Dual stimulatory receptors for immunotherapy

Abstract

Disclosed herein, in some aspects, are dual stimulatory receptors (DSRs), polynucleotides encoding the same, immune cells comprising the same, and compositions comprising the polypeptides, polynucleotides, and / or immune cells. In some aspects, DSRs enhance immune cell activation and / or anti-tumor activity. Also disclosed are methods for disease treatment, such as cancer treatment, comprising administering such immune cells and / or compositions to a subject in need thereof.

Description

DUAL STIMULATORY RECEPTORS FOR IMMUNOTHERAPYCLAIM OF PRIORITY

[0001] This Application claims the benefit of U.S. Provisional Patent Application No. 63 / 631,256, filed on 08 April 2024, which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on 07 April 2025, is named BAYM_P0420WO_Sequence_Listing.xml and is 173,239 bytes in size.TECHNICAL FIELD AND BACKGROUNDI. Technical Field

[0003] Aspects of this disclosure relate to at least the fields of immunology, immunotherapeutics, cancer biology, and medicine.IL Background

[0004] Adoptive cell therapy using immune cells manipulated ex vivo to recognize antigens on cancer cells can be curative in individuals with some hematologic malignancies. Ample clinical evidence shows that those infused immune cells need to persist longer in an individual’s body to achieve a complete response. When immune cells recognize target cells, e.g., cancer cells, they need three types of signals to become fully activated and persist longer in an individual’s body: signal 1 - antigen stimulation, signal 2 - costimulation, and signal 3 - cytokine stimulation. However, most cancer cells lack costimulatory ligands to provide signals 2 and 3 to immune cells, leading to lack of proper immune cell activation. To overcome this limitation, immune cells can be genetically modified ex vivo to supplement required signaling, such as by expressing synthetic chimeric antigen receptors (CARs) containing costimulatory endodomain. However, additional modifications of CARs to provide signal 2 and signal 3 can produce continuous costimulatory signaling resulting in overstimulation, exhaustion, or potentially indefinite growth of transgenic cells posing safety concerns. Thus, desirable signal 2 and signal 3 should be provided transiently and concomitantly with signal 1.SUMMARY

[0005] This disclosure describes a solution to at least some of the problems associated with activation of immune cells, e.g., CAR cells for immunotherapy. In some aspects, the solution resides in engineered polypeptides, receptors, and / or cells comprising one or more extracellular domains of a costimulatory ligand, one or more transmembrane domains of a membrane bound protein, and one or more intracellular cytokine signaling domains of a cytokine receptor. In some aspects, disclosed are at least compositions, kits, uses of said compositions and kits, and methods to activate immune cells using engineered receptors that mimic physiological signaling and methods of using the same to treat immune-related conditions, such as cancer. This disclosure describes at least novel compositions, kits, and methods comprising dualstimulatory receptors (DSRs) — synthetic molecules engineered to trigger costimulation (signal 2) and cytokine signaling (signal 3) transiently and simultaneously, when a cell gets antigen stimulation (signal 1) — and uses thereof. DSRs mimic physiologic timing of signals 2 and 3, which are delivered to immune cells together with signal 1, thus providing optimal responses without the risk of immune cell exhaustion and / or transformation.

[0006] DSRs can be specifically engineered to indirectly induce signal 2 and directly provide signal 3 in the cell in which they are expressed without providing signal 1 directly or indirectly. Thus, a DSR can lack components to provide signal 1, such as immunoreceptor tyrosine-based activation motifs (ITAMs), CD3 domains, and / or CD3zeta domains. In addition, a DSR can lack intracellular components that directly provide signal 2, such as an intracellular domain of a co-stimulatory receptor. Instead, a DSR can comprise an extracellular binding domain that binds endogenous co-stimulatory receptors for clustering and induction of native signal 2, such as an extracellular domain of a co-stimulatory ligand. A DSR can include an intracellular cytokine signaling domain which can directly provide signal 3 to the cell.

[0007] Without being bound by theory, a DSR can induce aggregation of costimulatory receptors in the same cell in which the DSR is expressed. The aggregation can facilitate crossactivation of co-stimulatory receptors and induction of signal 2. At the same time, aggregation of co-stimulatory receptor-DSR complexes can facilitate aggregation or increase the physical proximity of DSRs in the same cell facilitating cross-activation of the intracellular cytokine signaling domains of DSRs and induction of signal 3.

[0008] Aspects of the present disclosure address certain needs by providing at least compositions, nucleic acids, cells, proteins, kits, and methods useful in immunotherapy. Aspects of the present disclosure include nucleic acids encoding novel engineered polypeptidestermed herein as dual stimulatory receptors (DSRs) that can provide stimulation, e.g., costimulation and cytokine signaling, to a cell.

[0009] In certain aspects, a DSR may comprise an extracellular binding domain, a transmembrane domain, and an intracellular cytokine signaling domain. In certain aspects, an extracellular binding domain binds a co-stimulatory receptor. In certain aspects, an extracellular binding domain binds a co-stimulatory receptor of the same cell in which it is expressed. In certain aspects, an extracellular binding domain aggregates two or more co- stimulatory receptors of the same cell in which it is expressed. In certain aspects, an extracellular binding domain can aggregate two or more co-stimulatory receptors of the same cell in which it is expressed to induce co-stimulatory signaling in the cell. In certain aspects, an extracellular binding domain does not bind a target antigen on a diseased cell and / or a target on a different cell than the cell in which it is expressed. In certain aspects, an extracellular binding domain does not induce co-stimulatory signaling in a different cell than the cell in which it is expressed. In certain aspects, an intracellular cytokine signaling domain does not comprise a co-stimulatory domain. In certain aspects, an intracellular cytokine signaling domain does not comprise a co-stimulatory domain derived from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and / or HVEM. In certain aspects, an intracellular cytokine signaling domain does not comprise a CD3 domain. In certain aspects, an intracellular cytokine signaling domain does not comprise a CD3zeta domain. In certain aspects, an intracellular cytokine signaling domain does not comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more immunoreceptor tyrosine-based activation motifs (ITAM). In certain aspects, an intracellular cytokine signaling domain can initiate JAK-STAT signaling. In certain aspects, an intracellular cytokine signaling domain can initiate JAK-STAT signaling and RAS / RAF signaling. Without limitation, an extracellular binding domain may comprise, consist of, or consist essentially of an antibody (e.g., an agonistic antibody), or fragment thereof, or an extracellular costimulatory ligand domain. An extracellular binding domain, such as an antibody or fragment thereof, may bind and activate a costimulatory receptor. In some aspects, an antibody or fragment thereof may be an agonist of a costimulatory receptor selected from the group comprising, consisting essentially of, or consisting of cluster of differentiation 28 (CD28), inducible T cell costimulator (ICOS), cluster of differentiation 27 (CD27), TNF receptor superfamily member 14 (TNFRSF14, also known as HVEM), cluster of differentiation 40 (CD40), tumor necrosis factor receptor superfamily, member 9 (Tnfrsf9, also known as 4-1BB), TNF receptor superfamily member 4 (TNFRSF4, also known as 0X40), TNF receptor superfamily member 25 (TNFRSF25, also known as DR3), tumor necrosis factor receptor superfamily, member 18(Tnfrsfl8, also known as GITR), cluster of differentiation 30 (CD30), hepatitis A virus cellular receptor 1 (HAVCR1, also known as TIM-1), cluster of differentiation 150 (CD150, also known as SLAM), cluster of differentiation 2 (CD2), and cluster of differentiation 226 (CD226; also known as DNAM-1). In certain aspects an extracellular costimulatory ligand domain may be derived from tumor necrosis factor (ligand) superfamily, member 9 (Tnfsf9, also known as 4-1BBL), cluster of differentiation 80 (CD80, also known as B7-1), cluster of differentiation 86 (CD86, also known as B7-2), inducible T cell costimulator ligand (ICOS-L, also known as B7-H2), cluster of differentiation 70 (CD70, also known as CD27L), TNF superfamily member 14 (TNFSF14, also known as LIGHT), CD40 ligand (CD40L), TNF superfamily member 4 (TNFSF4, also known as OX40L), TNF superfamily member 15 (TNFSF15, also known as TL1A), TNF superfamily member 18 (TNFSF18, also known as GITRL), CD30 ligand (CD30L), T cell immunoglobulin and mucin domain containing 4 (TIM4), cluster of differentiation 150 (CD 150, also known as SLAM), cluster of differentiation 48 (CD48, also known as SLAMF2), cluster of differentiation 58 (CD58, also known as LFA-4), cluster of differentiation 155 (CD155, also known as PVR), and / or cluster of differentiation 112 (CD112, also known as Nectin-2). In certain aspects, an extracellular costimulatory ligand domain may be derived from 4-1BBL. In certain aspects, an extracellular costimulatory ligand domain may be derived from OX40L. In certain aspects, an extracellular costimulatory ligand domain may comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 8, 96, or 98. In certain aspects, an extracellular costimulatory ligand domain may comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8. In certain aspects, an extracellular costimulatory ligand domain may be encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 7, 95, or 97. In certain aspects, an extracellular costimulatory ligand domain may be encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 7.

[0010] In certain aspects, a transmembrane domain may be derived from cluster of differentiation 28 (CD28), cluster of differentiation 8 (CD8), cluster of differentiation (CD25), cluster of differentiation 4 (CD4), cluster of differentiation 3 zeta (CD3Q, cluster of differentiation 134 (CD 134), or cluster of differentiation 7 (CD7). In certain aspects, a transmembrane domain may be derived from CD28. In certain aspects, a transmembrane domain may comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 6, or 14. In certain aspects, a transmembrane domain may comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical toSEQ ID NO: 14. In certain aspects, a transmembrane domain may be encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 5, or 13. In certain aspects, a transmembrane domain may be encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13.

[0011] In certain aspects, a cytokine signaling domain may be derived from interleukin 7 receptor (IL7R, also known as IL-7Ra, or CD127), interleukin 2 receptor subunit beta (IL-2RP, also known as CD122, or IL-15RP), myeloid differentiation primary response protein 88 (MyD88), thrombopoietin receptor (cMPL, also known as TPO-R), interleukin 2 receptor subunit gamma (IL-2Ry, also known as CD132, or yc), interleukin 1 receptor type 1 (IL-1R), interleukin 12 receptor subunit beta 1 (IL-12RP1), interleukin 12 receptor subunit beta 2 (IL- 12RP2), interleukin 6 cytokine family signal transducer (IL6ST, also known as gpl30, or CD 130), interleukin 4 receptor (IL-4R), interleukin 9 receptor (IL-9R), interleukin 10 receptor (IL-10R), interleukin 13 receptor (IL-13R), interleukin 15 receptor (IL-15R), interleukin 21 receptor (IL-21R), interleukin 23 receptor (IL-23R), interferon alpha and beta receptor subunit 2 (IFNAR2, also known as IFNa / pR, or IFN-R), interleukin 10 receptor (IL-10R), interleukin 6 receptor (IL-6R), interleukin 11 receptor (IL- HR), ciliary neurotrophic factor receptor (CNTF-R), oncostatin M receptor (OSM-R), LIF receptor subunit alpha (LIF-R), calcitonin receptor (CT-R), interleukin 3 receptor (IL-3R), interleukin 5 receptor (IL-5R), granulocytemacrophage colony-stimulating factor receptor (GM-CSF-R), growth hormone receptor (GH- R), erythropoietin receptor (EPO-R), and / or prolactin receptor (PRL-R). In certain aspects, a cytokine signaling domain may be derived from IL-7Ra (CD 127), IL-2RP (CD122 / IL-15RP), MyD88, or cMPL (TPO-R). In certain aspects, a cytokine signaling domain may be derived from cMPL (TPO-R). In certain aspects, a cytokine signaling domain may be derived from IL- 7Ra (CD127), IL-2RP (CD122 / IL-15RP), MyD88, and / or cMPL (TPO-R). In certain aspects, a cytokine signaling domain may comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 20, 24, 28, or 32. In certain aspects, a cytokine signaling domain may comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 32. In certain aspects, a cytokine signaling domain may be encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 19, 23, 27, or 31. In certain aspects, a cytokine signaling domain may be encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 31.

[0012] In certain aspects, an extracellular costimulatory ligand domain, a transmembrane domain, and a cytokine signaling domain may be operably linked in a 5' to 3' order.

[0013] In certain aspects, a DSR may comprise, consist of, or consist essentially of an extracellular costimulatory ligand domain derived from 4-1BBL, a transmembrane domain derived from CD28, and a cytokine signaling domain derived from IL-7Ra.

[0014] In certain aspects, a DSR may comprise, consist of, or consist essentially of an extracellular costimulatory ligand domain derived from 4-1BBL, a transmembrane domain derived from CD28, and a cytokine signaling domain derived from IL-2Rp.

[0015] In certain aspects, a DSR may comprise, consist of, or consist essentially of an extracellular costimulatory ligand domain derived from 4-1BBL, a transmembrane domain derived from CD28, and a cytokine signaling domain derived from MyD88.

[0016] In certain aspects, a DSR may comprise, consist of, or consist essentially of an extracellular costimulatory ligand domain derived from 4-1BBL, a transmembrane domain derived from CD28, and a cytokine signaling domain derived from cMPL.

[0017] In certain aspects, a DSR may comprise, consist of, or consist essentially of an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 18, 22, 26, or 30. In certain aspects, a DSR may be encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 17, 21, 25, or 29. In certain aspects, a polynucleotide may comprise a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 82-85. In certain aspects, a polynucleotide may comprise a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the open reading frame of any one of SEQ ID NOs: 89-92. In certain aspects, a polynucleotide may comprise, consist essentially of, or consist of a DSR encoding sequence. In certain aspects, a polynucleotide may not comprise a sequence encoding an intracellular co-stimulatory domain and / or a signaling domain. In certain aspects, a polynucleotide may not comprise a sequence encoding an intracellular CD3z, CD28, or 4- 1BBL derived domain.

[0018] In certain aspects, a cell may be engineered to comprise a polynucleotide of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 95, and / or 97. In some aspects, an engineered cell may naturally or artificially expresses a full or partial TCR complex. In some aspects, an engineered cell may naturally or artificially expresses a full or partial CD3 complex. In some aspects, an engineeredcell may naturally or artificially expresses a full or partial CD3^ chain. In some aspects, an engineered cell may naturally or artificially comprise antigen stimulation. In some aspects, an engineered cell may comprise, consist of, or consist essentially of an immune cell. In some aspects, an immune cell may be autologous or allogeneic. In some aspects, an immune cell may comprise, consist of, or consist essentially of a T cell, NK cell, dendritic cell (DC), B cell, macrophage, or iNKT cell. In some aspects, a T cell may comprise, consist of, or consist essentially of aP T cells, virus-specific T cell, tumor-specific T cell, and / or y5 T cells. In some aspects, a macrophage may comprise, consist of, or consist essentially of an Ml macrophage and / or an M2 macrophage. In some aspects, a DC may comprise, consist of, or consist essentially of a conventional DC and / or a plasmacytoid DC. In some aspects, a cell may be derived from a pluripotent stem cell, a precursor immune cell, a cell line, cord blood, and / or a mature immune cell. In some aspects, a pluripotent stem cell may be an embryonic stem cell or an induced pluripotent stem cell.

[0019] In some aspects, a cell may be further engineered to comprise a chimeric antigen receptor (CAR), a chimeric T cell receptor (cTCR), a transgenic T cell receptor, and / or a T cell engager (TCE). In some aspects, a cTCR may target GD2 or TdT. In some aspects, a cTCR may comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 34, or 58. In some aspects, a cTCR may be encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 33, or 57. In some aspects, a TCE may target CD123 and CD3. In some aspects, a TCE may comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 64. In some aspects, a TCE may be encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 63. In some aspects, an engineered cell or composition may comprise an exogenously provided antibody.

[0020] In some aspects, a composition comprising a polynucleotide, polypeptide, and / or cell, may be comprised in a pharmaceutically acceptable carrier. In some aspects, a composition comprising a polynucleotide, polypeptide, and / or cell, may be comprised in a delivery device.

[0021] In some aspects, disclosed is a method of treating a disease in an individual. In some aspects, a method of treating a disease in an individual may comprise, consist of, or consist essentially of a step of administering to an individual in need thereof a therapeutically effective amount of a composition disclosed herein. In some aspects, an individual may have been diagnosed, may be suspected of having, or may have one or more symptoms of an autoimmunedisease, infection, and / or cancer. In some aspects, a cancer may be of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testes, endometrium, prostate, rectum, anus, cervix, or hematological. In some aspects, a cancer may be chronic myeloid leukemia (CML) or acute myeloid leukemia (AML).

[0022] In some aspects, an individual may be administered an immune cell. In some aspects, an individual may be administered an engineered immune cell. In some aspects, an engineered immune cell may display an increase in in vivo anti-cancer functionality relative to a non-engineered immune cell and / or an immune cell engineered to comprise a CAR, transgenic TCR, cTCR, or TCE without a DSR. In some aspects, an increase in in vivo anticancer functionality may comprise long-term cancer eradication, significant prolongation of an individual’s survival, and / or reduced toxicity or weight loss. In some aspects, an individual may be provided with an immune memory response against cancer by administering to the individual in need thereof a therapeutically effective amount of any one of a polynucleotide, polypeptide, cell, composition, or a combination thereof, described herein.

[0023] In certain aspects, a kit may comprise any one of a polynucleotide, polypeptide, cell, composition, or a combination thereof, described herein. In some aspects, a kit is disclosed that may be used for performing any of the methods disclosed herein.

[0024] In some aspects, disclosed herein are uses of a polynucleotide, polypeptide, composition, kit, cell, or method disclosed herein.

[0025] Certain aspects of the present disclosure are characterized through the following enumerated aspects.

[0026] Aspect 1 is a polynucleotide encoding a dual-stimulatory receptor (DSR), wherein the DSR comprises an extracellular binding domain, a transmembrane domain, and an intracellular cytokine signaling domain.

[0027] Aspect 1.1 is the polynucleotide according to aspect 1, wherein the extracellular binding domain binds a co-stimulatory receptor.

[0028] Aspect 1.2 is the polynucleotide according to aspect 1 or 1.1, wherein the extracellular binding domain binds a co-stimulatory receptor of the same cell in which it is expressed.

[0029] Aspect 1.3 is the polynucleotide according to any one of aspects 1-1.2, wherein the extracellular binding domain aggregates two or more co-stimulatory receptors of the same cell in which it is expressed.

[0030] Aspect 1.4 is the polynucleotide according to any one of aspects 1-1.3, wherein the extracellular binding domain can aggregate two or more co-stimulatory receptors of the same cell in which it is expressed to induce co-stimulatory signaling in the cell.

[0031] Aspect 1.5 is the polynucleotide according to any one of aspects 1-1.4, wherein the extracellular binding domain does not bind a target antigen on a diseased cell and / or a target on a different cell than the cell in which it is expressed.

[0032] Aspect 1.6 is the polynucleotide according to any one of aspects 1-1.5, wherein the extracellular binding domain does not induce co-stimulatory signaling in a different cell than the cell in which it is expressed.

[0033] Aspect 1.7 is the polynucleotide according to any one of aspects 1-1.6, wherein the intracellular cytokine signaling domain does not comprise a co-stimulatory domain.

[0034] Aspect 1.8 is the polynucleotide according to any one of aspects 1-1.7, wherein the intracellular cytokine signaling domain does not comprise a co-stimulatory domain derived from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and / or HVEM.

[0035] Aspect 1.9 is the polynucleotide according to any one of aspects 1-1.8, wherein the intracellular cytokine signaling domain does not comprise a CD3 domain.

[0036] Aspect 1.91 is the polynucleotide according to any one of aspects 1-1.9, wherein the intracellular cytokine signaling domain does not comprise a CD3zeta domain.

[0037] Aspect 1.92 is the polynucleotide according to any one of aspects 1-1.91, wherein the intracellular cytokine signaling domain does not comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 immunoreceptor tyrosine-based activation motifs (IT AM).

[0038] Aspect 1.93 is the polynucleotide according to any one of aspects 1-1.92, wherein the intracellular cytokine signaling domain can initiate JAK-STAT signaling.

[0039] Aspect 1.94 is the polynucleotide according to any one of aspects 1-1.93, wherein the intracellular cytokine signaling domain can initiate JAK-STAT signaling and RAS / RAF signaling.

[0040] Aspect 2 is the polynucleotide according to any one of aspects 1-1.94, wherein the extracellular binding domain comprises an antibody, a fragment thereof, or an extracellular costimulatory ligand domain.

[0041] Aspect 3 is the polynucleotide according to any one of aspects 1-2, wherein the extracellular costimulatory ligand domain is derived from tumor necrosis factor ligand superfamily, member 9 (Tnfsf9, also known as 4-1BBL), cluster of differentiation 80 (CD80, also known as B7-1), cluster of differentiation 86 (CD86, also known as B7-2), inducible T cell costimulator ligand (ICOS-L, also known as B7-H2), cluster of differentiation 70 (CD70,also known as CD27L), TNF superfamily member 14 (TNFSF14, also known as LIGHT), CD40 ligand (CD40L), TNF superfamily member 4 (TNFSF4, also known as OX40L), TNF superfamily member 15 (TNFSF15, also known as TL1A), TNF superfamily member 18(TNFSF18, also known as GITRL), CD30 ligand (CD30L), T cell immunoglobulin and mucin domain containing 4 (TIM4), cluster of differentiation 150 (CD 150, also known as SLAM), cluster of differentiation 48 (CD48, also known as SLAMF2), cluster of differentiation 58 (CD58, also known as LFA-4), cluster of differentiation 155 (CD155, also known as PVR), and / or cluster of differentiation 112 (CD112, also known as Nectin-2).

[0042] Aspect 4 is the polynucleotide according to any one of aspects 1-3, wherein the extracellular costimulatory ligand domain is derived from 4-1BBL.

[0043] Aspect 5 is the polynucleotide according to any one of aspects 1-4, wherein the extracellular costimulatory ligand domain is derived from OX40L.

[0044] Aspect 6 is the polynucleotide according to any one of aspects 1-5, wherein the encoded extracellular costimulatory ligand domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 8, 96, or 98.

[0045] Aspect 7 is the polynucleotide according to any one of aspects 1-6, wherein the encoded extracellular costimulatory ligand domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8.

[0046] Aspect 8 is the polynucleotide according to any one of aspects 1-7, wherein the extracellular costimulatory ligand domain is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 7, 95, or 97.

[0047] Aspect 9 is the polynucleotide according to aspect 8, wherein the extracellular costimulatory ligand domain is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 7.

[0048] Aspect 10 is the polynucleotide according to any one of aspects 1-9, wherein the transmembrane domain is derived from cluster of differentiation 28 (CD28), cluster of differentiation 8 (CD8), cluster of differentiation (CD25), cluster of differentiation 4 (CD4), cluster of differentiation 3 zeta (CD3Q, cluster of differentiation 134 (CD134), or cluster of differentiation 7 (CD7).

[0049] Aspect 11 is the polynucleotide according to aspect 10, wherein the transmembrane domain is derived from CD28.

[0050] Aspect 12 is the polynucleotide according to any one of aspects 1-11, wherein the encoded transmembrane domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 6, or 14.

[0051] Aspect 13 is the polynucleotide according to aspect 12, wherein the encoded transmembrane domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 14.

[0052] Aspect 14 is the polynucleotide according to any one of aspects 1-13, wherein the transmembrane domain is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 5, or 13.

[0053] Aspect 15 is the polynucleotide according to aspect 14, wherein the transmembrane domain is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13.

[0054] Aspect 16 is the polynucleotide according to any one of aspects 1-15, wherein the cytokine signaling domain is derived from interleukin 7 receptor (IL7R, also known as IL-7Ra, or CD127), interleukin 2 receptor subunit beta (IL-2RP, also known as CD122, or IL-15RP), myeloid differentiation primary response protein 88 (MyD88), thrombopoietin receptor (cMPL, also known as TPO-R), interleukin 2 receptor subunit gamma (IL-2Ry, also known as CD132, or yc), interleukin 1 receptor type 1 (IL-1R), interleukin 12 receptor subunit beta 1 (IL- 12Rpi), interleukin 12 receptor subunit beta 2 (IL-12RP2), interleukin 6 cytokine family signal transducer (IL6ST, also known as gpl30, or CD130), interleukin 4 receptor (IL-4R), interleukin 9 receptor (IL-9R), interleukin 10 receptor (IL-10R), interleukin 13 receptor (IL- 13R), interleukin 15 receptor (IL-15R), interleukin 21 receptor (IL-21R), interleukin 23 receptor (IL-23R), interferon alpha and beta receptor subunit 2 (IFNAR2, also known as IFNa / pR, or IFN-R), interleukin 10 receptor (IL-10R), interleukin 6 receptor (IL-6R), interleukin 11 receptor (IL-11R), ciliary neurotrophic factor receptor (CNTF-R), oncostatin M receptor (OSM-R), LIF receptor subunit alpha (LIF-R), calcitonin receptor (CT-R), interleukin 3 receptor (IL-3R), interleukin 5 receptor (IL-5R), granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R), growth hormone receptor (GH-R), erythropoietin receptor (EPO- R), and / or prolactin receptor (PRL-R).

[0055] Aspect 17 is the polynucleotide according to aspect 16, wherein the cytokine signaling domain is derived from IL-7Ra (CD127), IL-2RP (CD122 / IL-15RP), MyD88, and / or cMPL (TPO-R).

[0056] Aspect 18 is the polynucleotide according to any one of aspects 1-17, wherein the encoded cytokine signaling domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 20, 24, 28, or 32.

[0057] Aspect 19 is the polynucleotide according to aspect 18, wherein the encoded cytokine signaling domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 32.

[0058] Aspect 20 is the polynucleotide according to any one of aspects 1-19, wherein the cytokine signaling domain is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 19, 23, 27, or 31.

[0059] Aspect 21 is the polynucleotide according to aspect 20, wherein the cytokine signaling domain is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 31.

[0060] Aspect 22 is the polynucleotide according to any one of aspects 1-21, wherein the extracellular costimulatory ligand domain, the transmembrane domain, and the cytokine signaling domain are operably linked in a 5' to 3' order.

[0061] Aspect 23 is the polynucleotide according to any one of aspects 1-22, wherein the DSR comprises the extracellular costimulatory ligand domain derived from 4-1BBL, the transmembrane domain derived from CD28, and the cytokine signaling domain derived from IL-7Ra.

[0062] Aspect 24 is the polynucleotide according to any one of aspects 1-22, wherein the DSR comprises the extracellular costimulatory ligand domain derived from 4-1BBL, the transmembrane domain derived from CD28, and the cytokine signaling domain derived from IL-2RP.

[0063] Aspect 25 is the polynucleotide according to any one of aspects 1-22, wherein the DSR comprises the extracellular costimulatory ligand domain derived from 4-1BBL, the transmembrane domain derived from CD28, and the cytokine signaling domain derived from MyD88.

[0064] Aspect 26 is the polynucleotide according to any one of aspects 1-22, wherein the DSR comprises the extracellular costimulatory ligand domain derived from 4-1BBL, the transmembrane domain derived from CD28, and the cytokine signaling domain derived from cMPL.

[0065] Aspect 27 is the polynucleotide according to any one of aspects 1-26, wherein the encoded DSR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 18, 22, 26, or 30.

[0066] Aspect 28 is the polynucleotide according to any one of aspects 1-26, wherein the DSR is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 17, 21, 25, or 29.

[0067] Aspect 29 is the polynucleotide according to any one of aspects 1-28, wherein the polynucleotide comprises a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 82-85.

[0068] Aspect 30 is the polynucleotide according to aspect 29, wherein the polynucleotide comprises a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the open reading frame of any one of SEQ ID NOs: 89- 92.

[0069] Aspect 31 is the polynucleotide according to any one of aspects 1-30, wherein the polynucleotide consists essentially of, or consists of, the DSR encoding sequence.

