 55-73, 82-87, 151-156, 184-191 or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto. 60 24. The immune cell of any one of claims 4, 6, 7, 9, or 11-21, wherein the second polypeptide does not comprise at least one second switch domain.

25. The immune cell of claim 24, wherein the second polypeptide comprises SEQ ID NO:183 , or a sequence having at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity thereto, or that is 100% identical thereto.2165 26. The immune cell of any one of claims 1-25, wherein the immune cell is a T cell.

27. The immune cell of any one of claims 1-25, wherein the immune cell is a natural killer(NK) cell.

28. A pharmaceutical composition comprising a population of immune cells of any one of claims 1-27. 70 29. A method of expanding an immune cell population, the method comprising providing a population of immune cells according to any one of claims 1-27 and contacting the immune cells with a small molecule from Table 1 to boost activation of the immune cells, thereby expanding the immune cell population.

30. A method of treating or preventing cancer in a subject in need thereof, the method 75 comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 28; and further administering a small molecule from Table 1 to boost activation of the immune cells.

31. The method of claim 30, wherein the cancer is a solid tumor.

32. A polynucleotide or a plurality of polynucleotides encoding an artificial receptor, a 80 multipartite chimeric antigen receptor (CAR), a multipartite T cell receptor (TCR) or a signal booster as disclosed herein or one or more of the polypeptide chains thereof.

33. A vector comprising the polynucleotide or the plurality of polynucleotides of any one of claims 1-27.

34. The vector of claim 33, wherein the vector is a lentiviral vector. 85 35. A host cell comprising the polynucleotide or the plurality of polynucleotides of claim 28.

36. A method of making a plurality of immune cells, comprising, a. providing a plurality of immune cell, and b. transforming the plurality of immune cells with poly nucleotide system of claim 32 or the vector of claims 33 or 34. 90

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