[0070] Aspect 32 is the polynucleotide according to any one of aspects 1-31, wherein the polynucleotide does not comprise a sequence encoding an intracellular co-stimulatory domain and / or signaling domain.

[0071] Aspect 33 is the polynucleotide according to any one of aspects 1-32, wherein the polynucleotide does not comprise a sequence encoding an intracellular CD3z, CD28, or 4- 1BBL derived domain.

[0072] Aspect 34 is a polypeptide encoded by the polynucleotide of any one of aspects 1- 33.

[0073] Aspect 35 is a composition comprising a cell engineered to comprise the polynucleotide or polypeptide of any one of aspects 1-34.

[0074] Aspect 36 is the composition according to aspect 35, wherein the engineered cell naturally or artificially expresses a full or partial TCR complex.

[0075] Aspect 37 is the composition according to aspect 35 or 36, wherein the engineered cell naturally or artificially expresses a full or partial CD3 complex.

[0076] Aspect 38 is the composition according to any one of aspects 35-37, wherein the engineered cell naturally or artificially expresses a full or partial CD3^ chain.

[0077] Aspect 39 is the composition according to any one of aspects 35-38, wherein the engineered cell naturally or artificially comprises antigen stimulation functionality.

[0078] Aspect 40 is the composition according to any one of aspects 35-39, wherein the cell comprises an immune cell.

[0079] Aspect 41 is the composition according to aspect 40, wherein the immune cell is autologous or allogeneic.

[0080] Aspect 42 is the composition according to aspects 40 or 41, wherein the immune cell comprises a T cell, NK cell, dendritic cell (DC), B cell, macrophage, or iNKT cell.

[0081] Aspect 43 is the composition according to any one of aspects 40-42, wherein the T cell comprises aP T cells and / or y5 T cells.

[0082] Aspect 44 is the composition according to any one of aspects 40-42, wherein the macrophage comprises Ml macrophages and / or M2 macrophages.

[0083] Aspect 45 is the composition according to any one of aspects 40-42, wherein the DC comprises a conventional DC and / or a plasmacytoid DC.

[0084] Aspect 46 is the composition according to any one of aspects 35-42, wherein the cell is derived from a pluripotent stem cell, a precursor immune cell, a cell line, cord blood, and / or a mature immune cell.

[0085] Aspect 47 is the composition according to aspect 46, wherein the pluripotent stem cell is an embryonic stem cell or an induced pluripotent stem cell.

[0086] Aspect 48 is the composition according to any one of aspects 35-47, wherein the cell is further engineered to comprise a chimeric antigen receptor (CAR), a chimeric T cell receptor (cTCR), a transgenic T cell receptor, and / or a T cell engager (TCE).

[0087] Aspect 49 is the composition according to aspect 48, wherein the cTCR targets survivin, GD2, or TdT.

[0088] Aspect 50 is the composition according to aspect 48 or 49, wherein the cTCR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 34, or 58.

[0089] Aspect 51 is the composition according to any one of aspects 48-50, wherein the cTCR is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 33, or 57.

[0090] Aspect 52 is the composition according to aspect 48, wherein the TCE targets CD 123 and CD3.

[0091] Aspect 53 is the composition according to aspect 48 or 52, wherein the TCE comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 64.

[0092] Aspect 54 is the composition according to aspect 48, 52, or 53, wherein the TCE is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 63.

[0093] Aspect 55 is the composition according to any one of aspects 35-54, further comprising an exogenously provided antibody.

[0094] Aspect 56 is a composition comprising the polynucleotide, polypeptide, or composition of any one of aspects 1-55, and a pharmaceutically acceptable carrier.

[0095] Aspect 57 is the composition according to aspect 56, wherein the composition is comprised in a delivery device.

[0096] Aspect 58 is a method of treating a disease in an individual, the method comprising the step of administering to the individual in need thereof a therapeutically effective amount of the composition of any one of aspects 56-57.

[0097] Aspect 59 is the method according to aspect 58, wherein the individual has been diagnosed, is suspected of having, or has one or more symptoms of an autoimmune disease, infection, and / or cancer.

[0098] Aspect 60 is the method according to aspect 59, wherein the cancer is of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testes, endometrium, prostate, rectum, anus, cervix, or is hematological.

[0099] Aspect 61 is the method according to aspect 60, wherein the cancer is chronic myeloid leukemia (CML) or acute myeloid leukemia (AML).

[0100] Aspect 62 is the method according to any one of aspects 58-61, wherein the individual is administered an immune cell.

[0101] Aspect 63 is the method according to aspect 62, wherein the immune cell displays an increase in in vivo anti-cancer functionality relative to a non-engineered immune cell and / or an immune cell engineered to comprise a CAR, transgenic TCR, cTCR, or TCE without a DSR.

[0102] Aspect 64 is the method according to aspect 63, wherein the increase in in vivo anticancer functionality comprises long-term cancer eradication, significant prolongation of the individual’s survival, and / or reduced toxicity or weight loss.

[0103] Aspect 65 is a method of providing an individual with an immune memory response against cancer, the method comprising administering to an individual in need thereof a therapeutically effective amount of the polynucleotide, polypeptide, or composition of any one of aspects 1-57.

[0104] Aspect 66 is a kit comprising the polynucleotide, polypeptide, or composition of any one of aspects 1-57.

[0105] Aspect 67 is a kit for the means of performing the methods of any one of aspects 58-65.

[0106] Aspect 68 is a use of the polynucleotide, polypeptide, composition, kit, or method of any one of the preceding aspects in the manufacture of a medicament.

[0107] Aspect 69 is a DSR polypeptide comprising an extracellular binding domain derived from 4-1BBL, a transmembrane domain derived from CD28, and an intracellular cytokine signaling domain derived from cMPL.

[0108] Aspect 69.1 is the DSR polypeptide according to aspect 69, wherein the extracellular binding domain binds a co-stimulatory receptor.

[0109] Aspect 69.2 is the DSR polypeptide according to aspect 69 or 69.1, wherein the extracellular binding domain binds a co-stimulatory receptor of the same cell in which it is expressed.

[0110] Aspect 69.3 is the DSR polypeptide according to any one of aspects 69-69.2, wherein the extracellular binding domain aggregates two or more co-stimulatory receptors of the same cell in which it is expressed.

[0111] Aspect 69.4 is the DSR polypeptide according to any one of aspects 69-69.3, wherein the extracellular binding domain can aggregate two or more co-stimulatory receptors of the same cell in which it is expressed to induce co-stimulatory signaling in the cell.

[0112] Aspect 69.5 is the DSR polypeptide according to any one of aspects 69-69.4, wherein the extracellular binding domain does not bind a target antigen on a diseased cell and / or a target on a different cell than the cell in which it is expressed.

[0113] Aspect 69.6 is the DSR polypeptide according to any one of aspects 69-69.5, wherein the extracellular binding domain does not induce co-stimulatory signaling in a different cell than the cell in which it is expressed.

[0114] Aspect 69.7 is the DSR polypeptide according to any one of aspects 69-69.6, wherein the intracellular cytokine signaling domain does not comprise a co-stimulatory domain.

[0115] Aspect 69.8 is the DSR polypeptide according to any one of aspects 69-69.7, wherein the intracellular cytokine signaling domain does not comprise a co-stimulatory domain derived from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and / or HVEM.

[0116] Aspect 69.9 is the DSR polypeptide according to any one of aspects 69-69.8, wherein the intracellular cytokine signaling domain does not comprise a CD3 domain.

[0117] Aspect 69.91 is the DSR polypeptide according to any one of aspects 69-69.9, wherein the intracellular cytokine signaling domain does not comprise a CD3zeta domain.

[0118] Aspect 69.92 is the DSR polypeptide according to any one of aspects 69-69.91, wherein the intracellular cytokine signaling domain does not comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 immunoreceptor tyrosine-based activation motifs (IT AM).

[0119] Aspect 69.93 is the DSR polypeptide according to any one of aspects 69-69.92, wherein the intracellular cytokine signaling domain can initiate JAK-STAT signaling.

[0120] Aspect 69.94 is the DSR polypeptide according to any one of aspects 69-69.93, wherein the intracellular cytokine signaling domain can initiate JAK-STAT signaling and RAS / RAF signaling.

[0121] Aspect 70 is a human T cell comprising: (a) a DSR polypeptide comprising an extracellular binding domain derived from 4-1BBL, a transmembrane domain derived from CD28, and an intracellular cytokine signaling domain derived from cMPL; and (b) a cTCR targeting survivin or TdT.

[0122] Aspect 71 is a human T cell comprising: (a) a DSR polypeptide comprising an extracellular binding domain derived from 4-1BBL, a transmembrane domain derived from CD28, and an intracellular cytokine signaling domain derived from cMPL; and (b) a TCE that targets CD 123 and CD3.

[0123] Aspect 72 is a method of treating leukemia in an individual in need thereof comprising the step of administering to the individual a therapeutically effective amount of a human T cell comprising: (a) a DSR polypeptide comprising an extracellular binding domain derived from 4-1BBL, a transmembrane domain derived from CD28, and an intracellular cytokine signaling domain derived from cMPL; and (b) a cTCR targeting survivin or TdT.

[0124] Aspect 73 is a method of treating leukemia in an individual in need thereof comprising the step of administering to the individual a therapeutically effective amount of a human T cell comprising: (a) a DSR polypeptide comprising an extracellular binding domain derived from 4-1BBL, a transmembrane domain derived from CD28, and an intracellular cytokine signaling domain derived from cMPL; and (b) a TCE that targets CD 123 and CD3.

[0125] It is specifically contemplated that any limitation discussed with respect to one aspect of the invention may apply to any other aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Any aspectdiscussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. For example, any step in a method described herein can apply to any other method. Moreover, any method described herein may have an exclusion of any step or combination of steps. Aspects of an aspect set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Brief Description of the Drawings.

[0126] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0127] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0128] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0129] FIG. 1A shows three signaling events required for full T-cell activation, including 1) T-Cell Receptor (TCR) signaling (“signal 1”), 2) co-stimulation (“signal 2”), and 3) cytokine receptor signaling (“signal 3”). FIG. IB shows an example of a state-of-the-art strategy to induce full T-cell activation, including a Costimulatory Chimeric Antigen Receptor (CAR) only with co-stimulatory function (signal 2; e.g., though activation of an intracellular CD28 domain) and constitutively active cytokine receptors (signal 3). Constitutive cytokine stimulation, however, is undesired as it can lead to immune cell disorders, such as lymphocytosis.

[0130] FIG. 2A shows a schematic of a novel construct termed Dual-Stimulatory Receptor (DSR) of certain aspects of the disclosure. A DSR comprises, consists of, or consists essentially of i) an extracellular binding domain, e.g., the extracellular domain of a co-stimulatory ligandthat engages an endogenous co-stimulatory receptor, operably linked to, 2) a transmembrane domain, itself operably linked to, 3) an immunostimulatory endodomain capable of transmitting cytokine or cytokine-like signaling. FIG. 2B shows a schematic depicting a mode of action of DSRs. Upon production of signal 1 in a cell, co-stimulatory receptors, e.g., 4-1BB, are upregulated (“Initiation”). Thereafter, the co-stimulatory receptors are engaged by one or more DSRs — via the extracellular binding domain of the DSR — leading to multimerization and production of signals 2 and 3 (“Signaling”). Once co-stimulatory receptors are degraded or downregulated, production of signals 2 and 3 ceases (“Termination”). In such a way, all three signals required for full T-cell activation can be produced in a temporal manner that is dependent on signal 1, which can be endogenous, e.g., antigen signaling, or artificial, e.g., CAR or cTCR, ensuring robust and safe full T-cell activation.

[0131] FIG. 3A shows a schematic of native and chimeric TCRs (cTCR), both of which can produce signal 1 and could each work with DSRs, merely as examples. FIG. 3B shows a schematic of examples of different DSR constructs generated and disclosed herein. Each of these illustrative DSR construct comprise an extracellular co-stimulatory ligand domain, e.g. 4-1 -BBL, and an endodomain capable of cytokine signaling (“cytokine-like signaling domain”), such as one derived from interleukin 7 receptor subunit alpha (IL7Ra), interleukin 2 receptor subunit beta (IL2RP), myeloid differentiation primary response protein 88 (MyD88), or thrombopoietin receptor (cMPL), termed “4-lBBL / IL7Ra”, “4-1BBL / IL2RP”, “4- lBBL / MyD88”, and “4-lBBL / cMPL”, respectively. A DSR without an endodomain (“A4- 1BBL” or “delta4-lBBL”) was generated to be used as a control. A heterologous full length 4- 1BBL (“4-1BBL”) was sometimes also used as a control. FIG. 3C shows flow cytometry results of cytotoxicity and T-cell proliferation assays in which CHLA-255 neuroblastoma cells (“Target”) were co-cultured with GD2-targeting cTCR T cells (termed “cTCR” in FIGs. 3C- 3D) which also co-expressed an illustrative DSR, as specified in each figure. The results showed a dramatic expansion of cTCR + 4-lBBL / cMPL T-cells (5,634 cells at day 0 to 11,666 cells at day 6), compared to controls and all other DSR groups. In addition, these results showed a particularly dramatic increase of cytotoxicity against neuroblastoma cells by cTCR + 4- IBBL / cMPL T cells (1,897 neuroblastoma cells at day 0 down to 919 cells at day 6), compared to controls and all other DSR groups (e.g., negative control (-) showed 2,534 neuroblastoma cells at day 0 and 380,515 cells at day 6). FIG. 3D shows the effect of DSRs on T cell proliferation. cTCR T cells were stimulated with irradiated tumor cells to provide signal 1 every three days and T cell fold expansion was assessed. The results showed that 4-lBBL / cMPL DSR induced the greatest magnitude of T cell expansion (peak of about 150-fold expansion atday 9) compared to the other constructs (e.g., cTCR+4-lBBL control showed a peak of about 20-fold expansion at day 9). Lines at day 12 from top to bottom represent cTCR+4- IBBL / cMPL, cTCR+4-lBBL / MyD88, cTCR+4-lBBL, cTCR+4-lBBL / IL7Ra, cTCR+4- lBBL / IL2Rb, and cTCR. FIG. 3E shows results of experiments to test signal transduction of 4-lBBL / cMPL DSR (termed “DSR” in FIG. 3E) by stimulating cells with anti-CD3 antibody (“Antigen stim (+)”) and assessing phosphorylation of STAT3 or STAT5, which are downstream of cMPL signaling, by Phospho Flow Cytometry. The results showed that phosphorylation of both STAT3 and STAT5 increased only when DSR-expressing T cells received antigenic stimulation, indicating that DSR signal transduction depended on antigen stimulation (signal 1). Together, these results indicated that DSRs promoted T cell expansion and cytotoxicity and suggested that 4-lBBL / cMPL could be a particularly effective construct for in vivo applications. FIG. 3F shows the effect of DSRs on T cell proliferation.

[0132] FIG. 4A shows a schematic of the experimental design for in vivo testing of DSRs. NSG™ mice were intravenously (i.v.) inoculated with 3xl06BV173 cells (leukemia cell line) expressing green fluorescent protein (GFP) and Firefly Luciferase (FL) (“BV173-GFP / FL”). Five days later (“Day 0”), mice were i.v. inoculated with 5xl06thawed T cells. Four different groups of thawed T cells were tested: non-transduced (NT), engineered, e.g., transduced, to express a cTCR targeting Terminal deoxynucleotidyl transferase peptide / HLA complex (termed “cTCR” in FIGs. 4B-4D), a cTCR + 4-1BBL (“cTCR-41BBL”), or a cTCR + 4- IBBL / cMPL (“cTCR-DSR”). No treatment controls (“(-)”) did not receive T cell inoculation. Tumor cell growth was monitored by bioluminescent (“IVIS”™) imaging and T cell proliferation / persistence was evaluated by tail vein bleeding. FIG. 4B shows average bioluminescence radiance as indicative of tumor burden over time. The results showed that treatment with cTCR and cTCR-41BBL only slightly delayed tumor growth compared to NT and (-) controls. In contrast, cTCR-DSR drastically reduced tumor burden and maintained the mice tumor free until the end of the study. FIG. 4C shows the amount of inoculated T cells in peripheral blood (PB) over time as a measure of T cell proliferation / persistence. The results showed a sharp decrease of PB T cells within the first 21 days in the NT, cTCR, and cTCR- 41BBL groups. Contrastingly, the cTCR-DSR group showed a continuous increase of PB T cells up until day 35 and a maintenance of T cells thereafter until at least day 63. FIG. 4D shows a Kaplan-Meier survival curve of mice from FIGs. 4B-4C. The results showed that mice in the (-) (broken line), NT, cTCR, and cTCR-41BBL groups reached a median survival of about 28-39 days. Strikingly, mice from the cTCR-DSR group maintained 100% survival until at least day 63. Together, these results indicated that cTCR-DSR T cells had improvedproliferation, persistence, and tumor-elimination capabilities which translated into prolonged survival.

[0133] FIG. 5A shows flow cytometry density plots for assessing cytotoxicity against the acute myeloid leukemia cell line MOLM-13 (“Target”) and proliferation of T cells expressing a CD 123 T-cell engager (TCE) and a DSR. The results showed that T cells expressing only CD123 TCE (“(-)”) showed cytotoxicity against MOLM-13 (“Target”) compared to controls, but lacked T cell expansion (372 T cells at day 0 compared to 139 T cells at day 9). Coexpression of 4-1BBL or A4-1BBL, i.e., providing signal 2, mildly enhanced TCE T cell expansion (e.g., +4-1BBL showed 364 T cells at day 0 and 2,431 at day 9). In contrast, a striking increase in T cell expansion and persistence was observed when CD123 TCE-modified T cells were engineered to co-express 4-lBBL / cMPL DSR (“DSR” in FIGs. 5B-5C) (268 T cells at day 0 and 9,145 cells at day 9). FIG. 5B shows quantification of Target cell expansion. The results showed an inhibition of Target cell expansion by T cells expressing a TCE, including those co-expressing 4-1BBL, A4-1BBL, and a DSR. FIG. 5C shows quantification of T cell expansion. The results showed that T cells co-expressing TCE+DSR showed dramatic expansion (about 30-fold at day 9 (“D9”)) compared to all other groups (e.g., TCE+4-1BBL showed about 10-fold expansion at D9). Together, these results indicated that DSRs can supplement immune cell activation across diverse therapeutic strategies to ensure a robust and persistent activation of anti-tumor immune cells.

[0134] FIG. 6A shows a schematic of the experimental design for in vivo testing of the effect of DSRs on CD123 TCE engineered T cells. FIG. 6B shows average bioluminescence radiance from tumor cells as indicative of tumor burden over time. FIG. 6C shows average bioluminescence radiance from T cells as indicative of T cell proliferation and persistence over time. FIG. 6D shows a Kaplan-Meier survival curve of mice from FIGs. 6B-6C.

[0135] FIG. 7A shows tumor cell fold change and FIG. 7B shows T cell fold expansion after coculture with T cells engineered with a TCR targeting survivin (“survTCR) and, optionally, 4-1BBL, A4-1BBL or 4-lBBL / cMPL (“DSR”).

[0136] FIG. 8 shows the effect of DSR on proliferation of Epstein-Barr virus (EBV)- specific T cells (EBVST).DETAILED DESCRIPTION

[0137] It is to be understood that the present disclosure is not limited to particular aspects described, as such may, of course, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0138] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this technology belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present technology, the preferred methods, devices and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety.

[0139] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Green and Sambrook eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition; the series Ausubel et al. eds. (2015) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (2015) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield ed. (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Herdewijn ed. (2005) Oligonucleotide Synthesis: Methods and Applications; Hames and Higgins eds. (1984) Transcription and Translation; Buzdin and Lukyanov ed. (2007) Nucleic Acids Hybridization: Modern Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi ed. (2007) In Vitro Transcription and Translation Protocols, 2nd edition; Guisan ed. (2006) Immobilization of Enzymes and Cells; Perbal (1988) A Practical Guide to Molecular Cloning, 2nd edition; Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald eds. (2010) Handbook of Biochemistry and Molecular Biology, 4th edition; and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology, 5th edition.

[0140] It also is to be understood, although not always explicitly stated, that the reagents described herein are merely illustrative and that equivalents of such are known in the art. It is to be inferred without explicit recitation and unless otherwise intended, that when the presenttechnology relates to a polypeptide, protein, polynucleotide, cell, or antibody, an equivalent or a biologically equivalent of such is intended within the scope of the present technology.

[0141] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0142] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0143] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0144] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0145] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed inventions.

[0146] The term “engineered” as used herein refers to an entity that is generated by the hand of man, including a cell, nucleic acid, polypeptide, vector, and so forth. In at least some cases, an engineered entity is synthetic and comprises elements that are not naturally present or configured in the manner in which it is utilized in the disclosure. In specific aspects, a vector is engineered through recombinant nucleic acid technologies, and a cell is engineered, e.g., through transfection or transduction of an engineered vector. Cells may be engineered to express heterologous proteins that are not naturally expressed by the cells, either because the heterologous proteins are recombinant or synthetic or because the cells do not naturally express the proteins.

[0147] As used herein, “derived” refers to a segment, domain, or portion of an amino acid sequence that is obtained from a larger protein, such as but not limited to a wild-type protein. This term encompasses any fragment, subsequence, or isolated part of the original protein which may retain specific functional or structural characteristics. The derived segment may beobtained through various methods, including but not limited to, enzymatic digestion, chemical cleavage, or genetic manipulation. It is contemplated that a derived segment can maintain portions of at least one biological activity of the original protein while also possibly exhibiting enhanced or modified properties.

[0148] “Individual,” “subject,” and “patient” are used interchangeably and can refer to a human or non-human.

[0149] In certain aspects, the present disclosure describes the generation of novel synthetic molecules termed dual-stimulatory receptors (DSRs) that provide costimulation (signal 2) and cytokine signaling (signal 3) transiently and simultaneously, or nearly simultaneously, when immune cells recognize a target and receive antigen stimulation (signal 1), or when immune cells are artificially stimulated with signal 1. In certain aspects, a DSR comprises an extracellular binding domain, e.g., the extracellular domain of a costimulatory ligand or an antibody, that engages an endogenous costimulatory receptor, linked by a transmembrane domain to an immunostimulatory endodomain capable of transmitting cytokine receptor or cytokine receptor-like signaling. In certain aspect, the DSR is engineered to comprise an extracellular domain derived from 4-1BBL. In certain aspects, the DSR is engineered to comprise a transmembrane domain derived from CD28. In certain aspects, the DSR is engineered to comprise an endodomain derived from a cytokine receptor. In some aspects, an endodomain is derived from interleukin 7 receptor subunit alpha (IL7Ra), interleukin 2 receptor subunit beta (IL2RP), myeloid differentiation primary response protein 88 (MyD88), or thrombopoietin receptor (cMPL). In certain aspects, the DSR is encoded by a polynucleotide or vector. In certain aspects, the vector comprises a viral vector, e.g., a retrovirus or lentivirus.

[0150] In certain aspects, a cell is engineered, e.g., transduced, to comprise a polynucleotide encoding a DSR. In certain aspects, a cell is engineered to express a DSR. In certain aspects, a DSR provides supplemental costimulatory signaling (signal 2) to a cell. In certain aspects, a DSR provides supplemental cytokine signaling (signal 3) to a cell. In certain aspects, a DSR provides supplemental costimulatory signaling (signal 2) and cytokine signaling (signal 3) to a cell. In certain aspects, the cell is an immune cell, such as a T-cell. In certain aspects, the immune cell, e.g., a T-cell, is engineered to generate artificial antigen stimulation (signal 1). Some aspects of the present disclosure are based, at least in part, on the generation of therapeutic immune effector cells (e.g., NK cells, T Cells, etc.) expressing one or more DSRs, and optionally one or more molecules comprising antigen binding affinity (e.g., antigen receptors, antibodies, T-cell engagers (TCEs), Chimeric Antigen Receptors (CARs), chimeric T Cell Receptors (cTCRs), transgenic T cell receptors (tTCR), etc.). In certain aspects,the immune cell, e.g., a T-cell, is engineered to comprise a DSR and a cTCR or TCE. In certain aspects, the cTCR recognizes disialoganglioside GD2 (GD2). In some aspects, the cTCR recognizes terminal deoxynucleotidyl transferase (TdT). In some aspects, the TCE engagers recognizes cluster of differentiation 123 (CD123). In some aspects, the TCE engagers recognizes cluster of differentiation 123 (CD123) and CD3.

[0151] In certain aspects, a cell is engineered to comprise a DSR for increasing immune cell activation. In certain aspects, a cell is engineered to comprise a DSR for increasing T cell activation. In certain aspects, a cell is engineered to comprise a DSR for increasing immune cell cytotoxicity. In certain aspects, a cell is engineered to comprise a DSR for increasing immune cell cytotoxicity against cancer. In certain aspects, a cell is engineered to comprise a DSR for increasing T cell cytotoxicity. In certain aspects, a cell is engineered to comprise a DSR for increasing T cell cytotoxicity against cancer. In some aspects, the cancer is blood cancer. In certain aspects, a cell is engineered to comprise a DSR for increasing immune cell proliferation or persistence. In certain aspects, a cell is engineered to comprise a DSR for increasing T cell proliferation or persistence.

[0152] Accordingly, disclosed herein are polynucleotides, polypeptides, compositions, and / or immune cells comprising and / or encoding a DSR, as well as methods for use of the same in treatment of disease (e.g., cancer, infectious diseases, autoimmune diseases, etc.). In various aspects, the one or more DSRs are heterologous to the cell in which it is comprised. In various aspects, the one or more DSRs are heterologous to the cell in which it is comprised and are not expressed from the genome of the cell. In various aspects, the one or more DSRs are expressed from a synthetic vector generated by the hand of man. In some aspects, the one or more DSRs may integrate into the genome of the host cell.

[0153] In some aspects, a protein, peptide, domain, derived domain, derived amino acid sequence, polynucleotide, nucleic acid sequence, and / or gene product can be obtained or be substantially identical to a protein, peptide, domain, amino acid sequence, nucleic acid sequence, and / or gene product of a gene recited in the disclosure, including but not limited to 4-1BBL, IL7Ra, IL2RP, MyD88, cMPL, OX40L, CD28, or a combination thereof. Any gene referenced in the disclosure can refer to a mammalian gene, e.g., human or murine, or to a homolog or analog in any other species.

[0154] 4-1BBL: An example of a TNF superfamily member 9 (4-1BB-L; TNFSF9;CD137L; TNLG5A) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 8744 (TNFSF9) on chromosome 19pl3.3, at NC 000019.10 Reference GRCh38.pl4Primary Assembly (range, 6531026 to 6535924) for the genomic sequence which is incorporated herein by reference in its entirety. A 4-1BB-L mRNA transcript can be identified as NM_003811.4, and the associated protein product can be identified as NP_003802.1, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is a bidirectional signal transducer that acts as a ligand for TNFRSF9 / 4-1BB.

[0155] IL7Ra: An example of an interleukin 7 receptor (IL7Ra; IL7R; ILRA; CD127; IL7RA; CDW127; IMD104; sIL-7R; lnc-IL7R; IL7Ralpha; IL-7Ralpha; IL-7R-alpha) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 3575 (IL7R) on chromosome 5pl3.2, at NC_000005.10 Reference GRCh38.pl4 Primary Assembly (range, 35856891 to 35879603) for the genomic sequence which is incorporated herein by reference in its entirety. An IL7Ra mRNA transcript can be identified as NM_001410734.1 or NM_002185.5, and the associated protein product can be identified as NP_001397663.1 or NP_002176.2, respectively, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is a receptor for interleukin 7 (IL7).

[0156] IL2R]J: An example of an interleukin 2 receptor subunit beta (IL2RB; CD122; IMD63; IL15RB; P70-75) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 3560 (IL2RB) on chromosome 22ql2.3, at NC_000022. l l Reference GRCh38.pl4 Primary Assembly (range, 37125838 to 37175118, complement) for the genomic sequence which is incorporated herein by reference in its entirety. A CD122 mRNA transcript can be identified as NM_000878.5, NM_001346222.1, or NM_001346223.2, and the associated protein product can be identified as NP 000869.1, NP 001333151.1, or NP_001333152.1, respectively, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is involved in T cell-mediated immune responses.

[0157] MyD88: An example of a MYD88 innate immune signal transduction adaptor (MYD88; WM1; IMD68; MYD88D) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 4615 (MYD88) on chromosome 3p22.2, atNC_000003.12 Reference GRCh38.pl4 Primary Assembly (range, 38138661 to 38143022) for the genomic sequence which is incorporated herein by reference in its entirety. A MYD88 mRNA transcript can be identified as NM_001172566.2, NM_001172567.2, NM_001172568.2, NM_001172569.3, NM_001365876.1, NM_001365877.1, NM_001374787.1, or NM_002468.5, and the associated protein product can be identified as NP_001166037.2, NP_001166038.2,NP_001166039.2, NP_001166040.2, NP_001352805.1, NP_001352806.1, NP_001361716.1, or NP_002459.3, respectively, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is a cytosolic adapter protein that plays a central role in the innate and adaptive immune response.

[0158] cMPL: An example of a MPL proto-oncogene, thrombopoietin receptor (cMPL, MPL; MPLV; TPOR; C-MPL; CD110; THPOR; THCYT2) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 4352 (MPL) on chromosome lp34.2, at NC_000001.11 Reference GRCh38.pl4 Primary Assembly (range, 43337818 to 43354466) for the genomic sequence which is incorporated herein by reference in its entirety. A cMPL mRNA transcript can be identified as NM 005373.3, and the associated protein product can be identified as NP 005364.1, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is a 635 amino acid transmembrane domain, with two extracellular cytokine receptor domains and two intracellular cytokine receptor box motifs involved in platelet generation.

[0159] OX40L: An example of a TNF superfamily member 4 (OX40L; TNFSF4; GP34; CD252; TXGP1; CD134L; OX-40L; TNLG2B) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 7292 (OX40L) on chromosome lq25.1, at NC_000001. l l Reference GRCh38.pl4 Primary Assembly (range, 173172870 to 173450733, complement) for the genomic sequence which is incorporated herein by reference in its entirety. A OX40L mRNA transcript can be identified as NM_001297562.2 or NM_003326.5, and the associated protein product can be identified as NP 001284491.1 or NP 003317.1, each of which are incorporated by reference herein in their entirety. The protein encoded by this gene is a cytokine of the tumor necrosis factor (TNF) ligand family.

[0160] CD28: An example of a CD28 molecule (CD28; Tp44; IMD123) gene sequence can be found in the US NIH, National Library of Medicine, National Center for Biotechnology Information (NCBI) GENBANK® Database under Gene ID: 940 (CD28) on chromosome 2q33.2, at NC_000002.12 Reference GRCh38.pl4 Primary Assembly (range, 203706482 to 203738912) for the genomic sequence which is incorporated herein by reference in its entirety. A CD28 mRNA transcript can be identified as NM_001243077.2, NM_001243078.2, NM_001410981.1, or NM_006139.4, and the associated protein product can be identified as NP_001230006.1, NP_001230007.1, NP_001397910.1 , or NP_006130.1, respectively, each of which are incorporated by reference herein in their entirety. The protein encoded by this geneis essential for T-cell proliferation and survival, cytokine production, and T-helper type-2 development.I. Proteins

[0161] As used herein, a “protein” or “polypeptide” refers to a molecule comprising at least five amino acid residues. As used herein, a “peptide” refers to a molecule comprising at least three amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some aspects, wild-type versions of a protein or polypeptide are employed, however, in aspects of the engineered DSR protein of the disclosure, a modified protein or polypeptide is employed to act as cell stimulatory agent, such as increasing costimulation (signal 2) and / or cytokine signaling (signal 3). A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some aspects, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects. In some aspects, a modified / variant protein or polypeptide may be a chimeric polypeptide, i.e., a combination of two or more domains of wild-type proteins.

[0162] Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein or, optionally, a protein in which any signal sequence has been removed or inserted. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA / exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., a dual-stimulatory receptor (DSR), an antibody or fragment thereof, a chimeric antigen receptor (CAR), a chimeric T-cell receptor (cTCR), a T-cell engager (TCE), or a combination thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.

[0163] In certain aspects the size of a protein, domain, derived amino acid sequence, derived domain, or polypeptide (wild-type or modified) may comprise, but is not limited to, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or greater, and any range derivable therein, or derivative of a corresponding amino sequence described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g., for targeting or localization (e.g., a marker, such as dsRed mono), for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term “domain” refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.

[0164] The polypeptides, domain, derived amino acid sequence, derived domain, or proteins of the disclosure may comprise, consist essentially of, or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acids, or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% (or any derivable range therein) similar, identical, or homologous with at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329,330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348,349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405,406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450,475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or 750, or more contiguous amino acids, or any range derivable therein, of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 79, 94, 96, and / or 98-99, and / or of a protein product associated with CD122, CD25, CD132, CTLA-4, IL-10, IL-35, TGF-P, TIGIT, PD-1, CD28, or a combination thereof

[0165] In some aspects, a protein, domain, derived amino acid sequence, derived domain, or polypeptide may comprise, consist essentially of, or consist of amino acids 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295,296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314,315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 351, 352, 353,354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575,600, 625, 650, 675, 700, 725, or 750 (or any derivable range therein) of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 79, 94, 96, and / or 98-99.

[0166] In some aspects, a protein, domain, derived amino acid sequence, derived domain, or polypeptide may comprise, consist essentially of, or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317,318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336,337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355,356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393,394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412,413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625,650, 675, 700, 725, or 750 (or any derivable range therein) contiguous amino acids of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 79, 94, 96, and / or 98-99.

[0167] In some aspects, the polypeptide, domain, derived amino acid sequence, derived domain, or protein may comprise, consist essentially of, or consist of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311,312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330,331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387,388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406,407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 450, 475,500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or 750 (or any derivable range therein) contiguous amino acids of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 79, 94, 96, and / or 98-99, that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 79, 94, 96, and / or 98-99.

[0168] In some aspects there is a polypeptide, domain, derived amino acid sequence, derived domain, starting at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,4, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,3, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,2, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,1, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,0, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,9, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,8, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246,7, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,6, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,5, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,4, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322,3, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341,2, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360,1, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379,0, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398,9, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417,8, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or0 of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 79, 94, 96, and / or 98-, and comprising at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,3, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,2, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,1, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,0, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,9, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,8, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283,284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378,379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397,398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, or 750 (or any derivable range therein) contiguous amino acids of any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 79, 94, 96, and / or 98-99.

[0169] In some aspects, provided herein are nucleotide as well as the protein, polypeptide, and peptide sequences for various genes. In certain aspects, wild type copies of certain proteins, polypeptides, and / or nucleotides described herein have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and / or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.

[0170] It is contemplated that in some compositions of the disclosure, there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and / or protein per mL. The concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / mL or more (or any range derivable therein).

[0171] In some aspects, a protein may comprise cleavage sites, e.g., a Furin cleavage site. In some aspects, a protein may comprise a “self-cleaving” sequence, e.g., a 2A sequence. In some aspects, a protein may comprise a tag, e.g., a C-myc tag.

[0172] In certain aspects, a cleavage site comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 47-48.

[0173] In certain aspects, a self-cleaving sequence comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 49-50.

[0174] In certain aspects, a tag comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 53-54.

[0175] SEQ ID NO: 47. Furin cleavage site (in GDl.cTCR, and TdT.cTCR) - DNA: AGAGCCAAAAGA

[0176] SEQ ID NO: 48. Furin cleavage site (in GDl.cTCR, and TdT.cTCR) - AA: RAKR

[0177] SEQ ID NO: 49. P2A (in GDl.cTCR, and TdT.cTCR) - DNA:AGTGGTTCTGGCGCAACAAATTTCTCACTGCTCAAACAGGCGGGAGACGTGGAAGAGAATCC CGGTCCC

[0178] SEQ ID NO: 50. P2A (in GD2.cTCR, and TdT.cTCR) - AA:SGSGATNFSLLKQAGDVEENPGP

[0179] SEQ ID NO: 53. C-myc tag (in GD2.cTCR, and TdT.cTCR) - DNA: GAACAAAAAT T GAT T T CAGAGGAAGAT C T C

[0180] SEQ ID NO: 54. C-myc tag (in GD2.cTCR, and TdT.cTCR) - AA: EQKLISEEDL

[0181] In certain aspects, a protein may comprise, consist essentially of, or consist of a heterologous 4-1BBL protein. In certain aspects, a protein may comprise, consist essentially of, or consist of a extracellular domain of 4-1BBL. In certain aspects, a protein may comprise, consist essentially of, or consist of a truncated form of 4-1BBL protein.

[0182] In certain aspects, a heterologous 4-1BBL, comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 1-2, or 80.

[0183] In certain aspects, an extracellular domain of 4-1BBL comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 3-4.

[0184] In certain aspects, a truncated form of 4-1BBL comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 9-10, or 81.

[0185] SEQ ID NO: 1. 4-1BBL - DNA:ATGGAGTACGCTAGCGATGCTTCTCTCGACCCCGAGGCGCCCTGGCCCCCCGCCCCGCGGGC TAGAGCCTGCAGAGTGCTGCCCTGGGCCTTGGTAGCCGGCCTGCTCCTTCTGCTCCTCCTGG CCGCCGCTTGTGCCGTGTTTCTTGCCTGCCCCTGGGCGGTTAGTGGAGCTAGAGCTAGCCCG GGGTCCGCCGCTAGCCCGCGACTCAGAGAGGGCCCCGAGTTAAGCCCCGACGATCCGGCCGG CCTGCTGGACCTGAGACAAGGCATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCG ACGGACCCTTAAGCTGGTACAGCGATCCCGGACTCGCTGGCGTGAGCCTGACCGGCGGCCTG AGCTACAAGGAGGACACCAAGGAGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTT TCAGCTGGAGCTGCGTAGAGTGGTGGCCGGCGAGGGAAGCGGCTCTGTGTCACTGGCACTGC ATCTGCAGCCTCTCAGATCGGCCGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCG CCCGCTAGCAGCGAGGCTAGAAACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAG CGCCGGGCAGAGACTGGGCGTGCACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGC TGACCCAAGGCGCCACCGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTG CCTAGCCCTAGAAGCGAG

[0186] SEQ ID NO: 2. 4-1BBL - AA:MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASP GSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGL SYKEDTKELWAKAGVYYVFFQLELRRWAGEGSGSVSLALHLQPLRSAAGAAALALTVDLP PASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPE I PAGL PSPRSE

[0187] SEQ ID NO: 80. 5'LTR-4-lBBL-3'LTR - DNA:G C GAG TCATATTAATTACTAGT C AAT TAGTTGATTTTTATTTTT GAG AT AT AC AT G T GAAAG ACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAAT ACATAAC T GAGAATAGAAAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATAT GG GCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAA CAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCA AGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTT TCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCG CTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTC ACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCC TCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATT GACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCC AGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGAT TGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTG TATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGAC GTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTT AGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCG CGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTC TGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGA AAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACC TTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCG AGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACC AGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCC TTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACC TCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCC CCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCT GAG AT GAG AAGAG T T AC T AAC AG CCCCTCTCTC C AAG C T GAG T TAG AG GCTCTCTACTTAGT CCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGG TACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTA GAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGA CGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCAT AACTCGAGGCCACGATGGAGTACGCTAGCGATGCTTCTCTCGACCCCGAGGCGCCCTGGCCC CCCGCCCCGCGGGCTAGAGCCTGCAGAGTGCTGCCCTGGGCCTTGGTAGCCGGCCTGCTCCT TCTGCTCCTCCTGGCCGCCGCTTGTGCCGTGTTTCTTGCCTGCCCCTGGGCGGTTAGTGGAG CTAGAGCTAGCCCGGGGTCCGCCGCTAGCCCGCGACTCAGAGAGGGCCCCGAGTTAAGCCCC GACGATCCGGCCGGCCTGCTGGACCTGAGACAAGGCATGTTCGCTCAGCTGGTGGCTCAGAA CGTGCTGCTGATCGACGGACCCTTAAGCTGGTACAGCGATCCCGGACTCGCTGGCGTGAGCC TGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGGAGCTGGTGGTGGCCAAGGCCGGCGTG TACTACGTGTTCTTTCAGCTGGAGCTGCGTAGAGTGGTGGCCGGCGAGGGAAGCGGCTCTGT GTCACTGGCACTGCATCTGCAGCCTCTCAGATCGGCCGCCGGCGCCGCCGCCCTGGCCCTTA CTGTGGACTTACCGCCCGCTAGCAGCGAGGCTAGAAACAGCGCCTTCGGCTTCCAAGGCAGA CTGCTGCACCTGAGCGCCGGGCAGAGACTGGGCGTGCACCTGCACACCGAGGCTAGAGCTAG ACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCTGGGCCTGTTCAGAGTGACCCCCGAGA TCCCTGCCGGCCTGCCTAGCCCTAGAAGCGAGTGAGCATGCAACCTCGATCCGGATTAGTCC AAT T T G T T AAAGAC AG GAT AT GAG T G G T C GAG GCTCTAGTTTT GAG T C AAC AAT AT C AC C AG C T GAAG C C T AT AGAG TAG GAG C C AT AGAT AAAAT AAAAGAT TTTATTTAGTCTC C AGAAAAA GGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCA AGGCAT GGAAAAATACATAAC T GAGAATAGAGAAGT T CAGAT CAAGGT CAGGAACAGAT GGA ACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCC AAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCC CCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAG AACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACT AACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGA GCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTG TATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTC TCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTT

[0188] SEQ ID NO: 3. cytoplasmic domain in 4-1 BBL DNA:ATGGAGTACGCTAGCGATGCTTCTCTCGACCCCGAGGCGCCCTGGCCCCCCGCCCCGCGGGCTAGAGCCTGCAGAGTGCTGCCC

[0189] SEQ ID NO: 4. cytoplasmic domain in 4-1BBL - AA:MEYASDASLDPEAPWPPAPRARACRVLP

[0190] SEQ ID NO: 9. Truncated 4-1BBL (A4-1BBL) - DNA:ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCCTGCCCCTGGGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGCCGCTAGCCCGCGACTCAGAGAGGGCCCCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGGACCTGAGACAAGGCATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCCTTAAGCTGGTACAGCGATCCCGGACTCGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGGAGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAGCTGCGTAGAGTGGTGGCCGGCGAGGGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAGCCTCTCAGATCGGCCGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAGCAGCGAGGCTAGAAACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGCAGAGACTGGGCGTGCACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCCTAGAAGCGAGACGCGTTTTTGGGTGCTGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCTTACTGGTGACTGTTGCCTTTATTATTTTTTGGGTTCGAAGTAAGCGTTCCCGGCTG

[0191] SEQ ID NO: 10. Truncated 4-1BBL (A4-1BBL) - AA:MALPVTALLLPLALLLHAARPACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQG MFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELWAKAGVYYVFFQLELRRV VAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV HLHTEARARHAWQLTQGATVLGLFRVTPE I PAGLPSPRSETRFWVLVWGGVLACYSLLVTV AFI I FWVRSKRSRL

[0192] SEQ ID NO: 81. 5'LTR- Truncated 4-1BBL (A4-1BBL) -3'LTR- DNA:G C GAG TCATATTAATTACTAGT C AAT TAGTTGATTTTTATTTTT GAG AT AT AC AT G T GAAAG ACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAAT ACATAAC T GAGAATAGAAAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATAT GG GCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAA CAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCA AGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTT TCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCG CTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTC ACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCC TCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATT GACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCC AGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGAT TGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTG TATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGAC GTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTT AGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACC TAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCG CGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTC TGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGA AAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACC TTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCG AGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACC AGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCC TTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACC TCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCC CCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCT GAG AT GAG AAGAG T T AC T AAC AG CCCCTCTCTC C AAG C T C AC T TAG AG GCTCTCTACTTAGT CCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGG TACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTA GAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGA CGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATAACTCGAGGCCACGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTC CACGCCGCCAGGCCGGCCTGCCCCTGGGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGC CGCTAGCCCGCGACTCAGAGAGGGCCCCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGG ACCTGAGACAAGGCATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCC TTAAGCTGGTACAGCGATCCCGGACTCGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAA GGAGGACACCAAGGAGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG AGCTGCGTAGAGTGGTGGCCGGCGAGGGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAG CCTCTCAGATCGGCCGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAG CAGCGAGGCTAGAAACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGC AGAGACTGGGCGTGCACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAA GGCGCCACCGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCC TAGAAGCGAGACGCGTTTTTGGGTGCTGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCTTACTGGTGACTGTTGCCTTTATTATTTTTTGGGTTCGAAGTAAGCGTTCCCGGCTGTAAGCA TGCAACCTCGATCCGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTA G T T T T GAG T C AAC AAT AT GAG GAG C T GAAG C C T AT AGAG TAG GAG C C AT AGAT AAAAT AAAA GATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGC TAGC T TAAGTAACGCCAT T T T GCAAGGCAT GGAAAAAT AGAT AAC T GAGAATAGAGAAGT T C AGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAG CAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGA TATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGG TCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAA ATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTT CTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATT GACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTT

[0193] In certain aspects, a protein may comprise a truncated intracellular domain of CD28.

[0194] In certain aspects, a truncated form of 4-1BBL comprises, consists essentially of, or consists of a sequence at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 15-16.

[0195] SEQ ID NO: 15. AICD of CD28 (in A4-1BBL) - DNA:CGAAGTAAGCGTTCCCGGCTG

[0196] SEQ ID NO: 16. AICD of CD28 (in A4-1BBL) - AA:RSKRSRL

[0197] In certain aspects, a protein may comprise a reporter protein. In some aspects a reporter protein is dNGFR.

[0198] In certain aspects, a reporter protein comprises, consists essentially of, or consists of a sequence at least or exactly 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 78-79.

[0199] SEQ ID NO: 78. dNGFR (in CD123-BiTE-IRES-NGFR (CD123 TCE)) - DNA:ATGGGAGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCT GGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTG AGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACC GTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTG CAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGACG ACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGAGGCG TGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGAACACCGT GTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCACGTGGACCCGTGCCTGC CCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCC GAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGACAG CACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACGG TGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACC GACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGCCTTGTGGCCTA CATAGCCTTCAAGAGGTGGAACAGC

[0200] SEQ ID NO: 79. dNGFR (in CD123-BiTE-IRES-NGFR (CD123 TCE)) - AA: MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQT VCEPCLDSVTFSDWSATEPCKPCTECVGLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEA CRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADA ECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPWTRGTT DNL I PVYCS I LAAVWGLVAY I AFKRWNS

[0201] In certain aspects, a protein may comprise, consist essentially of, or consist of a heterologous OX40L protein. In certain aspects, a protein may comprise, consist essentially of, or consist of a truncated form of OX40L protein.

[0202] In certain aspects, a heterologous OX40L protein comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 93-94.

[0203] In certain aspects, a truncated form of OX40L protein comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 95-96.

[0204] SEQ ID NO: 93. Full QX40L - DNA:ATGGAGAGAGTGCAGCCCCTGGAGGAGAACGTGGGCAACGCCGCTAGACCTAGATTCGAGAG AAACAAGCTGCTGCTGGTGGCTAGCGTGATCCAAGGCCTGGGCCTGCTGCTGTGCTTCACCT ACATCTGCCTGCACTTCAGCGCCCTGCAAGTGAGCCACAGATACCCTAGAATTCAGAGCATC AAGGTGCAGTTCACCGAGTACAAGAAGGAGAAGGGCTTCATCCTGACATCTCAGAAGGAGGA CGAGATCATGAAGGTGCAGAACAACAGCGTGATCATCAACTGCGACGGCTTCTACCTGATCA GCCTGAAGGGCTACTTCAGCCAAGAGGTGAACATCAGCCTGCACTATCAGAAGGACGAGGAG CCCCTGTTTCAGCTGAAGAAGGTGAGAAGCGTGAACAGCCTGATGGTGGCTAGCCTGACCTAC AAG GAG AAG G T G T AC C T GAAC G T GAG GAG C GAG AAC AC AAG C C T G GAC GAC T T C C AC G T GA ACGGCGGCGAGCTGATCCTGATCCATCAGAACCCCGGCGAGTTCTGCGTGCTG

[0205] SEQ ID NO: 94. Full OX40L - AA:MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQS I KVQFTEYKKEKGFILTSQKEDE IMKVQNNSVI INCDGFYLI SLKGYFSQEVNI SLHYQKDEE PL FQLKKVRS VNS LMVAS L T YKDKVYLNVT T DNT S LDD FHVNGGE L I L I HQNPGE FCVL

[0206] SEQ ID NO: 95. OX40L-TM+Extracellular - DNA:CTGCTGCTGGTGGCTAGCGTGATCCAAGGCCTGGGCCTGCTGCTGTGCTTCACCTACATCTG CCTGCACTTCAGCGCCCTGCAAGTGAGCCACAGATACCCTAGAATTCAGAGCATCAAGGTGC AGTTCACCGAGTACAAGAAGGAGAAGGGCTTCATCCTGACATCTCAGAAGGAGGACGAGATC ATGAAGGTGCAGAACAACAGCGTGATCATCAACTGCGACGGCTTCTACCTGATCAGCCTGAA GGGCTACTTCAGCCAAGAGGTGAACATCAGCCTGCACTATCAGAAGGACGAGGAGCCCCTGT TTCAGCTGAAGAAGGTGAGAAGCGTGAACAGCCTGATGGTGGCTAGCCTGACCTACAAGGAC AAGGTGTACCTGAACGTGACCACCGACAACACAAGCCTGGACGACTTCCACGTGAACGGCGG CGAGCTGATCCTGATCCATCAGAACCCCGGCGAGTTCTGCGTGCTG

[0207] SEQ ID NO: 96. OX40L-TM+Extracellular - AA:LLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQS IKVQFTEYKKEKGFILTSQKEDE I MKVQNNSVI INCDGFYLI SLKGYFSQEVNI SLHYQKDEEPLFQLKKVRSVNSLMVASLTYKD KVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLA. Dual Stimulatory Receptor (DSR)

[0208] As used herein, a “dual stimulatory receptor”, or “dual-stimulatory receptor”, or “engineered dual stimulatory receptor”, or “DSR” refers to an engineered molecule capable of producing in a cell two or more signals required for immune cell activation, e.g., costimulatory signaling and cytokine signaling.

[0209] Certain aspects of the present disclosure include an engineered molecule that can generate immune costimulatory signaling (signal 2) and cytokine signaling (signal 3). In certain aspects, the engineered molecule is an engineered polypeptide. In certain aspects, the engineered polypeptide is a DSR. In certain aspects, an engineered polypeptide comprises an extracellular binding domain, e.g., a domain of a costimulatory ligand, a transmembrane domain, and an intracellular signaling domain, e.g., a cytokine or cytokine-like signaling endodomain. In certain aspects, an engineered polypeptide comprises an extracellular domain of a costimulatory ligand, a transmembrane domain of a membrane bound protein, and an intracellular cytokine signaling domain of a cytokine receptor. In certain aspects, the extracellular domain may be derived from tumor necrosis factor ligand superfamily, member 9 (TNFRSF9 ligand, also known as 4- IBB ligand (4-1 BBL), Ly63I, or CD137L; herein referred to as 4-1BBL). In certain aspects, the extracellular domain may be derived from TNF superfamily member 4 (TNFSF4, also known as 0X40 ligand (OX40L), GP34, CD252, TXGP1, CD134L, OX4OL, or TNLG2B; herein referred to as OX40L). In certain aspects, thetransmembrane domain may be derived from cluster of differentiation 28 (CD28). In certain aspects, the intracellular cytokine signaling domain may be derived from interleukin 7 receptor subunit alpha (IL7Ra; also known as ILRA; CD127; IL7RA; CDW127; IMD104; sIL-7R; Inc- IL7R; IL7Ralpha; IL-7Ralpha; IL-7R-alpha), interleukin 2 receptor subunit beta (IL2RP; also known as CD122; IMD63; IL15RB; P70-75; IL-2Rbeta), myeloid differentiation primary response protein 88 (MyD88; also known as WM1; IMD68; MYD88D), or thrombopoietin receptor (cMPL; also known as MPLV; TPOR; MPL; CD110; THPOR; THCYT2). In certain aspects, an engineered polypeptide can comprise, consist of, or consist essentially of any one or more of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 79, 94, 96, and / or 98.

[0210] Certain aspects of the present disclosure include an engineered DSR, nucleic acids encoding an engineered DSR, and / or cells comprising an engineered DSR.

[0211] In certain aspects, an engineered DSR polypeptide comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NO: 18 (4-lBBL / IL7Ra).

[0212] In certain aspects, an engineered DSR polypeptide comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NO: 22 (4-1BBL / IL2RP).

[0213] In certain aspects, an engineered DSR polypeptide comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NO: 26 (4-lBBL / MyD88).

[0214] In certain aspects, an engineered DSR polypeptide comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NO: 30 (4-lBBL / cMPL).

[0215] SEQ ID NO: 18. 4-lBBL-CD28TM-IL7Ra (4-lBBL / IL7Ra) - AA:MALPVTALLLPLALLLHAARPACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQG MFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELWAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSETRFWVLVWGGVLACYSLLVTV AFI I FWVKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDWITPESFGRDSSLTCLAGNVSACDAPI LSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGS NQEEAYVTMS S FYQNQ

[0216] SEQ ID NO: 22. 4-1BBL-CD28TM-IL2RP (4-1BBL / IL2RP) - AA:MALPVTALLLPLALLLHAARPACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQG MFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELWAKAGVYYVFFQLELRRV VAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSETRFWVLVWGGVLACYSLLVTV AFI I FWVNCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAP EISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV

[0217] SEQ ID NO: 26. 4-lBBL-CD28TM-MyD88 (4-lBBL / MyD88) - AA:MALPVTALLLPLALLLHAARPACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQG MFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELWAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSETRFWVLVWGGVLACYSLLVTV AF 11 FWVMAAGG P GAG S AAP VS S T S S L P LAALNMRVRRRL S L FLNVRT QVAADW T ALAE EMD FEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPS IEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI

[0218] SEQ ID NO: 30. 4-lBBL-CD28TM-cMPL (4-lBBL / cMPL) - AA:MALPVTALLLPLALLLHAARPACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQG MFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELWAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGV HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSETRFWVLVWGGVLACYSLLVTV AFI I FWVRWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLL EILPKSSERTPLPLCSSQAQMDYRRLQPSCLGTMPLSVCPPMAESGSCCTTHIANHSYLPLSYWQQPB. Extracellular Binding Domain

[0219] As used herein, an “extracellular binding domain” refers to any protein or polypeptide domain localized to the extracellular space that can specifically bind to an antigen target, including but not limited to, antibodies, and / or receptor ligands.1. Antibodies

[0220] As used herein, the terms “antibody” or “antibodies” collectively refer to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats,rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins. Unless specifically noted otherwise, the term “antibody” includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103M'1greater, at least 104M'1greater or at least 105M'1greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, murine or humanized non-primate antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Owen et al., Kuby Immunology, 7th Ed., W.H. Freeman & Co., 2013; Murphy, Janeway's Immunobiology, 8th Ed., Garland Science, 2014; Male et al., Immunology (Raitt), 8thEd., Saunders, 2012; Parham, The Immune System, 4th Ed., Garland Science, 2014.

[0221] In some aspects, the extracellular binding domain of an engineered polypeptide of the disclosure may comprise an antibody, or a fragment thereof. The antibody or fragment thereof, may bind and activate a costimulatory receptor. In some aspects, the antibody or fragment thereof is an agonist of a costimulatory receptor selected from the group comprising, consisting essentially of, or consisting of cluster of differentiation 28 (CD28), inducible T cell costimulatory (ICOS), cluster of differentiation 27 (CD27), TNF receptor superfamily member 14 (TNFRSF14, also known as HVEM), cluster of differentiation 40 (CD40), tumor necrosis factor receptor superfamily, member 9 (Tnfrsf9, also known as 4-1BB), TNF receptor superfamily member 4 (TNFRSF4, also known as 0X40), TNF receptor superfamily member 25 (TNFRSF25, also known as DR3), tumor necrosis factor receptor superfamily, member 18 (Tnfrsfl8, also known as GITR), cluster of differentiation 30 (CD30), hepatitis A virus cellular receptor 1 (HAVCR1, also known as TIM-1), cluster of differentiation 150 (CD150, also known as SLAM), cluster of differentiation 2 (CD2), and cluster of differentiation 226 (CD226; also known as DNAM-1). In some aspects, an antibody or fragment thereof comprises specificity or binding affinity to a tumor associated antigen (TAA). In some aspects, a TAA comprises, consists of, or consists essentially of CD123.

[0222] In certain aspects, an antibody or fragment thereof comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 37-42, 63-64, 67-72, and / or 75-76.

[0223] SEQ ID NO: 63. CD123-BiTE-IRES-NGFR (CD123 TCE)-Up to IRES - DNA:ATGGATTGGATCTGGCGCATCCTGTTTCTCGTGGGAGCCGCCACAGGCGCCCATTCTCAGGT GCAGCTGCAGCAGCCTGGCGCTGAACTCGTGCGGCCAGGCGCTTCTGTGAAGCTGAGCTGTA AAGCCAGCGGCTACACCTTCACCAGCTACTGGATGAACTGGGTCAAGCAGCGGCCCGACCAG GGCCTGGAATGGATCGGCAGAATCGACCCCTACGACAGCGAGACACACTACAACCAGAAGTT CAAGGACAAGGCCATCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGCAGCTGTCCA GCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGGCAACTGGGACGACTACTGG GGCCAGGGCACAACCCTGACAGTGTCTAGCGGAGGCGGAGGAAGTGGCGGCGGAGGATCTGG GGGAGGCGGATCTGATGTGCAGATCACCCAGAGCCCCAGCTACCTGGCTGCCTCTCCTGGCG AGACAATCACCATCAACTGCCGGGCCAGCAAGAGCATCTCCAAGGACCTGGCCTGGTATCAG GAAAAGCCCGGCAAGACCAACAAGCTGCTGATCTACAGCGGCAGCACCCTGCAGAGCGGCAT CCCCAGCAGATTTTCCGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCTCCCTGG AACCCGAGGACTTTGCCATGTACTATTGCCAGCAGCACAACAAGTACCCTTACACCTTCGGC GGAGGCACCAAGCTGGAAATCAAGTCCGGAGGTGGTGGATCCGATATCAAACTGCAGCAGTC AGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGACTTCTGGCTACA CCTTTACTAGGTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATT GGATACATTAATCCTAGCCGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCAC AT T GAG TACAGACAAAT CC T CCAGCACAGCC TACAT GCAAC T GAGCAGCC T GACAT C T GAGG ACTCTGCAGTCTATTACTGTGCAAGATATTATGATGATCATTACTGCCTTGACTACTGGGGC CAAGGCACCACTCTCACAGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGG TGGTGGTTCTGACATTCAGCTGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGA AGGTCACCATGACCTGCAGAGCCAGTTCAAGTGTAAGTTACATGAACTGGTACCAGCAGAAG TCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAAGTGGCTTCTGGAGTCCCTTA TCGCTTCAGTGGCAGTGGGTCTGGGACCTCATACTCTCTCACAATCAGCAGCATGGAGGCTG AAGATGCTGCCACTTATTACTGCCAACAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGG ACCAAGCTGGAGCTGAAATCC

[0224] SEQ ID NO: 64. CD123-BiTE-IRES-NGFR (CD123 TCE)- Up to IRES - AA:MDWIWRILFLVGAATGAHSQVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPDQ GLEWIGRIDPYDSETHYNQKFKDKAILTVDKSSSTAYMQLSSLTSEDSAVYYCARGNWDDYW GQGTTLTVSSGGGGSGGGGSGGGGSDVQI TQSPSYLAASPGET I T INCRASKS I SKDLAWYQ EKPGKTNKLLIYSGSTLQSGI PSRFSGSGSGTDFTLT I SSLEPEDFAMYYCQQHNKYPYTFG GGTKLE IKSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWI GYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWG QGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQK SGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLT I SSMEAEDAATYYCQQWSSNPLTFGAG TKLELKS

[0225] SEQ ID NO: 67. CD123 scFv clone 292 (in CD123-BiTE-IRES-NGFR (CD123 TCE)) - DNA:CAGGTGCAGCTGCAGCAGCCTGGCGCTGAACTCGTGCGGCCAGGCGCTTCTGTGAAGCTGAG CTGTAAAGCCAGCGGCTACACCTTCACCAGCTACTGGATGAACTGGGTCAAGCAGCGGCCCG ACCAGGGCCTGGAATGGATCGGCAGAATCGACCCCTACGACAGCGAGACACACTACAACCAG AAGTTCAAGGACAAGGCCATCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGCAGCT GTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGGCAACTGGGACGACT ACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGCGGAGGCGGAGGAAGTGGCGGCGGAGGA TCTGGGGGAGGCGGATCTGATGTGCAGATCACCCAGAGCCCCAGCTACCTGGCTGCCTCTCC TGGCGAGACAATCACCATCAACTGCCGGGCCAGCAAGAGCATCTCCAAGGACCTGGCCTGGT ATCAGGAAAAGCCCGGCAAGACCAACAAGCTGCTGATCTACAGCGGCAGCACCCTGCAGAGC GGCATCCCCAGCAGATTTTCCGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCTCCCTGGAACCCGAGGACTTTGCCATGTACTATTGCCAGCAGCACAACAAGTACCCTTACACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGTCC

[0226] SEQ ID NO: 68. CD123 scFv clone 292 (in CD123-BiTE-IRES-NGFR (CD123 TCE)) - AA:QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPDQGLEWIGRIDPYDSETHYNQ KFKDKAILTVDKSSSTAYMQLSSLTSEDSAVYYCARGNWDDYWGQGTTLTVSSGGGGSGGGG SGGGGSDVQI TQSPSYLAASPGET I T INCRASKS I SKDLAWYQEKPGKTNKLLIYSGSTLQS GI PSRFSGSGSGTDFTLT I SSLEPEDFAMYYCQQHNKYPYTFGGGTKLE IKS

[0227] SEQ ID NO: 69. CD123 scFv clone 292 VH (in CD123-BiTE-IRES-NGFR (CD123 TCE)) - DNA:CAGGTGCAGCTGCAGCAGCCTGGCGCTGAACTCGTGCGGCCAGGCGCTTCTGTGAAGCTGAG CTGTAAAGCCAGCGGCTACACCTTCACCAGCTACTGGATGAACTGGGTCAAGCAGCGGCCCG ACCAGGGCCTGGAATGGATCGGCAGAATCGACCCCTACGACAGCGAGACACACTACAACCAG AAGTTCAAGGACAAGGCCATCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGCAGCT GTCCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGGCAACTGGGACGACT ACTGGGGCCAGGGCACAACCCTGACAGTGTCTAGC

[0228] SEQ ID NO: 70. CD123 scFv clone 292 VH (in CD123-BiTE-IRES-NGFR (CD123 TCE)) - AA:QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPDQGLEWIGRIDPYDSETHYNQ KFKDKAILTVDKSSSTAYMQLSSLTSEDSAVYYCARGNWDDYWGQGTTLTVSS

[0229] SEQ ID NO: 71. CD123 scFv clone 292 VL (in CD123-BiTE-IRES-NGFR (CD123 TCE)) - DNA:GATGTGCAGATCACCCAGAGCCCCAGCTACCTGGCTGCCTCTCCTGGCGAGACAATCACCAT CAACTGCCGGGCCAGCAAGAGCATCTCCAAGGACCTGGCCTGGTATCAGGAAAAGCCCGGCA AGACCAACAAGCTGCTGATCTACAGCGGCAGCACCCTGCAGAGCGGCATCCCCAGCAGATTT TCCGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCTCCCTGGAACCCGAGGACTT TGCCATGTACTATTGCCAGCAGCACAACAAGTACCCTTACACCTTCGGCGGAGGCACCAAGC TGGAAATCAAGTCC

[0230] SEQ ID NO: 72. CD123 scFv clone 292 VL (in CD123-BiTE-IRES-NGFR (CD123 TCE)) - AA:DVQI TQSPSYLAASPGET I T INCRASKS I SKDLAWYQEKPGKTNKLLIYSGSTLQSGI PSRF SGSGSGTDFTLT I SSLEPEDFAMYYCQQHNKYPYTFGGGTKLE IKS

[0231] SEQ ID NO: 75. CD3 OKT3(in CD123 TCE))-DNA:GATATCAAACTGCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTC CTGCAAGACTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTAAAACAGAGGCCTG GACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCCGTGGTTATACTAATTACAATCAG AAGTTCAAGGACAAGGCCACATTGACTACAGACAAATCCTCCAGCACAGCCTACATGCAACT GAGCAGCC T GACAT C T GAGGAC T C T GCAGT C TAT TAG T GT GCAAGATAT TAT GAT GAT CAT T ACTGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGTGGTGGTGGTTCT GGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATTCAGCTGACCCAGTCTCCAGCAATCAT GTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGAGCCAGTTCAAGTGTAAGTTACA TGAACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAAGTGGCTTCTGGAGTCCCTTATCGCTTCAGTGGCAGTGGGTCTGGGACCTCATACTCTCTCAC AATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAACAGTGGAGTAGTAACC CGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAATCC

[0232] SEQ ID NO: 76. CD3 OKT3 (in CD123 TCE)) - AA:DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQ KFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGS GGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWI YDTSK VASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKS2. Costimulatory Ligand Domain

[0233] As used herein, a “costimulatory ligand domain”, or “extracellular costimulatory ligand domain” refers to a molecule comprising the extracellular domain of a ligand of a costimulatory receptor. As used herein “Costimulatory receptor” refers to a cell-surface molecule capable of positively modulating activation of T-cell receptor signaling. Many costimulatory receptors and their ligands are known in the art, such as described in Chen and Flies, Nat Rev Immunol. 2013 April; 13(4): 227-242, which is incorporated herein by reference. It is contemplated that the extracellular domain of any costimulatory ligand may be used in certain aspects of the present disclosure.

[0234] Certain aspects of the present disclosure include a protein comprising an extracellular costimulatory ligand domain, nucleic acids encoding an extracellular costimulatory ligand domain, and cells comprising a protein comprising an extracellular costimulatory ligand domain.

[0235] In certain aspects, the extracellular costimulatory ligand domain retains its ability to bind a cognate receptor. In certain aspects, the extracellular costimulatory ligand domain retains its ability to bind and activate a cognate receptor. In certain aspects, binding of the extracellular costimulatory ligand domain to a cognate receptor induces multimerization of cognate receptors, extracellular costimulatory ligands, or proteins containing extracellular costimulatory ligand domains (e.g., DSRs). In certain aspects, binding of the extracellular costimulatory ligand domain to a cognate receptor induces activation of the cognate receptor producing a costimulatory signaling (signal 2).

[0236] In certain aspects, the extracellular costimulatory ligand domain may be derived from tumor necrosis factor ligand superfamily, member 9 (Tnfsf9, also known as 4-1BBL), cluster of differentiation 80 (CD80, also known as B7-1), cluster of differentiation 86 (CD86, also known as B7-2), inducible T cell costimulator ligand (ICOS-L, also known as B7-H2), cluster of differentiation 70 (CD70, also known as CD27L), TNF superfamily member 14 (TNFSF14, also known as LIGHT), CD40 ligand (CD40L), TNF superfamily member 4 (TNFSF4, also known as OX40L), TNF superfamily member 15 (TNFSF15, also known as TL1A), TNF superfamily member 18 (TNFSF18, also known as GITRL), CD30 ligand (CD30L), T cell immunoglobulin and mucin domain containing 4 (TIM4), cluster ofdifferentiation 150 (CD 150, also known as SLAM), cluster of differentiation 48 (CD48, also known as SLAMF2), cluster of differentiation 58 (CD58, also known as LFA-4), cluster of differentiation 155 (CD155, also known as PVR), and / or cluster of differentiation 112 (CD112, also known as Nectin-2). In certain aspects, the extracellular costimulatory ligand domain is derived from 4-1BBL. In some aspects, the extracellular co-stimulatory ligand domain can preferentially bind 4-1BB (also known as CD137), CD28 (also known as Tp44), cytotoxic T- lymphocyte-associated protein 4 (CTLA-4), ICOS (also known as CD278), CD27 (also known as TNFRSF7), TNFRSF14 (also known as CD270), CD40 (also known as TNFRSF5), 0X40 (also known as CD 134), TNFRSF25 (also known as APO-3), TNFRSF18 (also known as GITR), CD30 (also known as TNFRSF8), TIM1 (also known as HAVCR1), SLAM family receptors, 2B4 (also known as CD244), CD2 (also known as LFA-2), DNAM-1 (also known as CD226), and / or TIGIT (also known as VSIG9),

[0237] In some aspects, an extracellular costimulatory ligand domain of 4-1BBL preferentially binds 4-1BB. In some aspects, an extracellular costimulatory ligand domain of CD80 preferentially binds CD28. In some aspects, an extracellular costimulatory ligand domain of CD86 preferentially binds CD28 and / or CTLA-4. In some aspects, an extracellular costimulatory ligand domain of ICOS-L preferentially binds ICOS. In some aspects, an extracellular costimulatory ligand domain of CD70 preferentially binds CD27. In some aspects, an extracellular costimulatory ligand domain of TNFSF14 preferentially binds TNFRSF14. In some aspects, an extracellular costimulatory ligand domain of CD40L preferentially binds CD40. In some aspects, an extracellular costimulatory ligand domain of OX40L preferentially binds 0X40. In some aspects, an extracellular costimulatory ligand domain of TNFSF15 preferentially binds TNFRSF25. In some aspects, an extracellular costimulatory ligand domain of TNFSF18 preferentially binds TNFRSF18. In some aspects, an extracellular costimulatory ligand domain of CD30L preferentially binds CD30. In some aspects, an extracellular costimulatory ligand domain of TIM4 preferentially binds TIM1. In some aspects, an extracellular costimulatory ligand domain of CD 150 preferentially binds SLAM family receptors. In some aspects, an extracellular costimulatory ligand domain of CD48 preferentially binds 2B4. In some aspects, an extracellular costimulatory ligand domain of CD58 preferentially binds CD2. In some aspects, an extracellular costimulatory ligand domain of CD155 preferentially binds DNAM-1 and / or TIGIT.

[0238] In certain aspects, an extracellular costimulatory ligand domain comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 7-8 (extracellular domain of 4-1BBL).

[0239] In certain aspects, an extracellular costimulatory ligand domain comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 97-98 (extracellular domain of OX40-L).

[0240] SEQ ID NO: 7. Extracellular domain of 4-1BBL - DNA:GCCTGCCCCTGGGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGCCGCTAGCCCGCGACT CAGAGAGGGCCCCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGGACCTGAGACAAGGCA TGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCCTTAAGCTGGTACAGC GATCCCGGACTCGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGGA GCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAGCTGCGTAGAGTGG TGGCCGGCGAGGGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAGCCTCTCAGATCGGCC GCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAGCAGCGAGGCTAGAAA CAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGCAGAGACTGGGCGTGC ACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCTG GGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCCTAGAAGCGAG

[0241] SEQ ID NO: 8. Extracellular domain of 4-1BBL - AA:ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYS DPGLAGVSLTGGLSYKEDTKELWAKAGVYYVFFQLELRRWAGEGSGSVSLALHLQPLRSA AGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVL GLFRVTPE I PAGLPSPRSE

[0242] SEQ ID NO: 97. OX40L-Extracellular - DNA:C AAG T GAG C C AC AGAT AC C C T AGAAT T C AGAG CAT C AAG G T G C AG T T C AC C GAG T AC AAGAA GGAGAAGGGC T T CAT CC T GACAT C T CAGAAGGAGGACGAGAT CAT GAAGGT GCAGAACAACA GCGTGATCATCAACTGCGACGGCTTCTACCTGATCAGCCTGAAGGGCTACTTCAGCCAAGAG GTGAACATCAGCCTGCACTATCAGAAGGACGAGGAGCCCCTGTTTCAGCTGAAGAAGGTGAG AAGCGTGAACAGCCTGATGGTGGCTAGCCTGACCTACAAGGACAAGGTGTACCTGAACGTGA CCACCGACAACACAAGCCTGGACGACTTCCACGTGAACGGCGGCGAGCTGATCCTGATCCAT CAGAACCCCGGCGAGTTCTGCGTGCTG

[0243] SEQ ID NO: 98. OX40L-Extracellular - AA:QVSHRYPRIQS IKVQFTEYKKEKGFILTSQKEDE IMKVQNNSVI INCDGFYLI SLKGYFSQE VNI SLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIH QNPGEFCVLC. Transmembrane (TM) Domain

[0244] Polypeptides of the present disclosure may comprise a transmembrane domain. In some aspects, a transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may result in different receptor stability.

[0245] In certain aspects, the transmembrane domain is interposed between the extracellular binding domain and the intracellular cytokine signaling domain.

[0246] In certain aspects, engineered DSR polypeptides may comprise a transmembrane domain sequence. In some aspects, a transmembrane domain sequence may have a functional impact on the expression, localization, and / or activity of a polypeptide comprising the same. In some aspects, a transmembrane domain sequence may be a cluster of differentiation 28 (CD28) derived sequence.

[0247] Any transmembrane domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. In some aspects, the transmembrane domain is derived from cluster of differentiation 28 (CD28), cluster of differentiation 8 (CD8), cluster of differentiation (CD25), cluster of differentiation 4 (CD4), cluster of differentiation 3 zeta (CD3Q, cluster of differentiation 134 (CD134), or cluster of differentiation 7 (CD7).

[0248] In certain aspects, a transmembrane domain comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 13-18 (CD28 transmembrane domain).

[0249] In certain aspects, a transmembrane domain comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 5 or 6.

[0250] SEQ ID NO: 5. TM domain in 4-1 BBL DNA:TGGGCCTTGGTAGCCGGCCTGCTCCTTCTGCTCCTCCTGGCCGCCGCTTGTGCCGTGTTTCT T

[0251] SEQ ID NO: 6. TM domain in 4-1 BBL AA:WALVAGLLLLLLLAAACAVFL

[0252] SEQ ID NO: 13. CD28 Transmembrane Domain - DNA:TTTTGGGTGCTGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCTTACTGGTGACTGTTGC CTTTATTATTTTTTGGGTT

[0253] SEQ ID NO: 14. CD28 Transmembrane Domain - AA: FWVLVWGGVLACYSLLVTVAFI I FWVD. Cytokine Signaling Domain

[0254] As used herein, a “cytokine signaling domain”, or “cytokine-like signaling domain”, or “endodomain” refers to a molecule comprising the intracellular domain of a cytokine receptor or a protein capable of cytokine-like signaling. Many cytokine receptors areknown in the art, such as described in Morris et al., Protein Sci. 2018 Dec;27(12): 1984-2009, which is incorporated herein by reference. It is contemplated that the intracellular domain of any protein capable of cytokine or cytokine-like signaling, including cytokine receptors, may be used in certain aspects of the present disclosure.

[0255] Certain aspects of the present disclosure include a protein comprising an intracellular cytokine signaling domain, nucleic acids encoding an intracellular cytokine signaling domain, and cells comprising a protein comprising an intracellular cytokine signaling domain.

[0256] In certain aspects, the intracellular cytokine signaling domain retains its ability to produce cytokine signaling (signal 3). In certain aspects, the intracellular cytokine signaling domain retains its ability to interact with other cytokine receptors. In certain aspects, the intracellular cytokine signaling domain retains its ability to interact with other molecules involved in cytokine signaling, such as JAK kinases and STAT transcription factors, for example. In certain aspects, the intracellular cytokine signaling domain retains its ability to interact with one or more of the same intracellular cytokine signaling domains to initiate cytokine or cytokine-like signaling (signal 3).

[0257] In certain aspects, the intracellular cytokine signaling domain may be derived from interleukin 7 receptor (IL7R, also known as IL-7Ra, or CD127), interleukin 2 receptor subunit beta (IL-2RP, also known as CD122, or IL-15RP), myeloid differentiation primary response protein 88 (MyD88), thrombopoietin receptor (cMPL, also known as TPO-R), interleukin 2 receptor subunit gamma (IL-2Ry, also known as CD 132, or yc), interleukin 1 receptor type 1 (IL-1R), interleukin 12 receptor subunit beta 1 (IL-12RP1), interleukin 12 receptor subunit beta 2 (IL-12RP2), interleukin 6 cytokine family signal transducer (IL6ST, also known as gpl30, or CD 130), interleukin 4 receptor (IL-4R), interleukin 9 receptor (IL-9R), interleukin 10 receptor (IL-10R), interleukin 13 receptor (IL-13R), interleukin 15 receptor (IL-15R), interleukin 21 receptor (IL-21R), interleukin 23 receptor (IL-23R), interferon alpha and beta receptor subunit 2 (IFNAR2, also known as IFNa / pR, or IFN-R), interleukin 10 receptor (IL- 10R), interleukin 6 receptor (IL-6R), interleukin 11 receptor (IL-11R), ciliary neurotrophic factor receptor (CNTF-R), oncostatin M receptor (OSM-R), LIF receptor subunit alpha (LIF- R), calcitonin receptor (CT-R), interleukin 3 receptor (IL-3R), interleukin 5 receptor (IL-5R), granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R), growth hormone receptor (GH-R), erythropoietin receptor (EPO-R), and / or prolactin receptor (PRL-R). In certain aspects, the intracellular cytokine signaling domain is derived from IL-7Ra (CD127),IL-2RP (CD122 / IL-15RP), MyD88, and / or cMPL (TPO-R). In certain aspects, the intracellular cytokine signaling domain is derived from cMPL (TPO-R).

[0258] In certain aspects, an intracellular cytokine signaling domain comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 19-20 (intracellular cytokine signaling domain of IL-7Ra).

[0259] In certain aspects, an intracellular cytokine signaling domain comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 23-24 (intracellular cytokine signaling domain of IL-2RP).

[0260] In certain aspects, an intracellular cytokine signaling domain comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 27-28 (intracellular cytokine signaling domain of MyD88).

[0261] In certain aspects, an intracellular cytokine signaling domain comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 31-32 (intracellular cytokine signaling domain of cMPL).

[0262] SEQ ID NO: 19. IL7Ra (in 4-lBBL / IL7Ra) - DNA:AAGAAAAGAATCAAGCCCATTGTCTGGCCAAGCCTGCCCGACCACAAGAAAACCCTGGAGCA TCTGTGCAAGAAACCCAGGAAAAACCTGAATGTGAGCTTCAACCCTGAATCCTTTCTGGATT GTCAGATCCACAGGGTGGACGATATTCAGGCTCGCGACGAGGTCGAAGGATTCCTGCAGGAT ACATTTCCCCAGCAGCTGGAGGAATCCGAGAAGCAGCGACTGGGAGGAGACGTGCAGTCTCC TAACTGCCCAAGTGAGGATGTGGTCATCACCCCTGAATCTTTCGGCCGAGACTCAAGCCTGA CATGCCTGGCAGGGAATGTCAGTGCATGTGACGCCCCTATCCTGTCCTCTAGTCGGTCACTG GATTGTAGAGAGAGCGGAAAGAACGGCCCACATGTGTACCAGGATCTGCTGCTGTCCCTGGG GACTACCAATTCTACCCTGCCCCCTCCATTTAGTCTGCAGTCAGGAATCCTGACTCTGAATC CAGTGGCCCAGGGCCAGCCCATTCTGACCAGCCTGGGCAGCAATCAGGAAGAGGCATACGTC ACAAT G T C T AGC T T T TAT CAGAAT GAG

[0263] SEQ ID NO: 20. IL7Ra (in 4-lBBL / IL7Ra) - AA:KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQD TFPQQLEESEKQRLGGDVQSPNCPSEDWITPESFGRDSSLTCLAGNVSACDAPILSSSRSL DCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYV TMSSFYQNQ

[0264] SEQ ID NO: 23. IL2R|J (in 4-1BBL / IL2RP) - DNA:AAC T G T C GAAAC AC T G G C C C T T G G C T GAAAAAAG T T C T AAAG T G T AAT AC AC C T GAC C C AAG TAAGTTTTTCTCTCAGTTATCCAGCGAGCATGGAGGAGATGTGCAGAAATGGCTGAGTTCCC CTTTCCCGTCCTCATCCTTTAGCCCGGGTGGTTTAGCTCCAGAGATCAGTCCTCTGGAGGTT C T G GAGAGAGAC AAG G T T AC T C AAT TGCTCCCCCT GAAT AC AGAC GCTTACCTGT C AC T T C A AGAGCTGCAGGGGCAGGACCCCACTCATCTCGTA

[0265] SEQ ID NO: 24. IL2R|J (in 4-1BBL / IL2RP) - AA:NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSS FSPGGLAPE I SPLEV LERDKVTQLLPLNTDAYLSLQELQGQDPTHLV

[0266] SEQ ID NO: 27. MyD88 (4-lBBL / MyD88) - DNA:ATGGCAGCAGGGGGCCCCGGCGCAGGCTCAGCGGCCCCGGTCTCTTCCACATCATCACTGCC TCTCGCCGCACTCAACATGCGTGTTCGCCGGCGGCTGTCACTGTTTCTCAATGTCAGGACCC AAGTGGCCGCCGACTGGACCGCTTTAGCTGAGGAGATGGATTTTGAGTATTTAGAAATCCGA CAACTGGAGACTCAGGCTGATCCTACCGGGAGGTTACTTGATGCCTGGCAAGGTAGACCAGG AGCAAGCGTGGGACGACTGCTGGAACTGCTGACTAAGCTGGGACGCGATGACGTCCTACTGG AACTTGGACCGAGTATCGAGGAAGATTGCCAGAAATATATTCTGAAGCAGCAACAGGAGGAA GCAGAGAAACCACTGCAGGTGGCAGCGGTGGACTCGTCCGTCCCCCGCACGGCCGAGCTAGC TGGAATTACTACGCTGGATGACCCTCTCGGCCACATGCCAGAGAGGTTTGACGCCTTTATAT GCTATTGCCCCTCCGACATT

[0267] SEQ ID NO: 28. MyD88 (4-lBBL / MyD88) - AA:MAAGG P GAG S AAP VS S T S S L P LAALNMRVRRRL S L FLNVRT QVAADW T ALAE EMD EE YLE I R QLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDVLLELGPS IEEDCQKYILKQQQEE AEKPLQVAAVDSSVPRTAELAGI TTLDDPLGHMPERFDAFICYCPSDI

[0268] SEQ ID NO: 31. cMPL (in 4-lBBL / cMPL) - DNA:AGATGGCAGTTCCCCGCGCACTACAGAAGGCTACGACACGCCCTGTGGCCCTCTCTGCCCGA CTTACACCGCGTTCTTGGCCAGTACCTTAGAGACACTGCCGCTTTGTCCCCACCCAAAGCTA CCGTATCGGATACCTGCGAAGAGGTGGAACCCAGCCTGCTTGAGATATTGCCCAAATCTAGT GAGCGGACGCCCCTACCCTTATGTTCCTCCCAGGCCCAGATGGATTATAGACGCCTGCAGCC CAGTTGTCTCGGCACAATGCCTCTATCCGTGTGTCCTCCTATGGCCGAAAGTGGATCTTGTT GCACCACTCACATCGCAAATCACAGCTACCTGCCACTCTCATACTGGCAACAACCA

[0269] SEQ ID NO: 32. cMPL (in 4-lBBL / cMPL) - AA:RWQFPAHYRRLRHALWPSLPDLHRVLGQYLRDTAALSPPKATVSDTCEEVEPSLLE ILPKSS ERTPLPLCSSQAQMDYRRLQPSCLGTMPLSVCPPMAESGSCCTTHIANHSYLPLSYWQQPE. Signal peptide

[0270] Polypeptides of the present disclosure may comprise a signal peptide. A “signal peptide” refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g., to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface. In some aspects, a signal peptide directs a nascent protein into the endoplasmic reticulum. This is essential if a receptor is to be glycosylated and anchored in the cell membrane. Generally, but not exclusively, the signal peptide natively attached to the amino-terminal most component is used (e.g., in an scFv with orientation light chain - linker - heavy chain, the native signal of the light-chain is used).

[0271] In certain aspects, engineered DSR polypeptides may comprise one or more signal peptides. In some aspects, a signal peptide may have a functional impact on the expression, localization, and / or activity of a polypeptide comprising the same. In some aspects, a signal peptide sequence may be a CD8 subunit alpha (CD8a) derived sequence.In some aspects, the signal peptide is cleaved after passage of the endoplasmic reticulum (ER), i.e., is a cleavable signal peptide. In some aspects, a signal peptide comprises a restriction site at the carboxyl end to facilitate cleavage.

[0272] In certain aspects, a signal peptide comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 11-12 (signal peptide of CD8a).

[0273] In certain aspects, a signal peptide comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 35-36, 51-52, 59-60, or 65-66.

[0274] SEQ ID NO: 11. CD8a Signal Peptide DNA:ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCC G

[0275] SEQ ID NO: 12. CD8a Signal Peptide - AA:MALPVTALLLPLALLLHAARP

[0276] SEQ ID NO: 35. Signal Peptide DNA:ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCTCTAG A

[0277] SEQ ID NO: 36. Signal Peptide AA:MEFGLSWLFLVAILKGVQCSR

[0278] SEQ ID NO: 51. Igk leader- DNA:ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGT

[0279] SEQ ID NO: 52. Igk leader- AA:METDTLLLWVLLLWVPGSTG

[0280] SEQ ID NO: 59. Leader- DNA:ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCC G

[0281] SEQ ID NO: 60. Leader- AA:MALPVTALLLPLALLLHAARP

[0282] SEQ ID NO: 65. Signal peptide for TCE - DNA:ATGGATTGGATCTGGCGCATCCTGTTTCTCGTGGGAGCCGCCACAGGCGCCCATTCT

[0283] SEQ ID NO: 66. Signal peptide for TCE - AA:MDW I WR I L FL VGAAT GAH SII. Nucleic Acids

[0284] Aspects of the present disclosure include nucleic acids. In certain aspects, nucleic acid sequences can exist in a variety of instances such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof, chimeric antigen receptor, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, anti-sense nucleic acids for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing described herein. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).

[0285] The term “polynucleotide” refers to a nucleic acid molecule that either is recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or noncoding sequences may, but need not, be present within a polynucleotide.

[0286] As will be understood by those in the art, the term “nucleic acid” or “polynucleotide” encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein.

[0287] In certain aspects, there are polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.

[0288] The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids can be any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and / or can comprise one or more additional sequences, for example, regulatory sequences, and / or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.

[0289] In certain aspects, a polynucleotide can comprise sequences encoded by any one or more of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 95, 97, and / or 100-101.

[0290] In certain aspects, an engineered DSR can comprise sequences encoded by any one or more of SEQ ID NOs: 17, 21, 25, 29, 82-85, or 89-92.

[0291] In certain aspects, an engineered DSR encoding polynucleotide comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 17, 82, or 89 v(4-lBBL / IL7Ra).

[0292] In certain aspects, an engineered DSR encoding polynucleotide comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 21, 83, or 90 (4-1BBL / IL2RP).

[0293] In certain aspects, an engineered DSR encoding polynucleotide comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 25, 84, or 91 (4-lBBL / MyD88).

[0294] In certain aspects, an engineered DSR encoding polynucleotide comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 29, 85, or 92 (4-lBBL / cMPL).

[0295] SEQ ID NO: 17. 4-lBBL-CD28TM-IL7Ra (4-lBBL / IL7Ra) - DNA:ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCC GGCCTGCCCCTGGGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGCCGCTAGCCCGCGAC TCAGAGAGGGCCCCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGGACCTGAGACAAGGC ATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCCTTAAGCTGGTACAG CGATCCCGGACTCGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGG AGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAGCTGCGTAGAGTG GTGGCCGGCGAGGGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAGCCTCTCAGATCGGC CGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAGCAGCGAGGCTAGAA ACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGCAGAGACTGGGCGTG CACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCT GGGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCCTAGAAGCGAGACGC GTTTTTGGGTGCTGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCTTACTGGTGACTGTT GCCTTTATTATTTTTTGGGTTAAGAAAAGAATCAAGCCCATTGTCTGGCCAAGCCTGCCCGA CCACAAGAAAACCCTGGAGCATCTGTGCAAGAAACCCAGGAAAAACCTGAATGTGAGCTTCA ACCCTGAATCCTTTCTGGATTGTCAGATCCACAGGGTGGACGATATTCAGGCTCGCGACGAG GTCGAAGGATTCCTGCAGGATACATTTCCCCAGCAGCTGGAGGAATCCGAGAAGCAGCGACT GGGAGGAGACGTGCAGTCTCCTAACTGCCCAAGTGAGGATGTGGTCATCACCCCTGAATCTT TCGGCCGAGACTCAAGCCTGACATGCCTGGCAGGGAATGTCAGTGCATGTGACGCCCCTATC CTGTCCTCTAGTCGGTCACTGGATTGTAGAGAGAGCGGAAAGAACGGCCCACATGTGTACCA GGATCTGCTGCTGTCCCTGGGGACTACCAATTCTACCCTGCCCCCTCCATTTAGTCTGCAGT CAGGAATCCTGACTCTGAATCCAGTGGCCCAGGGCCAGCCCATTCTGACCAGCCTGGGCAGC AAT CAGGAAGAGGCATACGT CACAAT GT C TAGC T T T TAT CAGAAT GAG

[0296] SEQ ID NO: 21. 4-1BBL-CD28TM-IL2RP (4-1BBL / IL2RP) - DNA:ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCC GGCCTGCCCCTGGGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGCCGCTAGCCCGCGAC TCAGAGAGGGCCCCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGGACCTGAGACAAGGC ATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCCTTAAGCTGGTACAG CGATCCCGGACTCGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGG AGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAGCTGCGTAGAGTG GTGGCCGGCGAGGGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAGCCTCTCAGATCGGC CGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAGCAGCGAGGCTAGAA ACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGCAGAGACTGGGCGTG CACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCT GGGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCCTAGAAGCGAGACGC GTTTTTGGGTGCTGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCTTACTGGTGACTGTT GCCTTTATTATTTTTTGGGTTAACTGTCGAAACACTGGCCCTTGGCTGAAAAAAGTTCTAAA G T G T AAT AC AC C T GAC C C AAG T AAG TTTTTCTCT C AG T T AT C C AG C GAG C AT G GAG GAGAT GTGCAGAAATGGCTGAGTTCCCCTTTCCCGTCCTCATCCTTTAGCCCGGGTGGTTTAGCTCCA GAGATCAGTCCTCTGGAGGTTCTGGAGAGAGACAAGGTTACTCAATTGCTCCCCCTGAATAC AGACGCTTACCTGTCACTTCAAGAGCTGCAGGGGCAGGACCCCACTCATCTCGTA

[0297] SEQ ID NO: 25. 4-lBBL-CD28TM-MyD88 (4-lBBL / MyD88) - DNA:ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCC GGCCTGCCCCTGGGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGCCGCTAGCCCGCGAC TCAGAGAGGGCCCCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGGACCTGAGACAAGGC ATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCCTTAAGCTGGTACAG CGATCCCGGACTCGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGG AGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAGCTGCGTAGAGTG GTGGCCGGCGAGGGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAGCCTCTCAGATCGGC CGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAGCAGCGAGGCTAGAA ACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGCAGAGACTGGGCGTG CACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCT GGGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCCTAGAAGCGAGACGC GTTTTTGGGTGCTGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCTTACTGGTGACTGTT GCCTTTATTATTTTTTGGGTTATGGCAGCAGGGGGCCCCGGCGCAGGCTCAGCGGCCCCGGT CTCTTCCACATCATCACTGCCTCTCGCCGCACTCAACATGCGTGTTCGCCGGCGGCTGTCAC TGTTTCTCAATGTCAGGACCCAAGTGGCCGCCGACTGGACCGCTTTAGCTGAGGAGATGGAT TTTGAGTATTTAGAAATCCGACAACTGGAGACTCAGGCTGATCCTACCGGGAGGTTACTTGA TGCCTGGCAAGGTAGACCAGGAGCAAGCGTGGGACGACTGCTGGAACTGCTGACTAAGCTGG GACGCGATGACGTCCTACTGGAACTTGGACCGAGTATCGAGGAAGATTGCCAGAAATATATT CTGAAGCAGCAACAGGAGGAAGCAGAGAAACCACTGCAGGTGGCAGCGGTGGACTCGTCCGT CCCCCGCACGGCCGAGCTAGCTGGAATTACTACGCTGGATGACCCTCTCGGCCACATGCCAG AGAGGTTTGACGCCTTTATATGCTATTGCCCCTCCGACATT

[0298] SEQ ID NO: 29. 4-lBBL-CD28TM-cMPL (4-lBBL / cMPL) - DNA:ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCC GGCCTGCCCCTGGGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGCCGCTAGCCCGCGAC TCAGAGAGGGCCCCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGGACCTGAGACAAGGC ATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCCTTAAGCTGGTACAG CGATCCCGGACTCGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGG AGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAGCTGCGTAGAGTG GTGGCCGGCGAGGGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAGCCTCTCAGATCGGC CGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAGCAGCGAGGCTAGAAACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGCAGAGACTGGGCGTGCACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCCTAGAAGCGAGACGCGTTTTTGGGTGCTGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCTTACTGGTGACTGTTGCCTTTATTATTTTTTGGGTTAGATGGCAGTTCCCCGCGCACTACAGAAGGCTACGACACGCCCTGTGGCCCTCTCTGCCCGACTTACACCGCGTTCTTGGCCAGTACCTTAGAGACACTGCCGCTTTGTCCCCACCCAAAGCTACCGTATCGGATACCTGCGAAGAGGTGGAACCCAGCCTGCTTGAGATATTGCCCAAATCTAGTGAGCGGACGCCCCTACCCTTATGTTCCTCCCAGGCCCAGATGGATTATAGACGCCTGCAGCCCAGTTGTCTCGGCACAATGCCTCTATCCGTGTGTCCTCCTATGGCCGAAAGTGGATCTTGTTGCACCACTCACATCGCAAATCACAGCTACCTGCCACTCTCA TACTGGCAACAACCA

[0299] SEQ ID NO: 82. 5'LTR-4-lBBL-CD28TM-IL7Ra(4-lBBL / IL7Ra)-3'LTR- DNA:G C GAG TCATATTAATTACTAGT C AAT TAGTTGATTTTTATTTTT GAG AT AT AC AT G T GAAAG ACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAAT ACATAAC T GAGAATAGAAAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATAT GG GCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAA CAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCA AGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTT TCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCG CTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTC ACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCC TCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATT GACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCC AGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGAT TGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTG TATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGAC GTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTT AGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACC TAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCG CGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTC TGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGA AAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACC TTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCG AGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACC AGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCC TTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACC TCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCC CCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCT GAG AT GAG AAGAG T T AC T AAC AG CCCCTCTCTC C AAG C T C AC T TAG AG GCTCTCTACTTAGT CCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGG TACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTA GAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGA CGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCAT AACTCGAGGCCACGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTC CACGCCGCCAGGCCGGCCTGCCCCTGGGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGC CGCTAGCCCGCGACTCAGAGAGGGCCCCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGG ACCTGAGACAAGGCATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCC TTAAGCTGGTACAGCGATCCCGGACTCGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAA GGAGGACACCAAGGAGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAGCTGCGTAGAGTGGTGGCCGGCGAGGGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAG CCTCTCAGATCGGCCGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAG CAGCGAGGCTAGAAACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGC AGAGACTGGGCGTGCACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAA GGCGCCACCGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCC TAGAAGCGAGACGCGTTTTTGGGTGCTGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCT TACTGGTGACTGTTGCCTTTATTATTTTTTGGGTTAAGAAAAGAATCAAGCCCATTGTCTGG CCAAGCCTGCCCGACCACAAGAAAACCCTGGAGCATCTGTGCAAGAAACCCAGGAAAAACCT GAATGTGAGCTTCAACCCTGAATCCTTTCTGGATTGTCAGATCCACAGGGTGGACGATATTC AGGCTCGCGACGAGGTCGAAGGATTCCTGCAGGATACATTTCCCCAGCAGCTGGAGGAATCC GAGAAGCAGCGACTGGGAGGAGACGTGCAGTCTCCTAACTGCCCAAGTGAGGATGTGGTCAT CACCCCTGAATCTTTCGGCCGAGACTCAAGCCTGACATGCCTGGCAGGGAATGTCAGTGCAT GTGACGCCCCTATCCTGTCCTCTAGTCGGTCACTGGATTGTAGAGAGAGCGGAAAGAACGGC CCACATGTGTACCAGGATCTGCTGCTGTCCCTGGGGACTACCAATTCTACCCTGCCCCCTCC ATTTAGTCTGCAGTCAGGAATCCTGACTCTGAATCCAGTGGCCCAGGGCCAGCCCATTCTGA CCAGCCTGGGCAGCAATCAGGAAGAGGCATACGTCACAATGTCTAGCTTTTATCAGAATCAG TGAGCATGCAACCTCGATCCGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAG GCTCTAGTTTT GAG T C AAC AAT AT GAG GAG C T GAAG C C T AT AGAG TAG GAG C C AT AGAT AAA ATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTG GCAAGC TAGC T TAAGTAACGCCAT T T T GCAAGGCAT GGAAAAAT AGAT AAC T GAGAAT AGAG AAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATAT GGGCCAAACAGGATAT C T GT GGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAA ACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAG ATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGA CCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGC GCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCT CCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGA CTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTT

[0300] SEQ ID NO: 83. 5'LTR- 4-1BBL-CD28TM-IL2RP (4-1BBL / IL2RP) -3'LTR - DNA:G C GAG TCATATTAATTACTAGT C AAT TAGTTGATTTTTATTTTT GAG AT AT AC AT G T GAAAG ACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAAT ACATAAC T GAGAATAGAAAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATAT GG GCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAA CAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCA AGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTT TCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCG CTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTC ACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCC TCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATT GACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCC AGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGAT TGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTG TATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGAC GTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTT AGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACC TAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCG CGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTC TGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACC TTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCG AGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACC AGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCC TTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACC TCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCC CCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCT GAG AT GAG AAGAG T T AC T AAC AG CCCCTCTCTC C AAG C T C AC T TAG AG GCTCTCTACTTAGT CCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGG TACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTA GAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGA CGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCAT AACTCGAGGCCACGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTC CACGCCGCCAGGCCGGCCTGCCCCTGGGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGC CGCTAGCCCGCGACTCAGAGAGGGCCCCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGG ACCTGAGACAAGGCATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCC TTAAGCTGGTACAGCGATCCCGGACTCGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAA GGAGGACACCAAGGAGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG AGCTGCGTAGAGTGGTGGCCGGCGAGGGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAG CCTCTCAGATCGGCCGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAG CAGCGAGGCTAGAAACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGC AGAGACTGGGCGTGCACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAA GGCGCCACCGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCC TAGAAGCGAGACGCGTTTTTGGGTGCTGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCT TACTGGTGACTGTTGCCTTTATTATTTTTTGGGTTAACTGTCGAAACACTGGCCCTTGGCTG AAAAAAG T T C T AAAG T G T AAT AC AC C T GAC C C AAG T AAG T T T T T C T C T C AG T T AT C C AG C GA GCATGGAGGAGATGTGCAGAAATGGCTGAGTTCCCCTTTCCCGTCCTCATCCTTTAGCCCGG GTGGTTTAGCTCCAGAGATCAGTCCTCTGGAGGTTCTGGAGAGAGACAAGGTTACTCAATTG CTCCCCCTGAATACAGACGCTTACCTGTCACTTCAAGAGCTGCAGGGGCAGGACCCCACTCA TCTCGTATAGGCATGCAACCTCGATCCGGATTAGTCCAATTTGTTAAAGACAGGATATCAGT GGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCAT AGATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGT AGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAG AATAGAGAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATAT GGGCCAAACAGGA TATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATAT GGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGG TCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCC CCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTC TGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGC CAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTG CATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTC AGCGGGGGTCTTT

[0301] SEQ ID NO: 84. 5'LTR- 4-lBBL-CD28TM-MyD88 (4-lBBL / MyD88) -3'LTR - DNA:G C GAG TCATATTAATTACTAGT C AAT TAGTTGATTTTTATTTTT GAG AT AT AC AT G T GAAAG ACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAAT ACATAAC T GAGAATAGAAAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATAT GG GCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAA CAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCA AGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCC TCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATT GACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCC AGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGAT TGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTG TATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGAC GTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTT AGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACC TAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCG CGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTC TGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGA AAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACC TTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCG AGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACC AGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCC TTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACC TCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCC CCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCT GAG AT GAG AAGAG T T AC T AAC AG CCCCTCTCTC C AAG C T GAG T TAG AG GCTCTCTACTTAGT CCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGG TACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTA GAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGA CGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCAT AACTCGAGGCCACGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTC CACGCCGCCAGGCCGGCCTGCCCCTGGGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGC CGCTAGCCCGCGACTCAGAGAGGGCCCCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGG ACCTGAGACAAGGCATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCC TTAAGCTGGTACAGCGATCCCGGACTCGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAA GGAGGACACCAAGGAGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG AGCTGCGTAGAGTGGTGGCCGGCGAGGGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAG CCTCTCAGATCGGCCGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAG CAGCGAGGCTAGAAACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGC AGAGACTGGGCGTGCACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAA GGCGCCACCGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCC TAGAAGCGAGACGCGTTTTTGGGTGCTGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCT TACTGGTGACTGTTGCCTTTATTATTTTTTGGGTTATGGCAGCAGGGGGCCCCGGCGCAGGC TCAGCGGCCCCGGTCTCTTCCACATCATCACTGCCTCTCGCCGCACTCAACATGCGTGTTCG CCGGCGGCTGTCACTGTTTCTCAATGTCAGGACCCAAGTGGCCGCCGACTGGACCGCTTTAG CTGAGGAGATGGATTTTGAGTATTTAGAAATCCGACAACTGGAGACTCAGGCTGATCCTACC GGGAGGTTACTTGATGCCTGGCAAGGTAGACCAGGAGCAAGCGTGGGACGACTGCTGGAACT GCTGACTAAGCTGGGACGCGATGACGTCCTACTGGAACTTGGACCGAGTATCGAGGAAGATT GCCAGAAATATATTCTGAAGCAGCAACAGGAGGAAGCAGAGAAACCACTGCAGGTGGCAGCG GTGGACTCGTCCGTCCCCCGCACGGCCGAGCTAGCTGGAATTACTACGCTGGATGACCCTCT CGGCCACATGCCAGAGAGGTTTGACGCCTTTATATGCTATTGCCCCTCCGACATTTGAGCAT GCAACCTCGATCCGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTAG T T T T GAG T C AAC AAT AT C AC C AG C T GAAG C C T AT AGAG TAG GAG C C AT AGAT AAAAT AAAAG ATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCT AGC T TAAGTAACGCCAT T T T GCAAGGCAT GGAAAAATACATAAC T GAGAATAGAGAAGT T CA GATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTT

[0302] SEQ ID NO: 85. 5'LTR- 4-lBBL-CD28TM-cMPL (4-lBBL / cMPL) -3'LTR - DNA:G C GAG TCATATTAATTACTAGT C AAT TAGTTGATTTTTATTTTT GAG AT AT AC AT G T GAAAG ACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAAT ACATAAC T GAGAATAGAAAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATAT GG GCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAA CAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCA AGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTT TCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCG CTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTC ACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCC TCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATT GACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCC AGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGAT TGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTG TATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGAC GTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTT AGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACC TAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCG CGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTC TGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGA AAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACC TTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCG AGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACC AGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCC TTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACC TCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCC CCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCT GAG AT GAG AAGAG T T AC T AAC AG CCCCTCTCTC C AAG C T C AC T TAG AG GCTCTCTACTTAGT CCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGG TACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTA GAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGA CGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCAT AACTCGAGGCCACGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTC CACGCCGCCAGGCCGGCCTGCCCCTGGGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGC CGCTAGCCCGCGACTCAGAGAGGGCCCCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGG ACCTGAGACAAGGCATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCC TTAAGCTGGTACAGCGATCCCGGACTCGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAA GGAGGACACCAAGGAGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGG AGCTGCGTAGAGTGGTGGCCGGCGAGGGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAG CCTCTCAGATCGGCCGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAG CAGCGAGGCTAGAAACAGCGCCTTCGGCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGC AGAGACTGGGCGTGCACCTGCACACCGAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCC TAGAAGCGAGACGCGTTTTTGGGTGCTGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCT TACTGGTGACTGTTGCCTTTATTATTTTTTGGGTTAGATGGCAGTTCCCCGCGCACTACAGA AGGCTACGACACGCCCTGTGGCCCTCTCTGCCCGACTTACACCGCGTTCTTGGCCAGTACCT TAGAGACACTGCCGCTTTGTCCCCACCCAAAGCTACCGTATCGGATACCTGCGAAGAGGTGG AACCCAGCCTGCTTGAGATATTGCCCAAATCTAGTGAGCGGACGCCCCTACCCTTATGTTCC TCCCAGGCCCAGATGGATTATAGACGCCTGCAGCCCAGTTGTCTCGGCACAATGCCTCTATC CGTGTGTCCTCCTATGGCCGAAAGTGGATCTTGTTGCACCACTCACATCGCAAATCACAGCT ACCTGCCACTCTCATACTGGCAACAACCATGAGCATGCAACCTCGATCCGGATTAGTCCAAT TTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCTG AAG C C T AT AGAG TAG GAG C C AT AGAT AAAAT AAAAGAT TTTATTTAGTCTC C AGAAAAAG G G GGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGG CAT GGAAAAAT AGAT AAG T GAGAATAGAGAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACA GCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAG AACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCG GCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAAC CATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAAC CAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCC CACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTAT CCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCC TCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTT

[0303] SEQ ID NO: 89. X-4-lBBL-CD28TM-IL7Ra (4-lBBL / IL7Ra-)-X - DNA:TTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCCAGGGACCACCGACCCACCACCGGGAG GTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTAT GCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTG ACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTT TGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTTAGGACTCTTTGGTGCACCCCCCTTAG AGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAA TTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCT GTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAAAATATGGGCCCGGGCTAGCCTG TTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAAC CAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTT TAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCA AGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAA GCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCC TCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCT CCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCCCCATATGGCCATATGAGATCTTATAT GGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCTGACATGACAAGAGTTACTAACAGCCC CTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGG CGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGAC ACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACAC AGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCG CCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATAACTCGAGGCCACGATGGCCTTACCA GTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCCTGCCCCTG GGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGCCGCTAGCCCGCGACTCAGAGAGGGCC CCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGGACCTGAGACAAGGCATGTTCGCTCAG CTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCCTTAAGCTGGTACAGCGATCCCGGACT CGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGGAGCTGGTGGTGG CCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAGCTGCGTAGAGTGGTGGCCGGCGAG GGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAGCCTCTCAGATCGGCCGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAGCAGCGAGGCTAGAAACAGCGCCTTCG GCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGCAGAGACTGGGCGTGCACCTGCACACC GAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCTGGGCCTGTTCAG AGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCCTAGAAGCGAGACGCGTTTTTGGGTGC TGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCTTACTGGTGACTGTTGCCTTTATTATT TTTTGGGTTAAGAAAAGAATCAAGCCCATTGTCTGGCCAAGCCTGCCCGACCACAAGAAAAC CCTGGAGCATCTGTGCAAGAAACCCAGGAAAAACCTGAATGTGAGCTTCAACCCTGAATCCT TTCTGGATTGTCAGATCCACAGGGTGGACGATATTCAGGCTCGCGACGAGGTCGAAGGATTC CTGCAGGATACATTTCCCCAGCAGCTGGAGGAATCCGAGAAGCAGCGACTGGGAGGAGACGT GCAGTCTCCTAACTGCCCAAGTGAGGATGTGGTCATCACCCCTGAATCTTTCGGCCGAGACT CAAGCCTGACATGCCTGGCAGGGAATGTCAGTGCATGTGACGCCCCTATCCTGTCCTCTAGT CGGTCACTGGATTGTAGAGAGAGCGGAAAGAACGGCCCACATGTGTACCAGGATCTGCTGCT GTCCCTGGGGACTACCAATTCTACCCTGCCCCCTCCATTTAGTCTGCAGTCAGGAATCCTGA CTCTGAATCCAGTGGCCCAGGGCCAGCCCATTCTGACCAGCCTGGGCAGCAATCAGGAAGAG GCATACGTCACAATGTCTAGCTTTTATCAGAATCAGTGAGCATGCAACCTCGATCCGGATTA G T C C AAT T T G T T AAAGAC AG GAT AT GAG T G G T C GAG GCTCTAGTTTT GAG T C AAC AAT AT GA C GAG C T GAAG C C T AT AGAG TAG GAG C C AT AGAT AAAAT AAAAGAT TTTATTTAGTCTC GAGA AAAAGGGGGGAA

[0304] SEQ ID NO: 90. X- 4-1BBL-CD28TM-IL2RP (4-1BBL / IL2RP) -X - DNA:TTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCCAGGGACCACCGACCCACCACCGGGAG GTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTAT GCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTG ACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTT TGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTTAGGACTCTTTGGTGCACCCCCCTTAG AGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAA TTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCT GTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAAAATATGGGCCCGGGCTAGCCTG TTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAAC CAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTT TAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCA AGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAA GCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCC TCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCT CCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCCCCATATGGCCATATGAGATCTTATAT GGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCTGACATGACAAGAGTTACTAACAGCCC CTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGG CGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGAC ACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACAC AGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCG CCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATAACTCGAGGCCACGATGGCCTTACCA GTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCCTGCCCCTG GGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGCCGCTAGCCCGCGACTCAGAGAGGGCC CCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGGACCTGAGACAAGGCATGTTCGCTCAG CTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCCTTAAGCTGGTACAGCGATCCCGGACT CGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGGAGCTGGTGGTGG CCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAGCTGCGTAGAGTGGTGGCCGGCGAG GGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAGCCTCTCAGATCGGCCGCCGGCGCCGC CGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAGCAGCGAGGCTAGAAACAGCGCCTTCG GCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGCAGAGACTGGGCGTGCACCTGCACACC GAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCCTAGAAGCGAGACGCGTTTTTGGGTGC TGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCTTACTGGTGACTGTTGCCTTTATTATT TTTTGGGTTAACTGTCGAAACACTGGCCCTTGGCTGAAAAAAGTTCTAAAGTGTAATACACC TGACCCAAGTAAGTTTTTCTCTCAGTTATCCAGCGAGCATGGAGGAGATGTGCAGAAATGGCTGAGTTCCCCTTTCCCGTCCTCATCCTTTAGCCCGGGTGGTTTAGCTCCAGAGATCAGTCCT CTGGAGGTTCTGGAGAGAGACAAGGTTACTCAATTGCTCCCCCTGAATACAGACGCTTACCT GTCACTTCAAGAGCTGCAGGGGCAGGACCCCACTCATCTCGTATAGGCATGCAACCTCGATC CGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAAC AAT AT GAG GAG C T GAAG C C T AT AGAG TAG GAG C C AT AGAT AAAAT AAAAGAT TTTATTTAGT C T CCAGAAAAAGGGGGGAA

[0305] SEQ ID NO: 91. X- 4-lBBL-CD28TM-MyD88 (4-lBBL / MyD88) -X - DNA:TTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCCAGGGACCACCGACCCACCACCGGGAG GTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTAT GCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTG ACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTTAGGACTCTTTGGTGCACCCCCCTTAG AGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAA TTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCT GTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAAC CAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTT TAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCA AGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCC TCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCT CCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCCCCATATGGCCATATGAGATCTTATAT GGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGG CGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGAC ACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACAC AGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATAACTCGAGGCCACGATGGCCTTACCA GTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCCTGCCCCTG GGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGCCGCTAGCCCGCGACTCAGAGAGGGCC CCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGGACCTGAGACAAGGCATGTTCGCTCAG CTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCCTTAAGCTGGTACAGCGATCCCGGACT CGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGGAGCTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAGCTGCGTAGAGTGGTGGCCGGCGAG GGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAGCCTCTCAGATCGGCCGCCGGCGCCGC CGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAGCAGCGAGGCTAGAAACAGCGCCTTCG GCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGCAGAGACTGGGCGTGCACCTGCACACC GAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCTGGGCCTGTTCAG AGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCCTAGAAGCGAGACGCGTTTTTGGGTGC TGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCTTACTGGTGACTGTTGCCTTTATTATT TTTTGGGTTATGGCAGCAGGGGGCCCCGGCGCAGGCTCAGCGGCCCCGGTCTCTTCCACATC ATCACTGCCTCTCGCCGCACTCAACATGCGTGTTCGCCGGCGGCTGTCACTGTTTCTCAATG TCAGGACCCAAGTGGCCGCCGACTGGACCGCTTTAGCTGAGGAGATGGATTTTGAGTATTTA GAAATCCGACAACTGGAGACTCAGGCTGATCCTACCGGGAGGTTACTTGATGCCTGGCAAGG TAGACCAGGAGCAAGCGTGGGACGACTGCTGGAACTGCTGACTAAGCTGGGACGCGATGACG TCCTACTGGAACTTGGACCGAGTATCGAGGAAGATTGCCAGAAATATATTCTGAAGCAGCAACAGGAGGAAGCAGAGAAACCACTGCAGGTGGCAGCGGTGGACTCGTCCGTCCCCCGCACGGC CGAGCTAGCTGGAATTACTACGCTGGATGACCCTCTCGGCCACATGCCAGAGAGGTTTGACG CCTTTATATGCTATTGCCCCTCCGACATTTGAGCATGCAACCTCGATCCGGATTAGTCCAAT TTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCTG AAG C C T AT AGAG TAG GAG C C AT AGAT AAAAT AAAAGAT TTTATTTAGTCTC C AGAAAAAG G GGGGAA

[0306] SEQ ID NO: 91. X- 4-lBBL-CD28TM-cMPL (4-lBBL / cMPL) -X - DNA:TTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCCAGGGACCACCGACCCACCACCGGGAG GTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGACTGATTTTAT GCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTG ACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTTAGGACTCTTTGGTGCACCCCCCTTAG AGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAA TTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCT GTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAAC CAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTT TAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCA AGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCC TCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCT CCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCCCCATATGGCCATATGAGATCTTATAT GGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTCTACTTAGTCCAGCACGAAGTCTGGAGACCTCTGG CGGCAGCCTACCAAGAACAACTGGACCGACCGGTGGTACCTCACCCTTACCGAGTCGGCGAC ACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTAGAACCTCGCTGGAAAGGACCTTACAC AGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCATAACTCGAGGCCACGATGGCCTTACCA GTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGCCTGCCCCTG GGCGGTTAGTGGAGCTAGAGCTAGCCCGGGGTCCGCCGCTAGCCCGCGACTCAGAGAGGGCC CCGAGTTAAGCCCCGACGATCCGGCCGGCCTGCTGGACCTGAGACAAGGCATGTTCGCTCAGCTGGTGGCTCAGAACGTGCTGCTGATCGACGGACCCTTAAGCTGGTACAGCGATCCCGGACT CGCTGGCGTGAGCCTGACCGGCGGCCTGAGCTACAAGGAGGACACCAAGGAGCTGGTGGTGG CCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAGCTGCGTAGAGTGGTGGCCGGCGAG GGAAGCGGCTCTGTGTCACTGGCACTGCATCTGCAGCCTCTCAGATCGGCCGCCGGCGCCGCCGCCCTGGCCCTTACTGTGGACTTACCGCCCGCTAGCAGCGAGGCTAGAAACAGCGCCTTCG GCTTCCAAGGCAGACTGCTGCACCTGAGCGCCGGGCAGAGACTGGGCGTGCACCTGCACACC GAGGCTAGAGCTAGACACGCCTGGCAGCTGACCCAAGGCGCCACCGTGCTGGGCCTGTTCAG AGTGACCCCCGAGATCCCTGCCGGCCTGCCTAGCCCTAGAAGCGAGACGCGTTTTTGGGTGCTGGTTGTGGTGGGAGGGGTACTGGCCTGCTATAGCTTACTGGTGACTGTTGCCTTTATTATT TTTTGGGTTAGATGGCAGTTCCCCGCGCACTACAGAAGGCTACGACACGCCCTGTGGCCCTC TCTGCCCGACTTACACCGCGTTCTTGGCCAGTACCTTAGAGACACTGCCGCTTTGTCCCCAC CCAAAGCTACCGTATCGGATACCTGCGAAGAGGTGGAACCCAGCCTGCTTGAGATATTGCCCAAATCTAGTGAGCGGACGCCCCTACCCTTATGTTCCTCCCAGGCCCAGATGGATTATAGACG CCTGCAGCCCAGTTGTCTCGGCACAATGCCTCTATCCGTGTGTCCTCCTATGGCCGAAAGTG GATCTTGTTGCACCACTCACATCGCAAATCACAGCTACCTGCCACTCTCATACTGGCAACAA CCATGAGCATGCAACCTCGATCCGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCTGAAGCCTATAGAGTACGAGCCATAGAT AAAAT AAAAGAT T T TAT T TAGT C T CCAGAAAAAGGGGGGAA

[0307] In some aspects, a nucleic acid may comprise an internal ribosomal entry site (IRES). In certain aspects, an IRES comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NO: 77.

[0308] SEQ ID NO: 77. IRES (in CD123-BiTE-IRES-NGFR (CD123 TCE)) - DNA: CGGGATCAATTCCGCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGA AACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATG CAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAAC GTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCC AAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGT TGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGA TGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACAT GTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT GAAAAAC AC GAT AAT AC C1. Hybridization

[0309] The nucleic acids that hybridize to other nucleic acids under particular hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, e.g., Current Protocols in Molecular Biology, John Wiley and Sons, N.Y. (1989), 6.3.1-6.3.6. As defined herein, a moderately stringent hybridization condition uses a prewashing solution containing 5* sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6* SSC, and a hybridization temperature of 55° C. (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of 42° C), and washing conditions of 60° C. in 0.5*SSC, 0.1% SDS. A stringent hybridization condition hybridizes in 6* SSC at 45° C., followed by one or more washes in 0.1 *SSC, 0.2% SDS at 68° C. Furthermore, one of skill in the art can manipulate the hybridization and / or washing conditions to increase or decrease the stringency of hybridization such that nucleic acids comprising nucleotide sequence that are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to each other typically remain hybridized to each other.

[0310] The parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by, for example, Sambrook, Fritsch, and Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11 (1989); Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4 (1995), both of which are incorporated herein by reference in their entirety for all purposes) and can be readilydetermined by those having ordinary skill in the art based on, for example, the length and / or base composition of the DNA.2. Mutation

[0311] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antibody or antibody derivative, chimeric antigen receptor, DSR, etc.) that it encodes. Mutations can be introduced using any technique known in the art. In one aspect, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another aspect, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. However it is made, a mutant polypeptide can be expressed and screened for a desired property.

[0312] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. See, e.g., Romain Studer et al., Biochem. J. 449:581-594 (2013), incorporated herein by reference in its entirety. For example, the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody.3. Probes

[0313] In another aspect, nucleic acid molecules are suitable for use as primers or hybridization probes for the detection of nucleic acid sequences. A nucleic acid molecule can comprise only a portion of a nucleic acid sequence encoding a full-length polypeptide, for example, a fragment that can be used as a probe or primer or a fragment encoding an active portion of a given polypeptide.

[0314] Probes based on the desired sequence of a nucleic acid can be used to detect the nucleic acid or similar nucleic acids, for example, transcripts encoding a polypeptide of interest. The probe can comprise a label group, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used to identify a cell that expresses the polypeptide.III. Cancer Therapy

[0315] In some aspects, the disclosed methods comprise administering a cancer therapy to a subject, individual, or patient. In some aspects, the disclosed methods are not limited to use as a cancer therapeutic, but may also be utilized in any immunotherapy in which modulation of the activation of an immune effector cell is desired, such as but not limited to, autoimmune disease and / or infectious disease (e.g., bacterial, viral, protozoan, etc.), The cancer therapy may be chosen based on an expression level measurement, alone or in combination with a clinical risk score calculated for the subject. The cancer therapy may be chosen based on a genotype of a subject. In some aspects, the cancer therapy comprises a local cancer therapy. In some aspects, the cancer therapy excludes a systemic cancer therapy. In some aspects, the cancer therapy excludes a local therapy. In some aspects, the cancer therapy comprises a local cancer therapy without the administration of a system cancer therapy. In some aspects, the cancer therapy comprises an immunotherapy, which may be a checkpoint inhibitor therapy. Any of these cancer therapies may also be excluded. Combinations of these therapies may also be administered.

[0316] The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. In certain aspects, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, skin, stomach, testis, tongue, or uterus. In some aspects, the cancer is a Stage I cancer. In some aspects, the cancer is a Stage II cancer. In some aspects, the cancer is a Stage III cancer. In some aspects, the cancer is a Stage IV cancer.

[0317] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchi olo-alveol ar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenalcortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget’s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; Kaposi’s sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing’s sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin’s disease; Hodgkin’s; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin’s lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia;monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0318] In some aspects, the cancer is breast cancer. In some aspects, the cancer is HER2+ breast cancer.

[0319] In some aspects the cancer is brain cancer. In some aspects, the cancer is glioblastoma and / or medulloblastoma.

[0320] In some aspects the cancer is a blood cancer. In some aspects, the cancer is leukemia, acute myeloid leukemia, chronic myeloid leukemia, lymphoma, Non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, myeloma, myelodysplastic syndromes, myelodysplastic neoplasms.

[0321] In some aspects, the cancer expresses relatively low tumor associated antigen (TAA) levels relative to other cancers expressing the same TAA. In some such aspects, use of an DSR that increases immune cell activation may be appropriate.

[0322] In some aspects, the cancer expresses relatively high tumor associated antigen (TAA) levels relative to other cancers expressing the same TAA. In some such aspects, use of DSR that increases immune cell activation may be appropriate.

[0323] Methods may involve the determination, administration, or selection of an appropriate cancer “management regimen” and predicting the outcome of the same. As used herein the phrase “management regimen” refers to a management plan that specifies the type of examination, screening, diagnosis, surveillance, care, and treatment (such as dosage, schedule and / or duration of a treatment) provided to a subject in need thereof (e.g., a subject diagnosed with cancer).A. Radiotherapy

[0324] In some aspects, a radiotherapy, such as ionizing radiation, is administered to a subject. As used herein, “ionizing radiation” means radiation comprising particles or photons that have sufficient energy or can produce sufficient energy via nuclear interactions to produce ionization (gain or loss of electrons). A non-limiting example of ionizing radiation is x- radiation. Means for delivering x-radiation to a target tissue or cell are well known in the art.

[0325] In some aspects, the radiotherapy can comprise external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiation therapy (IORT). In some aspects, the external radiotherapy comprises three-dimensional conformal radiation therapy (3D-CRT), intensity modulated radiation therapy (IMRT), proton beam therapy, image-guided radiation therapy (IGRT), or stereotactic radiation therapy. In some aspects, the internalradiotherapy comprises interstitial brachytherapy, intracavitary brachytherapy, or intraluminal radiation therapy. In some aspects, the radiotherapy is administered to a primary tumor.

[0326] In some aspects, the amount of ionizing radiation is greater than 20 gray (Gy) and is administered in one dose. In some aspects, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some aspects, the amount of ionizing radiation is at least, at most, or exactly 0.5, 1, 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 Gy (or any derivable range therein). In some aspects, the ionizing radiation is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 does (or any derivable range therein). When more than one dose is administered, the dose may be about1, 4, 8, 12, or 24 hours or 1, 2, 3, 4, 5, 6, 7, or 8 days or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks apart, or any derivable range therein.

[0327] In some aspects, the amount of radiotherapy administered to a subject may be presented as a total dose of radiotherapy, which is then administered in fractionated doses. For example, in some aspects, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some aspects, the total dose is 50-90 Gy, administered in 20-60 fractionated doses of 2-3 Gy each. In some aspects, the total dose of radiation is at least, at most, or about 0.5, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 Gy (or any derivable range therein). In some aspects, the total dose is administered in fractionated doses of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any derivable range therein). In some aspects, at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100 fractionated doses are administered (or any derivable range therein). In some aspects, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 (or any derivable range therein) fractionated doses are administered per day. In some aspects, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) fractionated doses are administered per week.B. Cancer Immunotherapy

[0328] In some aspects, the methods comprise administration of a cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated IO) is the use of the immune system to treat cancer. Immunotherapies can, in some cases, be categorized as active, passive or hybrid (i.e., active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and / or cytokines. Various immunotherapies are known in the art, and certain examples are described below.1. Checkpoint Inhibitors and Combination Treatment

[0329] Aspects of the disclosure may include administration of immune checkpoint inhibitors, examples of which are further described below. As disclosed herein, “checkpoint inhibitor therapy” (also “immune checkpoint blockade therapy,” “checkpoint blockade therapy,” “immune checkpoint therapy,” “ICT,” “checkpoint blockade immunotherapy,” or “CBI”), refers to cancer therapy comprising providing one or more immune checkpoint inhibitors to a subject suffering from or suspected of having cancer. a. PD-1, PDL1, and PDL2 inhibitors

[0330] PD -1 can act in the tumor microenvironment where T cells encounter an infection or tumor. Activated T cells upregulate PD-1 and continue to express it in the peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to the tissues during an immune response. Inhibitors of the disclosure may block one or more functions of PD-1 and / or PDL1 activity.

[0331] Alternative names for “PD-1” include CD279 and SLEB2. Alternative names for “PDL1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PDL2” include B7- DC, Btdc, and CD273. In some aspects, PD-1, PDL1, and PDL2 are human PD-1, PDL1 and PDL2.

[0332] In some aspects, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1and / or PDL2. In another aspect, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and / or B7- 1. In another aspect, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art such as described in U. S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all incorporated herein by reference.

[0333] In some aspects, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some aspects, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some aspects, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some aspects, the PDL1 inhibitor comprises AMP- 224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in W02009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in W02009 / 101611. AMP -224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in W02010 / 027827 and WO201 1 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0334] In some aspects, the immune checkpoint inhibitor is a PDL1 inhibitor such as Durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or combinations thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor such as rHIgM12B7.

[0335] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2 and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another aspect, the antibody competes forbinding with and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. b. CTLA-4, B7-1, and B7-2

[0336] Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4 or CTLA4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number LI 5006. CTLA-4 is found on the surface of T cells and acts as an “off’ switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA- 4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. Inhibitors of the disclosure may block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some aspects, the inhibitor blocks the CTLA-4 and B7- 1 interaction. In some aspects, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0337] In some aspects, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0338] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. For example, the anti- CTLA-4 antibodies disclosed in: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 also can be used. For example, a humanized CTLA-4 antibody is described in International Patent Application No. W02001 / 014424, W02000 / 037504, and U.S. Patent No. 8,017,114; all incorporated herein by reference.

[0339] A further anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX- 010, MDX- 101, and YERVOY®) or antigen binding fragments and variants thereof (see, e.g., WO 01 / 14424).

[0340] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another aspect, the antibody competes for binding with and / or binds to the same epitope on PD-1, B7-1, or B7- 2 as the above- mentioned antibodies. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. c. LAG3

[0341] Another immune checkpoint that can be targeted in the methods provided herein is the lymphocyte-activation gene 3 (LAG3), also known as CD223 and lymphocyte activating 3. The complete mRNA sequence of human LAG3 has the Genbank accession number NM 002286. LAG3 is a member of the immunoglobulin superfamily that is found on the surface of activated T cells, natural killer cells, B cells, and plasmacytoid dendritic cells. LAG3’s main ligand is MHC class II, and it negatively regulates cellular proliferation, activation, and homeostasis of T cells, in a similar fashion to CTLA-4 and PD-1, and has been reported to play a role in Treg suppressive function. LAG3 also helps maintain CD8+T cells in a tolerogenic state and, working with PD-1, helps maintain CD8 exhaustion during chronic viral infection. LAG3 is also known to be involved in the maturation and activation of dendritic cells. Inhibitors of the disclosure may block one or more functions of LAG3 activity.

[0342] In some aspects, the immune checkpoint inhibitor is an anti-LAG3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0343] Anti-human-LAG3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-LAG3 antibodies can be used. For example, the anti-LAG3 antibodies can include: GSK2837781, IMP321, FS-118, Sym022, TSR-033, MGD013, BI754111, AVA-017, or GSK2831781. The anti-LAG3 antibodies disclosed in: US 9,505,839 (BMS-986016, also known as relatlimab); US 10,711,060 (IMP-701, also known as LAG525); US 9,244,059 (IMP731, also known as H5L7BW); US 10,344,089 (25F7, also known as-n -LAG3.1); WO 2016 / 028672 (MK-4280, also known as 28G-10); WO 2017 / 019894 (BAP050); Burova E., et al., J. ImmunoTherapy Cancer, 2016; 4(Supp. 1):P195 (REGN3767); Yu, X., et al., mAbs, 2019; 11 :6 (LBL-007) can be used in the methods disclosed herein. These and other anti-LAG-3 antibodies useful in the claimed inventions can be found in, for example: WO 2016 / 028672, WO 2017 / 106129, WO 2017062888, WO 2009 / 044273, WO 2018 / 069500, WO 2016 / 126858, WO 2014 / 179664, WO 2016 / 200782, WO 2015 / 200119, WO 2017 / 019846, WO 2017 / 198741, WO 2017 / 220555, WO 2017 / 220569, WO 2018 / 071500, WO2017 / 015560; WO 2017 / 025498, WO 2017 / 087589, WO 2017 / 087901, WO 2018 / 083087, WO 2017 / 149143, WO 2017 / 219995, US 2017 / 0260271, WO 2017 / 086367, WO2017 / 086419, WO 2018 / 034227, and WO 2014 / 140180. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to LAG3 also can be used.

[0344] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-LAG3 antibody. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-LAG3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-LAG3 antibody. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range therein) variable region amino acid sequence identity with the above-mentioned antibodies. d. TIM-3

[0345] Another immune checkpoint that can be targeted in the methods provided herein is the T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2) and CD366. The complete mRNA sequence of human TIM-3 has the Genbank accession number NM_032782. TIM-3 is found on the surface IFNy- producing CD4+Thl and CD8+Tel cells. The extracellular region of TIM-3 consists of a membrane distal single variable immunoglobulin domain (IgV) and a glycosylated mucin domain of variable length located closer to the membrane. TIM-3 is an immune checkpoint and, together with other inhibitory receptors including PD-1 and LAG3, it mediates T-cell exhaustion. TIM-3 has also been shown as a CD4+Thl-specific cell surface protein that regulates macrophage activation. Inhibitors of the disclosure may block one or more functions of TIM-3 activity.

[0346] In some aspects, the immune checkpoint inhibitor is an anti-TIM-3 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[0347] Anti-human-TIM-3 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-TIM-3 antibodies can be used. For example, anti-TIM-3 antibodies including: MBG453, TSR-022 (also known as Cobolimab), and LY3321367 can be used in the methods disclosed herein. These and other anti-TIM-3 antibodies useful in the claimed inventions can be found in, for example: US 9,605,070, US 8,841,418, US2015 / 0218274, and US 2016 / 0200815. The teachings of each of the aforementioned publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies for binding to TIM-3 also can be used.

[0348] In some aspects, the inhibitor comprises the heavy and light chain CDRs or VRs of an anti-TIM-3 antibody. Accordingly, in one aspect, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of an anti-TIM-3 antibody, and the CDR1, CDR2 and CDR3 domains of the VL region of an anti-TIM-3 antibody. In another aspect, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or any derivable range or value therein) variable region amino acid sequence identity with the above-mentioned antibodies.2. Activator of co-stimulatory molecules

[0349] In some aspects, the immunotherapy comprises an activator (also “agonist”) of a co-stimulatory molecule. In some aspects, the agonist comprises an agonist of CD3, B7-1 (CD80), B7-2 (CD86), CD28, ICOS, 0X40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Agonists include activating antibodies, polypeptides, compounds, and nucleic acids.3. Dendritic cell therapy

[0350] Dendritic cell therapy provokes anti-tumor responses by causing dendritic cells to present tumor antigens to lymphocytes, which activates them, priming them to kill other cells that present the antigen. Dendritic cells are antigen presenting cells (APCs) in the mammalian immune system. In cancer treatment they aid cancer antigen targeting. One example of cellular cancer therapy based on dendritic cells is sipuleucel-T.

[0351] One method of inducing dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small parts of protein that correspond to the protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to increase the immune and anti-tumor responses. Otheradjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte macrophage colony-stimulating factor (GM-CSF).

[0352] Dendritic cells can also be activated in vivo by making tumor cells express GM- CSF. This can be achieved by either genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with an oncolytic virus that expresses GM-CSF.

[0353] Another strategy is to remove dendritic cells from the blood of a patient and activate them outside the body. The dendritic cells are activated in the presence of tumor antigens, which may be a single tumor-specific peptide / protein or a tumor cell lysate (a solution of broken down tumor cells). These cells (with optional adjuvants) are infused and provoke an immune response.

[0354] Dendritic cell therapies include the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibody and can induce the dendritic cells to mature and provide immunity to the tumor. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.4. Chimeric Immune Receptors (CIR)

[0355] Chimeric immune receptors (CIRs), including Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors), and chimeric T cell receptors (cTCRs; also known as artificial T cell receptors) are engineered receptors that combine a new specificity with an immune cell to target cancer cells. Typically, these receptors graft the specificity of an antigen binding domain, e.g., an antibody, onto a T cell, natural killer (NK) cell, or other immune cell. The receptors are called chimeric because they are fused of parts from different sources. CIR- immune cell therapy, including CAR-T cell therapy and cTCR-T cell therapy, refers to a treatment that uses such transformed cells for cancer therapy, where the transformed cells are immune cells. Similar therapies include, for example, CAR-NK or cTCR-NK or TCR-NK cell therapy, which uses engineered NK cells. In some aspects, a CAR of the disclosure does not comprise a costimulatory region.

[0356] The basic principle of CIR-T cell design involves recombinant receptors that combine antigen-binding and immune cell activating functions, e.g., T-cell activating functions. The general premise of CIR-T cells is to artificially generate immune cells targeted to markers found on cancer cells. Scientists can remove immune cells, e.g., T or NK cells, from a person, genetically alter them, and put them back into the patient for them to attack the cancer cells. Once the immune cell has been engineered to become a CIR-immune cell, it acts as a “living drug”. CIR-immune cells create a link between an extracellular ligand recognitiondomain to an intracellular signaling molecule which in turn activates immune cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CIR-immune cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CIR-immune cells is determined by the choice of molecule that is targeted. In certain aspects, the CIR-immune cells are autologous to a subject. In certain aspects, the CIR-immune cells are allogeneic to the subject.

[0357] Example CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtagene ciloleucel (Yescarta).

[0358] While CARs are encouraging therapies against cancer, CARs are limited to surface antigens and have limited stimulatory capabilities. TCRs have properties that may help overcome some of the deficiencies of CARs. For example, TCRs can recognize any peptide that is processed for antigen presentation, and TCR signaling is 10 to 100 times more sensitive than CAR signaling (Harris et al., J Immunol. 2018 Feb l;200(3): 1088-1100., incorporated herein in its entirety by reference). For at least these reasons, TCR-based immunotherapy is a promising anti-cancer therapy, especially towards solid tumors.

[0359] TCRs are composed of aP (or y5) chain heterodimers that assemble at the cell membrane with the CD3 signaling complex (CD3sy, CD3s5, and CD3 Q. The a and P chains each comprise an extracellular immunoglobulin (Ig)-like domain comprising a variable region that provides antigen binding specificity and a constant domain, a transmembrane domain, and a short cytoplasmic region that lacks intracellular signaling motifs. Upon TCR binding to an antigen, immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 complex undergo phosphorylation and activate a downstream T cell signaling cascade.

[0360] Chimeric TCRs (cTCRs) combine the specificity of antigen binding domains, e.g., antibody scFv, with the ability of TCRs to engage endogenous signaling complexes (e.g., CD3 complex) to activate immune cells, e.g., T cells. cTCRs may comprise various structural configurations (see e.g., Yue Liu et al., 2021, “Chimeric STAR receptors using TCR machinery mediate robust responses against solid tumors” Set Transl Med. which is incorporated herein by reference in its entirety for the purposes described herein). In general, the endogenous variable region of a and / or P chains are replaced with an antigen binding domain of interest (e.g., a scFv targeting a cancer antigen) that is linked directly to the constant region of the a or P chains. In some instances, the entire antigen binding domain may be linked to one or both a and P chains, although the antigen binding domain may also be split amongst the two chains (e.g., an antibody VL chain may be linked to the a-chain while the VH may be linked to the Pchain, or vice versa); see e.g., W02020 / 029774 Al published 2020 / 02 / 13, which is incorporated herein by reference in its entirety for the purposes described herein. In some instances, the a and / or p chains may be engineered to comprise one or more cysteine residues and / or one or more hydrophobic substitutions in the constant domain to enhance heterodimer stability and complex formation, such as those described in Cohen et al., Cancer Res. 2007 Apr 15; 67(8): 3898-3903. and / or Jin et al., JCI Insight. 2018 Apr 19;3(8):e99488., both hereby incorporated by reference in their entirety. The antigen binding domain may also be linked to other components of the TCR complex, such as the y5 chains or the CD3 complex subunits, including the CD3y, CD35, and / or CD3s subunits.

[0361] In certain aspects, a cTCR comprises, consists essentially of, or consists of a amino acid sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 34, or 58.

[0362] In certain aspects, a cTCR is encoded by a polynucleotide that comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 33, 57, 86, 87.

[0363] As disclosed herein, in certain aspects, immune effector cells expressing a tumor antigen targeting molecule, e.g., a cTCR, may be further modified to express a polynucleotide and / or polypeptide of the immediate disclosure, such as but not limited to, polypeptides 4- lBBL / IL7Ra (SEQ ID NO: 18), 4-1BBL / IL2RP (SEQ ID NO: 22), 4-lBBL / MyD88 (SEQ ID NO: 26), and / or 4-lBBL / cMPL (SEQ ID NO: 30).

[0364] SEQ ID NO: 33. GDl.cTCR - DNA:ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCTCTAG AGATATTTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCA T C T C T T G C AGAT C T AG T C AGAG T C T T G TAG AC C G T AAT G GAAAC AC C T AT T TAG AT T G G T AC CTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTCACAAAGTTTCCAACCGATTTTCTGG GGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAG TGGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAAGTACACATGTTCCTCCGCTCACG TTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTT CCCAGGCTCGGGCGGTGGTGGGTCGGGTGGCGAGGTGAAGCTTCAGCAGTCTGGACCTAGCC TGGTGGAGCCTGGCGCTTCAGTGATGATATCCTGCAAGGCTTCTGGTTCCTCATTCACTGGC TACAACAT GAAC T GGGT GAGGCAGAACAT T GGAAAGAGCC T T GAAT GGAT T GGAGC TAT T GA TCCTTACTATGGTGGAACTAGCTACAACCAGAAGTTCAAGGGCAGGGCCACATTGACTGTAG ACAAATCGTCCAGCACAGCCTACATGCACCTCAAGAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGCGGAATGGAGTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCAGTCTATGGAAGGGTCACCGTCTCTTCAGGATCCCCATACATTC AAAATCCAGAACCTGCGGTATACCAATTGAAAGACCCAAGAAGCCAGGATTCCACGCTGTGT C T G T T T AC AGAC T T C GAG T C AC AGAT C AAT G T T C C C AAGAC AAT G GAAAG C G G GAC G T T C AT CACCGATAAATGCGTTCTGGACATGAAGGCTATGGATTCTAAAAGTAATGGTGCTATAGCCT G GAG T AAC C AGAC CTCATTTACTTGC C AAGAC AT T T T C AAG GAGAC C AAT G C TAG AT AT C C A TCCTCCGACGTCCCTTGTGACGCCACTCTGACCGAGAAATCTTTTGAGACCGATATGAACCT GAACTTTCAGAACCTACTGGTTATCGTTCTGAGAATACTGTTGCTCAAAGTCGCCGGATTCA ACCTTCTCATGACTCTGAGACTGTGGAGTAGCAGAGCCAAAAGAAGTGGTTCTGGCGCAACA AATTTCTCACTGCTCAAACAGGCGGGAGACGTGGAAGAGAATCCCGGTCCCATGGAGACAGA CACACTCCTGctatgggtgctgctgctctgggttCCAGGTTCCACAGGTGGCGGCGGTGGGT CAGAACAAAAATTGATTTCAGAGGAAGATCTCGGAGGGGGAGGGTCAACGCGTGAGGACTTG AGGAATGTGACGCCACCGAAGGTCTCTTTGTTTGAGCCGTCTAAGGCAGAAATTGCTAATAA ACAAAAGGCCACCCTAGTGTGTCTCGCGAGGGGATTCTTTCCTGATCACGTTGAGTTGTCCT GGTGGGTGAATGGTAAGGAGGTTCATAGCGGTGTCTGCACCGACCCCCAGGCATACAAGGAG TCGAACTACTCCTACTGCCTGTCATCTAGACTCCGAGTCAGTGCCACGTTCTGGCATAACCC CCGTAACCATTTTAGGTGCCAGGTGCAATTCCACGGGCTTAGTGAAGAGGACAAGTGGCCAG AGGGGTCCCCAAAGCCGGTGACCCAAAATATCTCTGCGGAAGCTTGGGGGCGCGCCGATTGT GGGATTACCAGCGCGAGCTATCAGCAGGGAGTCCTGAGTGCAACGATACTCTACGAGATATT ACTGGGAAAGGCTACATTATATGCAGTGTTGGTGAGCACTCTGGTCGTCATGGCAATGGTTA AGAGAAAGAATAGC

[0365] SEQ ID NO: 34. GDl.cTCR - AA:MEFGLSWLFLVAILKGVQCSRDILLTQTPLSLPVSLGDQAS I SCRSSQSLVHRNGNTYLHWY LQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYFCSQSTHVPPLT FGAGTKLELKRADAAPTVS I FPGSGGGGSGGEVKLQQSGPSLVEPGASVMI SCKASGSS FTG YNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAV YYCVSGMEYWGQGTSVTVSSAKTTPPSVYGRVTVSSGSPYIQNPEPAVYQLKDPRSQDSTLC LFTDFDSQINVPKTMESGTFI TDKCVLDMKAMDSKSNGAIAWSNQTS FTCQDI FKETNATYP SSDVPCDATLTEKS FETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSSRAKRSGSGAT NFSLLKQAGDVEENPGPMETDTLLLWVLLLWVPGSTGGGGGSEQKLI SEEDLGGGGSTREDL RNVTPPKVSLFEPSKAE IANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKE SNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNI SAEAWGRADC GI TSASYQQGVLSAT ILYE ILLGKATLYAVLVSTLWMAMVKRKNS

[0366] SEQ ID NO: 57. TdT.cTCR - DNA:ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCC GGCTCAAGTTCAGCTGGTGCAGAGCGGAGCGGAGGTGAAGAAGCCAGGGTCTTCCGTTAAGG TCTCTTGTAAGGCCTCAGGCGGAACTTTCTCATCTTACGCTATTTCCTGGGTAAGGCAAGCA CCAGGACAGGGATTAGAATGGATGGGGGGGATTATCCCCATTTTCGGTACCGCGAACTACGC CCAAAAATTTCAAGGTCGGGTAACCATCACCGCCGATGAATCGACATCTACCGCCTACATGG AACTGAGCTCTCTTCGGAGCGAGGACACCGCAGTCTATTACTGCGCGAGAGACGGGTATAGT GGTAGCTACTACTATTATTACGGCATGGACGTTTGGGGCCAGGGGACCTTAGTGACGGTGAG CAGCGGAGGAGGCGGCGGGTCCCAGTCTGCTCTGACCCAACCTGCCAGCGTCTCCGGCAGTC CAGGACAAAGTATCACCATCAGCTGCACTGGGACTTCCAGCGATGTGGGGGGCTACAATTAC GTAAGTTGGTACCAGCAACACCCTGGGAAGGCCCCCAAGCTGATGATTTATGATGTCTCATA CAGACCGAGCGGAGTGAGCCACCGTTTTAGTGGCTCCAAGTCCGGAAACACCGCTTCACTAA CAATCAGTGGCTTACAAGCAGAGGACGAAGCCGATTATTACTGCTCATCTTACACCAGTTCC AGTACGCTTGTGTTTGGTACTGGAACAAAGTTGACTGTGCTGGGCGGATCCCCATACATTCA AAATCCAGAACCTGCGGTATACCAATTGAAAGACCCAAGAAGCCAGGATTCCACGCTGTGTC T G T T TAG AGAC T T C GAC T GAG AGAT C AAT G T T C C C AAGAC AAT G GAAAG C G G GAC G T T C AT C ACCGATAAATGCGTTCTGGACATGAAGGCTATGGATTCTAAAAGTAATGGTGCTATAGCCTGGAG T AC C AGAC CTCATTTACTTGC C AAGAC AT T T T C AAG GAGAC C AAT G C TAG AT AT C C AT CCTCCGACGTCCCTTGTGACGCCACTCTGACCGAGAAATCTTTTGAGACCGATATGAACCTG AACTTTCAGAACCTACTGGTTATCGTTCTGAGAATACTGTTGCTCAAAGTCGCCGGATTCAA CCTTCTCATGACTCTGAGACTGTGGAGTAGCAGAGCCAAAAGAAGTGGTTCTGGCGCAACAA ATTTCTCACTGCTCAAACAGGCGGGAGACGTGGAAGAGAATCCCGGTCCCATGGAGACAGAC ACACTCCTGctatgggtgctgctgctctgggttCCAGGTTCCACAGGTGGCGGCGGTGGGTC AGAACAAAAATTGATTTCAGAGGAAGATCTCGGAGGGGGAGGGTCAACGCGTGAGGACTTGA GGAATGTGACGCCACCGAAGGTCTCTTTGTTTGAGCCGTCTAAGGCAGAAATTGCTAATAAA CAAAAGGCCACCCTAGTGTGTCTCGCGAGGGGATTCTTTCCTGATCACGTTGAGTTGTCCTG GTGGGTGAATGGTAAGGAGGTTCATAGCGGTGTCTGCACCGACCCCCAGGCATACAAGGAGT CGAACTACTCCTACTGCCTGTCATCTAGACTCCGAGTCAGTGCCACGTTCTGGCATAACCCC CGTAACCATTTTAGGTGCCAGGTGCAATTCCACGGGCTTAGTGAAGAGGACAAGTGGCCAGA GGGGTCCCCAAAGCCGGTGACCCAAAATATCTCTGCGGAAGCTTGGGGGCGCGCCGATTGTG GGATTACCAGCGCGAGCTATCAGCAGGGAGTCCTGAGTGCAACGATACTCTACGAGATATTA CTGGGAAAGGCTACATTATATGCAGTGTTGGTGAGCACTCTGGTCGTCATGGCAATGGTTAA GAGAAAGAATAGC

[0367] SEQ ID NO: 58. TdT.cTCR - AA:MALPVTALLLPLALLLHAARPAQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI SWVRQA PGQGLEWMGGI I PI FGTANYAQKFQGRVT I TADESTSTAYMELSSLRSEDTAVYYCARDGYS GSYYYYYGMDVWGQGTLVTVSSGGGGGSQSALTQPASVSGSPGQS I T I SCTGTSSDVGGYNY VSWYQQHPGKAPKLMIYDVSYRPSGVSHRFSGSKSGNTASLT I SGLQAEDEADYYCSSYTSS STLVFGTGTKLTVLGGSPYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFI TDKCVLDMKAMDSKSNGAIAWSNQTS FTCQDI FKETNATYPSSDVPCDATLTEKS FETDMNL NFQNLLVIVLRILLLKVAGFNLLMTLRLWSSRAKRSGSGATNFSLLKQAGDVEENPGPMETD TLLLWVLLLWVPGSTGGGGGSEQKLI SEEDLGGGGSTREDLRNVTPPKVSLFEPSKAE IANK QKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNP RNHFRCQVQFHGLSEEDKWPEGSPKPVTQNI SAEAWGRADCGI TSASYQQGVLSAT ILYE IL LGKATLYAVLVS TLWMAMVKRKNS

[0368] SEQ ID NO: 86. 5'LTR- GDl.cTCR -3'LTR - DNA:G C GAG TCATATTAATTACTAGT C AAT TAGTTGATTTTTATTTTT GAG AT AT AC AT G T GAAAG ACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAAT ACATAAC T GAGAATAGAAAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATAT GG GCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAA CAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCA AGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTT TCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCG CTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTC ACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCC TCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATT GACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCC AGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGAT TGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTG TATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGAC GTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTT AGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACC TAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCG CGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTC TGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGA AAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCCCCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCT GAG AT GAG AAGAG T T AC T AAC AG CCCCTCTCTC C AAG C T GAG T TAG AG GCTCTCTACTTAGT CCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGG TACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTA GAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGA CGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCAT AACTCGAGGCCACGATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGT GTCCAGTGCTCTAGAGATATTTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGG AGAT C AAG C C T C C AT C T C T T G C AGAT C T AG T C AGAG T C T T G T AC AC C G T AAT G GAAAC AC C T ATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTCACAAAGTTTCC AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACT CAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAAGTACACATG TTCCTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCA ACTGTATCCATCTTCCCAGGCTCGGGCGGTGGTGGGTCGGGTGGCGAGGTGAAGCTTCAGCA GTCTGGACCTAGCCTGGTGGAGCCTGGCGCTTCAGTGATGATATCCTGCAAGGCTTCTGGTT CCTCATTCACTGGCTACAACATGAACTGGGTGAGGCAGAACATTGGAAAGAGCCTTGAATGG ATTGGAGCTATTGATCCTTACTATGGTGGAACTAGCTACAACCAGAAGTTCAAGGGCAGGGC C AC AT T GAC T G T AGAC AAAT C G T C C AG C AC AG C C TAG AT G C AC C T C AAGAG C C T GAC AT C T G AGGACTCTGCAGTCTATTACTGTGTAAGCGGAATGGAGTACTGGGGTCAAGGAACCTCAGTC ACCGTCTCCTCAGCCAAAACGACACCCCCATCAGTCTATGGAAGGGTCACCGTCTCTTCAGG AT C C C CAT AC AT T C AAAAT C C AGAAC CTGCGGTATAC C AAT T GAAAGAC C C AAGAAG C C AG G ATTCCACGCTGTGTCTGTTTACAGACTTCGACTCACAGATCAATGTTCCCAAGACAATGGAA AGCGGGACGTTCATCACCGATAAATGCGTTCTGGACATGAAGGCTATGGATTCTAAAAGTAA TGGTGCTATAGCCTGGAGTAACCAGACCTCATTTACTTGCCAAGACATTTTCAAGGAGACCA ATGCTACATATCCATCCTCCGACGTCCCTTGTGACGCCACTCTGACCGAGAAATCTTTTGAG ACCGATATGAACCTGAACTTTCAGAACCTACTGGTTATCGTTCTGAGAATACTGTTGCTCAA AGTCGCCGGATTCAACCTTCTCATGACTCTGAGACTGTGGAGTAGCAGAGCCAAAAGAAGTG GTTCTGGCGCAACAAATTTCTCACTGCTCAAACAGGCGGGAGACGTGGAAGAGAATCCCGGT CCCATGGAGACAGACACACTCCTGctatgggtgctgctgctctgggttCCAGGTTCCACAGG TGGCGGCGGTGGGTCAGAACAAAAATTGATTTCAGAGGAAGATCTCGGAGGGGGAGGGTCAA CGCGTGAGGACTTGAGGAATGTGACGCCACCGAAGGTCTCTTTGTTTGAGCCGTCTAAGGCA GAAATTGCTAATAAACAAAAGGCCACCCTAGTGTGTCTCGCGAGGGGATTCTTTCCTGATCA CGTTGAGTTGTCCTGGTGGGTGAATGGTAAGGAGGTTCATAGCGGTGTCTGCACCGACCCCC AGGCATACAAGGAGTCGAACTACTCCTACTGCCTGTCATCTAGACTCCGAGTCAGTGCCACG TTCTGGCATAACCCCCGTAACCATTTTAGGTGCCAGGTGCAATTCCACGGGCTTAGTGAAGA GGACAAGTGGCCAGAGGGGTCCCCAAAGCCGGTGACCCAAAATATCTCTGCGGAAGCTTGGG GGCGCGCCGATTGTGGGATTACCAGCGCGAGCTATCAGCAGGGAGTCCTGAGTGCAACGATA CTCTACGAGATATTACTGGGAAAGGCTACATTATATGCAGTGTTGGTGAGCACTCTGGTCGT CATGGCAATGGTTAAGAGAAAGAATAGCTAAGCATGCAACCTCGATCCGGATTAGTCCAATT TGTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCTGA AGCCTATAGAGTACGAGCCATAGATAAAATAAAAGAT T T TAT T TAGTCTCCAGAAAAAGGGG GGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGC AT GGAAAAATACATAAC T GAGAATAGAGAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAG CTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGA ACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGG CTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTT

[0369] SEQ ID NO: 87. 5'LTR- TdT-Bl -3'LTR - DNA:G C GAG TCATATTAATTACTAGT C AAT TAGTTGATTTTTATTTTT GAG AT AT AC AT G T GAAAG ACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAAT ACATAAC T GAGAATAGAAAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATAT GG GCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAA CAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCA AGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTT TCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCG CTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTC ACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCC TCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATT GACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCC AGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTCTGTCCGAT TGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAACTAGCTCTG TATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCCTGGGAGAC GTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCCGATCGTTT AGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGACGAGAACC TAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCCGCGCCGCG CGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTGTATTTGTC TGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGA AAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACC TTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCG AGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACC AGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCC TTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACC TCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCC CCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCCCTGACCCT GAG AT GAG AAGAG T T AC T AAC AG CCCCTCTCTC C AAG C T C AC T TAG AG GCTCTCTACTTAGT CCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCGACCGGTGG TACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTAAGAACCTA GAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTCAAAGTAGA CGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGGTGGACCAT AACTCGAGGCCACGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTC CACGCCGCCAGGCCGGCTCAAGTTCAGCTGGTGCAGAGCGGAGCGGAGGTGAAGAAGCCAGG GTCTTCCGTTAAGGTCTCTTGTAAGGCCTCAGGCGGAACTTTCTCATCTTACGCTATTTCCT GGGTAAGGCAAGCACCAGGACAGGGATTAGAATGGATGGGGGGGATTATCCCCATTTTCGGT ACCGCGAACTACGCCCAAAAATTTCAAGGTCGGGTAACCATCACCGCCGATGAATCGACATC TACCGCCTACATGGAACTGAGCTCTCTTCGGAGCGAGGACACCGCAGTCTATTACTGCGCGA GAGACGGGTATAGTGGTAGCTACTACTATTATTACGGCATGGACGTTTGGGGCCAGGGGACC TTAGTGACGGTGAGCAGCGGAGGAGGCGGCGGGTCCCAGTCTGCTCTGACCCAACCTGCCAG CGTCTCCGGCAGTCCAGGACAAAGTATCACCATCAGCTGCACTGGGACTTCCAGCGATGTGG GGGGCTACAATTACGTAAGTTGGTACCAGCAACACCCTGGGAAGGCCCCCAAGCTGATGATT TATGATGTCTCATACAGACCGAGCGGAGTGAGCCACCGTTTTAGTGGCTCCAAGTCCGGAAA CACCGCTTCACTAACAATCAGTGGCTTACAAGCAGAGGACGAAGCCGATTATTACTGCTCAT CTTACACCAGTTCCAGTACGCTTGTGTTTGGTACTGGAACAAAGTTGACTGTGCTGGGCGGAT C C C CAT AC AT T C AAAAT C C AGAAC CTGCGGTATAC C AAT T GAAAGAC C C AAGAAG C C AG GA TTCCACGCTGTGTCTGTTTACAGACTTCGACTCACAGATCAATGTTCCCAAGACAATGGAAA GCGGGACGTTCATCACCGATAAATGCGTTCTGGACATGAAGGCTATGGATTCTAAAAGTAAT GGTGCTATAGCCTGGAGTAACCAGACCTCATTTACTTGCCAAGACATTTTCAAGGAGACCAA TGCTACATATCCATCCTCCGACGTCCCTTGTGACGCCACTCTGACCGAGAAATCTTTTGAGA CCGATATGAACCTGAACTTTCAGAACCTACTGGTTATCGTTCTGAGAATACTGTTGCTCAAA GTCGCCGGATTCAACCTTCTCATGACTCTGAGACTGTGGAGTAGCAGAGCCAAAAGAAGTGG TTCTGGCGCAACAAATTTCTCACTGCTCAAACAGGCGGGAGACGTGGAAGAGAATCCCGGTC CCATGGAGACAGACACACTCCTGctatgggtgctgctgctctgggttCCAGGTTCCACAGGT GGCGGCGGTGGGTCAGAACAAAAATTGATTTCAGAGGAAGATCTCGGAGGGGGAGGGTCAAC GCGTGAGGACTTGAGGAATGTGACGCCACCGAAGGTCTCTTTGTTTGAGCCGTCTAAGGCAG AAATTGCTAATAAACAAAAGGCCACCCTAGTGTGTCTCGCGAGGGGATTCTTTCCTGATCAC GTTGAGTTGTCCTGGTGGGTGAATGGTAAGGAGGTTCATAGCGGTGTCTGCACCGACCCCCA GGCATACAAGGAGTCGAACTACTCCTACTGCCTGTCATCTAGACTCCGAGTCAGTGCCACGT TCTGGCATAACCCCCGTAACCATTTTAGGTGCCAGGTGCAATTCCACGGGCTTAGTGAAGAG GACAAGTGGCCAGAGGGGTCCCCAAAGCCGGTGACCCAAAATATCTCTGCGGAAGCTTGGGG GCGCGCCGATTGTGGGATTACCAGCGCGAGCTATCAGCAGGGAGTCCTGAGTGCAACGATAC TCTACGAGATATTACTGGGAAAGGCTACATTATATGCAGTGTTGGTGAGCACTCTGGTCGTC ATGGCAATGGTTAAGAGAAAGAATAGCTAAGCATGCAACCTCGATCCGGATTAGTCCAATTT GTTAAAGACAGGATATCAGTGGTCCAGGCTCTAGTTTTGACTCAACAATATCACCAGCTGAA GCCTATAGAGTACGAGCCATAGATAAAATAAAAGAT T T TAT T TAGTCTCCAGAAAAAGGGGG GAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCA T GGAAAAATACATAAC T GAGAATAGAGAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC TGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAA CAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGC TCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCA TCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCA ATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCA CAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCC AATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTC TGAGTGATTGACTACCCGTCAGCGGGGGTCTTT

[0370] In certain aspects, CIRs may comprise one or more, or a combination of the following components:(1) Signal peptide

[0371] Polypeptides of the present disclosure may comprise a signal peptide. A “signal peptide” refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g., to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface. In some aspects, a signal peptide directs the nascent protein into the endoplasmic reticulum. This is essential if a receptor is to be glycosylated and anchored in the cell membrane. Generally, the signal peptide natively attached to the amino-terminal most component is used (e.g., in an scFv with orientation light chain - linker - heavy chain, the native signal of the light-chain is used).

[0372] In certain aspects, engineered CAR and / or cTCR polypeptides may comprise one or more signal peptides. In some aspects, a signal peptide may have a functional impact on theexpression, localization, and / or activity of a polypeptide comprising the same. In some aspects, a signal peptide sequence may be an IgG derived sequence.

[0373] In some aspects, the signal peptide is cleaved after passage of the endoplasmic reticulum (ER), i.e., is a cleavable signal peptide. In some aspects, a signal peptide comprises a restriction site is at or near the carboxyl end to facilitate cleavage.

[0374] In certain aspects, a signal peptide comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 35-36, 51-52, 59-60, or 65-66.(2) Antigen binding domain

[0375] Polypeptides of the present disclosure may comprise one or more antigen binding domains. An “antigen binding domain” describes a region of a polypeptide capable of binding to an antigen under appropriate conditions. In some aspects, an antigen binding domain is a single-chain variable fragment (scFv) based on one or more antibodies (e.g., HER2 antibodies). In some aspects, an antigen binding domain comprise a variable heavy (VH) region and a variable light (VL) region, with the VH and VL regions being on the same polypeptide or on different polypeptides. In some aspects, the antigen binding domain comprises a linker between the VH and VL regions. A linker may enable the antigen binding domain to form a desired structure for antigen binding.

[0376] The variable regions of the antigen-binding domains of the polypeptides of the disclosure can be modified by mutating amino acid residues within the VH and / or VL CDR 1, CDR 2 and / or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term “CDR” refers to a complementarity-determining region that is based on a part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B cells and T cells respectively, where these molecules bind to their specific antigen. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered. The mutations may be amino acid substitutions, additions or deletions.

[0377] Framework modifications can be made to the antibodies to decrease immunogenicity, for example, by “backmutating” one or more framework residues to the corresponding germline sequence.

[0378] It is also contemplated that the antigen binding domain may be multi-specific or multivalent by multimerizing the antigen binding domain with VH and VL region pairs that bind either the same antigen (multi -valent) or a different antigen (multi-specific).

[0379] The binding affinity of the antigen binding region, such as the variable regions (heavy chain and / or light chain variable region), or of the CDRs may be at least 10'5M, 10'6M, 10'7M, 10'8M, 10'9M, 1O'1OM, 10-11M, 10'12M, or 10'13M. In some aspects, the KD of the antigen binding region, such as the variable regions (heavy chain and / or light chain variable region), or of the CDRs may be at least 10’5M, 10’6M, 10’7M, 10’8M, 10’9M, 10’10M, 101M, 10’12M, or 10'13M (or any derivable range therein).

[0380] Binding affinity, KA, or KD can be determined by methods known in the art such as by surface plasmon resonance (SRP)-based biosensors, by kinetic exclusion assay (KinExA), by optical scanner for microarray detection based on polarization-modulated oblique-incidence reflectivity difference (OI-RD), or by ELISA.

[0381] In certain aspects, an antigen binding domain comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 37-42, 61, and / or 62.

[0382] SEQ ID NO: 37. 14g2a (VL+VH) (in GD2.cTCR) - DNA:GATATTTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCAT C T C T T G C AGAT C T AG T C AGAG T C T T G TAG AC C G T AAT G GAAAC AC C T AT T TAG AT T G G T AC C TGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTCACAAAGTTTCCAACCGATTTTCTGGG GTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGT GGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAAGTACACATGTTCCTCCGCTCACGT TCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTC CCAGGCTCGGGCGGTGGTGGGTCGGGTGGCGAGGTGAAGCTTCAGCAGTCTGGACCTAGCCT GGTGGAGCCTGGCGCTTCAGTGATGATATCCTGCAAGGCTTCTGGTTCCTCATTCACTGGCT ACAACAT GAAC T GGGT GAGGCAGAACAT T GGAAAGAGCC T T GAAT GGAT T GGAGC TAT T GAT CCTTACTATGGTGGAACTAGCTACAACCAGAAGTTCAAGGGCAGGGCCACATTGACTGTAGA CAAATCGTCCAGCACAGCCTACATGCACCTCAAGAGCCTGACATCTGAGGACTCTGCAGTCT ATTACTGTGTAAGCGGAATGGAGTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCC AAAACGACACCCCCATCAGTCTATGGAAGGGTCACCGTCTCTTCA

[0383] SEQ ID NO: 38. 14g2a (VL+VH) (in GD2.cTCR) - AA:DILLTQTPLSLPVSLGDQAS I SCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSG VPDRFSGSGSGTDFTLKI SRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKRADAAPTVS I F PGSGGGGSGGEVKLQQSGPSLVEPGASVMI SCKASGSS FTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSAKTTPPSVYGRVTVSS

[0384] SEQ ID NO: 39. 14g2a (VL) (in GDl.cTCR) - DNA:GATATTTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCAT C T C T T G C AGAT C T AG T C AGAG T C T T G TAG AC C G T AAT G GAAAC AC C T AT T TAG AT T G G T AC C TGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTCACAAAGTTTCCAACCGATTTTCTGGG GTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGT GGAGGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAAGTACACATGTTCCTCCGCTCACGT TCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0385] SEQ ID NO: 40. 14g2a (VL) (in GD2.cTCR) - AA:DILLTQTPLSLPVSLGDQAS I SCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSG VPDRFSGSGSGTDFTLKI SRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK

[0386] SEQ ID NO: 41. 14g2a (VH) (in GD2.cTCR) - DNA:GAGGTGAAGCTTCAGCAGTCTGGACCTAGCCTGGTGGAGCCTGGCGCTTCAGTGATGATATC CTGCAAGGCTTCTGGTTCCTCATTCACTGGCTACAACATGAACTGGGTGAGGCAGAACATTG GAAAGAGCCTTGAATGGATTGGAGCTATTGATCCTTACTATGGTGGAACTAGCTACAACCAG AAGTTCAAGGGCAGGGCCACATTGACTGTAGACAAATCGTCCAGCACAGCCTACATGCACCT CAAGAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGTAAGCGGAATGGAGTACTGGG GTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCAGTCTATGGAAGG GTCACCGTCTCTTCA

[0387] SEQ ID NO: 42. 14g2a (VH) (in GD2.cTCR) - AA:EVKLQQSGPSLVEPGASVMI SCKASGSS FTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQ KFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMEYWGQGTSVTVSSAKTTPPSVYGR VTVSS

[0388] SEQ ID NO: 61. TdT-Bl (in TdT.cTCR) - DNA:GCTCAAGTTCAGCTGGTGCAGAGCGGAGCGGAGGTGAAGAAGCCAGGGTCTTCCGTTAAGGT CTCTTGTAAGGCCTCAGGCGGAACTTTCTCATCTTACGCTATTTCCTGGGTAAGGCAAGCAC CAGGACAGGGATTAGAATGGATGGGGGGGATTATCCCCATTTTCGGTACCGCGAACTACGCC CAAAAATTTCAAGGTCGGGTAACCATCACCGCCGATGAATCGACATCTACCGCCTACATGGA ACTGAGCTCTCTTCGGAGCGAGGACACCGCAGTCTATTACTGCGCGAGAGACGGGTATAGTG GTAGCTACTACTATTATTACGGCATGGACGTTTGGGGCCAGGGGACCTTAGTGACGGTGAGC AGCGGAGGAGGCGGCGGGTCCCAGTCTGCTCTGACCCAACCTGCCAGCGTCTCCGGCAGTCC AGGACAAAGTATCACCATCAGCTGCACTGGGACTTCCAGCGATGTGGGGGGCTACAATTACG TAAGTTGGTACCAGCAACACCCTGGGAAGGCCCCCAAGCTGATGATTTATGATGTCTCATAC AGACCGAGCGGAGTGAGCCACCGTTTTAGTGGCTCCAAGTCCGGAAACACCGCTTCACTAAC AATCAGTGGCTTACAAGCAGAGGACGAAGCCGATTATTACTGCTCATCTTACACCAGTTCCA GTACGCTTGTGTTTGGTACTGGAACAAAGTTGACTGTGCTGGGC

[0389] SEQ ID NO: 62. TdT-Bl (in TdT.cTCR) - AA:AQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAI SWVRQAPGQGLEWMGGI I PI FGTANYA QKFQGRVT I TADESTSTAYMELSSLRSEDTAVYYCARDGYSGSYYYYYGMDVWGQGTLVTVS SGGGGGSQSALTQPASVSGSPGQS I T I SCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSY RPSGVSHRFSGSKSGNTASLT I SGLQAEDEADYYCSSYTSSSTLVFGTGTKLTVLG(3) TCR chains

[0390] A TCR chain, such as the a, P, y, or 5 chains, may be used to generate cTCRs. In some aspects, the TCR chains are derived from mouse TCR chains. The TCR chains may be humanized. In some aspects, the TCR chains are derived from human TCR chains. In some aspects, the TCR chains are a and P chains. In some aspects, the chains are engineered to comprise one or more cysteine residues and / or one or more hydrophobic substitutions in the constant domain to enhance heterodimer stability and complex formation, such as those described in Cohen et al., Cancer Res. 2007 Apr 15; 67(8): 3898-3903. and / or Jin et al., JCI Insight. 2018 Apr 19;3(8):e99488., both hereby incorporated by reference in their entirety.

[0391] SEQ ID NO: 45. mTRAC (in GDl.cTCR, and TdT.cTCR) - DNA:C CAT AC AT T C AAAAT C C AGAAC CTGCGGTATAC C AAT T GAAAGAC C C AAGAAG C C AG GAT T C C AC G C T G T G T C T G T T T AC AGAC T T C GAC T C AC AGAT C AAT G T T C C C AAGAC AAT G GAAAG C G GGACGTTCATCACCGATAAATGCGTTCTGGACATGAAGGCTATGGATTCTAAAAGTAATGGT GCTATAGCCTGGAGTAACCAGACCTCATTTACTTGCCAAGACATTTTCAAGGAGACCAATGC TACATATCCATCCTCCGACGTCCCTTGTGACGCCACTCTGACCGAGAAATCTTTTGAGACCG ATATGAACCTGAACTTTCAGAACCTACTGGTTATCGTTCTGAGAATACTGTTGCTCAAAGTC GCCGGATTCAACCTTCTCATGACTCTGAGACTGTGGAGTAGC

[0392] SEQ ID NO: 46. mTRAC (in GDl.cTCR, and TdT.cTCR) - AA:PYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFI TDKCVLDMKAMDSKSNG AIAWSNQTS FTCQDI FKETNATYPSSDVPCDATLTEKS FETDMNLNFQNLLVIVLRILLLKV AGFNLLMTLRLWSS

[0393] SEQ ID NO: 55. mTrbcl (in GDl.cTCR, and TdT.cTCR) - DNA:GAGGACTTGAGGAATGTGACGCCACCGAAGGTCTCTTTGTTTGAGCCGTCTAAGGCAGAAAT TGCTAATAAACAAAAGGCCACCCTAGTGTGTCTCGCGAGGGGATTCTTTCCTGATCACGTTG AGTTGTCCTGGTGGGTGAATGGTAAGGAGGTTCATAGCGGTGTCTGCACCGACCCCCAGGCA TACAAGGAGTCGAACTACTCCTACTGCCTGTCATCTAGACTCCGAGTCAGTGCCACGTTCTG GCATAACCCCCGTAACCATTTTAGGTGCCAGGTGCAATTCCACGGGCTTAGTGAAGAGGACA AGTGGCCAGAGGGGTCCCCAAAGCCGGTGACCCAAAATATCTCTGCGGAAGCTTGGGGGCGC GCCGATTGTGGGATTACCAGCGCGAGCTATCAGCAGGGAGTCCTGAGTGCAACGATACTCTA CGAGATATTACTGGGAAAGGCTACATTATATGCAGTGTTGGTGAGCACTCTGGTCGTCATGG CAAT GGT TAAGAGAAAGAATAGC

[0394] SEQ ID NO: 56. mTrbcl (in GDl.cTCR, and TdT.cTCR) - AA:EDLRNVTPPKVSLFEPSKAE IANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQA YKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNI SAEAWGR ADCGI TSASYQQGVLSAT ILYE ILLGKATLYAVLVSTLWMAMVKRKNS(4) Peptide spacer

[0395] A peptide spacer, such as an extracellular spacer, may link an antigen-binding domain to a transmembrane domain. In some aspects, a peptide spacer is flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen binding.In some aspects, the spacer comprises the hinge region from IgG. In some aspects, the spacer comprises or further comprises the CH2CH3 region of immunoglobulin and portions of CD3.

[0396] In certain aspects, engineered polypeptides may comprise one or more hinges and / or linker sequences. In some aspects, a hinge and / or linker sequence may have a functional impact on the expression, localization, and / or activity of a polypeptide comprising the same.

[0397] As used herein, the term “hinge”, “linker”, or “hinge region” refers to a flexible polypeptide connector region providing structural flexibility and spacing to flanking polypeptide regions and can consist of natural or synthetic polypeptides. A “hinge” derived from an immunoglobulin (e.g., IgGl) is generally defined as stretching from Glu216 to Pro230 of human IgGl (Burton (1985) Molec. Immunol., 22: 161- 206). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain disulfide (S-S) bonds in the same positions. The hinge region may be of natural occurrence or non-natural occurrence, including but not limited to an altered hinge region as described in U.S. Pat. No. 5,677,425, incorporated by reference herein. The hinge region can include a complete hinge region derived from an antibody of a different class or subclass from that of the CHI domain. The term “hinge” can also include regions derived from CD8 and other receptors that provide a similar function in providing flexibility and spacing to flanking regions.

[0398] The extracellular spacer can have a length of at least, at most, or exactly 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 18, 19, 20, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110, 119, 120, 130, 140,150, 160, 170, 180, 190, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300,325, 350, or 400 amino acids (or any derivable range therein). In some aspects, the extracellular spacer consists of or comprises a hinge region from an immunoglobulin (e.g., IgG). Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87: 162; and Huck et al. (1986) Nucl. Acids Res., incorporated herein by reference in its entirety.

[0399] The length of an extracellular spacer may have effects on the CAR’s or cTCR’s signaling activity and / or the CAR- or cTCR-immune cells’ expansion properties in response to antigen-stimulated CAR or cTCR signaling. In some aspects, a shorter spacer such as less than 50, 45, 40, 30, 35, 30, 25, 20, 15, 14, 13, 12, 11, or 10 amino acids is used. In some aspects, a longer spacer, such as one that is at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 280, or 290 amino acids may have the advantage of increased expansion in vivo or in vitro.

[0400] When the extracellular spacer comprises multiple parts, there may be anywhere from 0-50 amino acids in between the various parts. For example, there may be at least, at most, or exactly 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 amino acids (or any derivable range therein) between the hinge and the CH2 or CH3 region or between the CH2 and CH3 region when both are present. In some aspects, the extracellular spacer consists essentially of a hinge, CH2, and / or CH3 region, meaning that the hinge, CH2, and / or CH3 region is the only identifiable region present and all other domains or regions are excluded, but further amino acids not part of an identifiable region may be present.

[0401] In certain aspects, a linker comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 43, 44, 73, and / or 74.

[0402] SEQ ID NO: 43. 14g2a linker (in GD2.cTCR) - DNA:CGGGCTGATGCTGCACCAACTGTATCCATCTTCCCAGGCTCGGGCGGTGGTGGGTCGGGTGG C

[0403] SEQ ID NO: 44. 14g2a linker (in GD2.cTCR) - AA:RADAAPTVS I FPGSGGGGSGG

[0404] SEQ ID NO: 73. CD123 scFv clone 292 Linker (in CD123-BiTE-IRES-NGFR (CD123 TCE)) - DNA:GGAGGCGGAGGAAGTGGCGGCGGAGGATCTGGGGGAGGCGGATCT

[0405] SEQ ID NO: 74. CD123 scFv clone 292 Linker (in CD123-BiTE-IRES-NGFR (CD123 TCE)) - AA:GGGGSGGGGSGGGGS(5) Transmembrane domain

[0406] Polypeptides of the present disclosure may comprise a transmembrane domain. In some aspects, a transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains may result in different receptor stability.

[0407] In some aspects, the transmembrane domain is interposed between the extracellular spacer and the cytoplasmic region. In some aspects, the transmembrane domain is interposedbetween the extracellular spacer and one or more costimulatory regions. In some aspects, a linker is between the transmembrane domain and the one or more costimulatory regions.

[0408] In certain aspects, engineered polypeptides may comprise a transmembrane domain sequence. In some aspects, a transmembrane domain sequence may have a functional impact on the expression, localization, and / or activity of a polypeptide comprising the same. In some aspects, a transmembrane domain sequence may be a CD28 derived sequence.

[0409] Any transmembrane domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell may be suitable for use. In some aspects, the transmembrane domain is derived from CD28, CD8, CD4, CD3-zeta (CD3Q, CD134, or CD7.

[0410] In certain aspects, a transmembrane domain comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 13 or 14.(6) Cytoplasmic region

[0411] After antigen recognition, receptors of the present disclosure may cluster and a signal transmitted to the cell through the cytoplasmic region. In some aspects, the costimulatory domains described herein are part of the cytoplasmic region. In some aspects, the cytoplasmic region comprises an intracellular signaling domain. An intracellular signaling domain may comprise a primary signaling domain and one or more costimulatory domains.

[0412] In certain aspects, engineered polypeptides may comprise a cytoplasmic signaling domain(s) sequence. In some aspects, a cytoplasmic signaling domain(s) sequence may have a functional impact on the expression, localization, and / or activity of a polypeptide comprising the same.

[0413] Cytoplasmic regions and / or costimulatory regions suitable for use in the polypeptides of the disclosure include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation by way of binding of the antigen to the antigen binding domain. In some aspects, the cytoplasmic region includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described herein. In some aspects, the cytoplasmic region includes DAP10 / CD28 type signaling chains.

[0414] Cytoplasmic regions suitable for use in the polypeptides of the disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. An IT AM motif is YX1X2(L / I), where XI and X2 are independently any amino acid. In some cases, the cytoplasmic region comprises 1, 2, 3, 4, or 5 ITAM motifs. In some cases, an ITAM motif is repeated twice in an endodomain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids, e.g., (YXlX2(L / I))(X3)n(YXlX2(L / I)), where n is an integer from 6 to 8, and each of the 6-8 X3 can be any amino acid.

[0415] A suitable cytoplasmic region may be an TTAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable cytoplasmic region can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable endodomain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, DAP10, FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3-zeta; and CD79A (antigen receptor complex-associated protein alpha chain).

[0416] Exemplary cytoplasmic regions are known in the art.

[0417] In some aspects, a suitable cytoplasmic region can comprise an ITAM motifcontaining portion of the full length DAP 12 amino acid sequence. In some aspects, the cytoplasmic region is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon R1 -gamma; fcRgamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In some aspects, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length FCER1G amino acid sequence.

[0418] In some aspects, the cytoplasmic region is derived from T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD35; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.). In some aspects, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 delta amino acid sequence. In some aspects, the cytoplasmic region is derived from T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, CD3s; T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3- epsilon, T3e, etc.). In some aspects, a suitable cytoplasmic region can comprise an ITAM motif-containing portion of the full length CD3 epsilon amino acid sequence. In some aspects,the cytoplasmic region is derived from T cell surface glycoprotein CD3 gamma chain (also known as CD3G, CD3y, T cell receptor T3 gamma chain, CD3 -GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In some aspects, a suitable cytoplasmic region can comprise an IT AM motif-containing portion of the full length CD3 gamma amino acid sequence. In some aspects, the cytoplasmic region is derived from T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, CD3(^, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In some aspects, a suitable cytoplasmic region can comprise an IT AM motif-containing portion of the full length CD3 zeta amino acid sequence.

[0419] In some aspects, the cytoplasmic region is derived from CD79A (also known as B- cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; Ig-alpha; membranebound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In some aspects, a suitable cytoplasmic region can comprise an IT AM motif-containing portion of the full length CD79A amino acid sequence.(7) Costimulatory region

[0420] Non-limiting examples of suitable costimulatory regions, such as those included in the cytoplasmic region, include, but are not limited to, polypeptides from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0421] A costimulatory region may have a length of at least, at most, or exactly 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids or any range derivable therein. In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; CDwl37; ILA; etc.). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD28 (also known as Tp44). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD 134, 0X40, TXGP1L). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD27 (also known as S 152, T14, TNFRSF7, and Tp55). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). In some aspects, the costimulatory region is derived from an intracellular portion of the transmembrane proteinGITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). In some aspects, the costimulatory region derived from an intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, AT AR, CD270, HVEA, HVEM, LIGHTR, and TR2).

[0422] A CAR of the disclosure may lack a costimulatory region. Such a CAR is typically referred to as a 1stgeneration CAR. First generation CARs can still provide signal 1 to a cell of the disclosure. A first generation CAR may lack a costimulatory region from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and / or HVEM.(8) Peptide linkers

[0423] In some aspects, the polypeptides of the disclosure include peptide linkers (sometimes referred to as a linker). A peptide linker may be used to separate any of the peptide domain / regions described herein. As an example, a linker may be between the signal peptide and the antigen binding domain, between the VH and VL of the antigen binding domain, between the antigen binding domain and the peptide spacer, between the peptide spacer and the transmembrane domain, flanking the costimulatory region or on the N- or C- region of the costimulatory region, and / or between the transmembrane domain and the endodomain. The peptide linker may have any of a variety of amino acid sequences. Domains and regions can be joined by a peptide linker that is generally of a flexible nature, although other chemical linkages are not excluded. A linker can be a peptide of between about 6 and about 40 amino acids in length, or between about 6 and about 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins.

[0424] Peptide linkers with a degree of flexibility can be used. The peptide linkers may have virtually any amino acid sequence, bearing in mind that suitable peptide linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art.

[0425] Suitable linkers can be readily selected and can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0426] Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0427] Example flexible linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n, (G4S)n, and (GGGS)n, where n is an integer of at least one). In some aspects, n is at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any derivable range therein). Glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains. Exemplary spacers can comprise amino acid sequences including, but not limited to, GGSG, GGSGG, GSGSG, GSGGG, GGGSG, or GSSSG.5. Cytokine therapy

[0428] Cytokines are proteins produced by many types of cells present within a tumor. They can modulate immune responses. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.

[0429] Interferons are produced by the immune system. They are usually involved in antiviral response, but also have use for cancer. They fall in three groups: type I (IFNa and IFNP), type II (IFNy) and type III (IFNk).

[0430] Interleukins have an array of immune system effects. IL-2 is an example interleukin used for cytokine therapy.6. Adoptive cell therapy

[0431] Adoptive cell therapy is a form of passive immunization by the transfusion of immune cells, such as T cells, NK cells, or other immune cells (also called “adoptive cell transfer”). Immune cells used for adoptive cell therapy include those found in normal tissue and those found in tumor tissue (where they are known as tumor infiltrating immune cells or tumor infiltrating lymphocytes). Although tumor infiltrating immune cells can attack a tumor,the environment within the tumor is generally highly immunosuppressive, preventing immune- mediated tumor death.

[0432] Multiple ways of producing and obtaining tumor targeted immune cells have been developed. Immune cells specific to a tumor antigen can be removed from a tumor sample or filtered from blood. Subsequent activation and culturing may be performed ex vivo, with the results reinfused. Activation can take place through gene therapy, by exposing the immune cells to tumor antigens, or by other methods known in the art.7. Oncolytic virus

[0433] In some aspects, the cancer therapy comprises an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumor. Oncolytic viruses are thought not only to cause direct destruction of the tumor cells, but also to stimulate host anti-tumor immune responses for long-term immunotherapy. One example of oncolytic virus therapy is teserpaturev (DELYTACT®).8. T-cell Engager (TCE)

[0434] T-cell engagers (TCEs) are chimeric antibodies comprising a target cell binding domain comprising an antigen binding domain such as a single-domain antibody (e.g., a VHH) that specifically binds to an antigen on a target cell, and an immune effector cell binding domain that specifically binds to an antigen on an immune effector cell. In this way, TCEs can redirect T cell to recognize and kill tumor cells. In certain aspects, bispecific antibodies comprising two scFvs targeting CD3 on T cells and a tumor antigen on cancer cells may be used.

[0435] As disclosed herein, in certain aspects, immune effector cells expressing a TCE, e.g., a TCE recognizing CD123, may be further engineered to express a polynucleotide and / or polypeptide of the immediate disclosure, such as but not limited to, polypeptides 4- lBBL / IL7Ra (SEQ ID NO: 18), 4-1BBL / IL2RP (SEQ ID NO: 22), 4-lBBL / MyD88 (SEQ ID NO: 26), and / or 4-lBBL / cMPL (SEQ ID NO: 30).

[0436] In some aspects, the TCE comprises, consists essentially of, or consists of an amino acid sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical SEQ ID NO: 64.

[0437] In some aspects, the TCE may be encoded by a polynucleotide that comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to anyone of SEQ ID NOs: 63, or 88.

[0438] SEQ ID NO: 88. 5'LTR- CD123-BiTE-IRES-NGFR (CD123 TCE) -3'LTR - DNA:AATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCAT GGAAAAATACATAAC T GAGAATAGAAAAGT T CAGAT CAAGGT CAGGAACAGAT GGAACAGC T GAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAAC AGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCT CAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCAT CAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAA TCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTATGCTCCCCGAGCTCAATAAAAGAGCCCAC AACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCA ATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCT GAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATTTGGGGGCTCGTCCGGGATCGGGAGAC CCCTGCCCAGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCCAGCAACTTATCTGTGTC TGTCCGATTGTCTAGTGTCTATGACTGATTTTATGCGCCTGCGTCGGTACTAGTTAGCTAAC TAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCGGAACACCCGGCCGCAACCC TGGGAGACGTCCCAGGGACTTCGGGGGCCGTTTTTGTGGCCCGACCTGAGTCCTAAAATCCC GATCGTTTAGGACTCTTTGGTGCACCCCCCTTAGAGGAGGGATATGTGGTTCTGGTAGGAGA CGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGGACCGAAGCC GCGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGTTTCTG TATTTGTCTGAAAATATGGGCCCGGGCTAGCCTGTTACCACTCCCTTAAGTTTGACCTTAGG TCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTT GGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACC TTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACA CCCAGACCAGGTGGGGTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGG TCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCC CTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCT AGGCGCCCCCATATGGCCATATGAGATCTTATATGGGGCACCCCCGCCCCTTGTAAACTTCC CTGACCCTGACATGACAAGAGTTACTAACAGCCCCTCTCTCCAAGCTCACTTACAGGCTCTC TACTTAGTCCAGCACGAAGTCTGGAGACCTCTGGCGGCAGCCTACCAAGAACAACTGGACCG ACCGGTGGTACCTCACCCTTACCGAGTCGGCGACACAGTGTGGGTCCGCCGACACCAGACTA AGAACCTAGAACCTCGCTGGAAAGGACCTTACACAGTCCTGCTGACCACCCCCACCGCCCTC AAAGTAGACGGCATCGCAGCTTGGATACACGCCGCCCACGTGAAGGCTGCCGACCCCGGGGG TGGACCATCCTCTAGACTGCCGTTTAAACGCCACGATGGATTGGATCTGGCGCATCCTGTTT CTCGTGGGAGCCGCCACAGGCGCCCATTCTCAGGTGCAGCTGCAGCAGCCTGGCGCTGAACT CGTGCGGCCAGGCGCTTCTGTGAAGCTGAGCTGTAAAGCCAGCGGCTACACCTTCACCAGCT ACTGGATGAACTGGGTCAAGCAGCGGCCCGACCAGGGCCTGGAATGGATCGGCAGAATCGAC CCCTACGACAGCGAGACACACTACAACCAGAAGTTCAAGGACAAGGCCATCCTGACCGTGGA CAAGAGCAGCAGCACCGCCTACATGCAGCTGTCCAGCCTGACCAGCGAGGACAGCGCCGTGT ACTACTGCGCCAGAGGCAACTGGGACGACTACTGGGGCCAGGGCACAACCCTGACAGTGTCT AGCGGAGGCGGAGGAAGTGGCGGCGGAGGATCTGGGGGAGGCGGATCTGATGTGCAGATCAC CCAGAGCCCCAGCTACCTGGCTGCCTCTCCTGGCGAGACAATCACCATCAACTGCCGGGCCA GCAAGAGCATCTCCAAGGACCTGGCCTGGTATCAGGAAAAGCCCGGCAAGACCAACAAGCTG CTGATCTACAGCGGCAGCACCCTGCAGAGCGGCATCCCCAGCAGATTTTCCGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCTCCCTGGAACCCGAGGACTTTGCCATGTACTATT GCCAGCAGCACAACAAGTACCCTTACACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGTCC GGAGGTGGTGGATCCGATATCAAACTGCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGC CTCAGTGAAGATGTCCTGCAAGACTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGG TAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCCGTGGTTAT AC T AAT T ACAAT CAGAAG T T CAAGGACAAGGC CACAT T GAG T ACAGACAAAT C C T C CAGCAC AGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAT ATTATGATGATCATTACTGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA GGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATTCAGCTGACCCA GTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGAGCCAGTT CAAGTGTAAGTTACATGAACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATT TATGACACATCCAAAGTGGCTTCTGGAGTCCCTTATCGCTTCAGTGGCAGTGGGTCTGGGAC CTCATACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAAC AGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAATCCTAACTC GAGCGGGATCAATTCCGCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCG GTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCC GGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGA ATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAAC AACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCG GCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTG AGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAA GGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTA CATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCC TTTGAAAAACACGATAATACCATGGGAGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCG CCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAG GCCTGTACACACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAG CCTTGTGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGT GGTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGG CGCCGTGCGTGGAGGCCGACGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAG ACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTG CCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCA ACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAG TGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCAC ACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAAC AAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCC GTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGT GGTTGTGGGCCTTGTGGCCTACATAGCCTTCAAGAGGTGGAACAGCTAAGCGGCCGCGACTC TAGAGTCGAGATCGATCCGGATTAGTCCAATTTGTTAAAGACAGGATATCAGTGGTCCAGGC TCTAGTTTT GAG T C AAC AAT AT GAG GAG C T GAAG C C T AT AGAG TAG GAG C C AT AGAT AAAAT AAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGC AAGC TAGC T TAAGTAACGCCAT T T T GCAAGGCAT GGAAAAAT AGAT AAC T GAGAATAGAGAA GTTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGG TAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAAC AGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGAT GCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACC TGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGC GCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCC GATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACT TGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGT CTTTCA9. Transgenic T cell Receptors (tTCR)

[0439] Transgenic T cell receptors (tTCR) are heterologous TCR that recognize specific antigens, e.g., cancer antigens, suitable for cell engineering and immunotherapy. tTCR may target mutation antigens (e.g., B-RAF mutation), tumor-specific antigens (e.g., NY-ESO-1), differentiation antigens (e.g., MART-1), and / or overexpressed antigens (e.g., p53). Antigens that may be targeted through molecules comprising antigen binding affinity described herein, such as but not limited to tTCR, include but are not limited to AFP, CMV, EBV, gplOO, HA- 1, HBV, HERV-E, HPV-16 E6, HPV-16 E7, KRAS G12D, KRAS G12V, MAGE-A10, MAGE-A3 / A6 (KITE-718), MAGE-A3-DP0401 / 0402, MAGE-A4, MART-1, MART-1 F5, MCPyV, NY-ESO-1, survivin, p53, PRAME, PRAME / COL6A3, Tyrosinase, WT-1, CEA, hTG, TGFpiII, and TRAIL bound to DR4.

[0440] In certain aspects, a TCR comprises, consists essentially of, or consists of a amino acid sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NO: 99.

[0441] In certain aspects, a TCR is encoded by a polynucleotide that comprises, consists essentially of, or consists of a sequence at least or exactly 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 100-101.10. Suicide genes

[0442] In some aspects, the ability to kill a modified cells may be desired, such as to terminate a treatment, to prevent the cells from becoming neoplastic, in research where the absence of the cells after their absence is of interest, or other event. For this purpose, the expression of certain gene products that can kill modified cells under controlled conditions is considered. These genes are usually referred to as suicide gene products and include, as nonlimiting examples, caspase 9 and inducible caspase 9.C. Chemotherapies

[0443] In some aspects, a therapy of the present disclosure comprises a chemotherapy. Suitable classes of chemotherapeutic agents include (a) Alkylating Agents, such as nitrogen mustards (e.g., mechlorethamine, cylophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates(e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozoticin, streptozocin) and triazines (e.g., dicarbazine), (b) Antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5 -fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related materials (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin), (c) Natural Products, such as vinca alkaloids (e.g., vinblastine, vincristine), epipodophylotoxins (e.g., etoposide, teniposide), antibiotics (e.g., dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin and mitoxanthrone), enzymes (e.g., L-asparaginase), and biological response modifiers (e.g., Interferon-a), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhy diazine derivatives (e.g., procarbazine), and adrenocortical suppressants (e.g., taxol and mitotane). In some aspects, cisplatin is a particularly suitable chemotherapeutic agent.

[0444] Cisplatin has been widely used to treat cancers such as, for example, metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes such as, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection.

[0445] Other suitable chemotherapeutic agents include antimicrotubule agents, e.g., Paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). Doxorubicin is absorbed poorly and is preferably administered intravenously. In certain aspects, appropriate intravenous doses for an adult include about 60 mg / m2to about 75 mg / m2at about 21-day intervals or about 25 mg / m2to about 30 mg / m2on each of 2 or 3 successive days repeated at about 3 week to about 4 week intervals or about 20 mg / m2once a week.

[0446] Nitrogen mustards are another suitable chemotherapeutic agent useful in the methods of the disclosure. A nitrogen mustard may include, but is not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN®) is available from Mead Johnson and NEOSTAR®, available from Adria, is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, intravenous doses include, for example, initially about 40 mg / kg to about 50 mg / kg in divided doses over a period of about 2 days to about 5 days or about 10 mg / kg to about 15 mg / kg about every 7 days to about 10 days or about 3 mg / kg to about 5 mg / kg twice a week or about 1.5 mg / kg / day to about 3 mg / kg / day. Because of adverse gastrointestinal effects, the intravenous route is preferred in certain cases. The drug also sometimes is administered intramuscularly, by infiltration or into body cavities.

[0447] Additional suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5 -fluorouracil (fluouracil; 5-FU) and floxuridine (fluorodeoxyuridine; FudR). 5-FU may be administered to a subject in a dosage of anywhere between about 7.5 to about 1000 mg / m2. Further, 5-FU dosing schedules may be for a variety of time periods, for example up to six weeks, or as determined by one of ordinary skill in the art to which this disclosure pertains.

[0448] The amount of the chemotherapeutic agent delivered to a patient may be variable. In one suitable aspect, the chemotherapeutic agent may be administered in an amount effective to cause arrest or regression of the cancer in a host, when the chemotherapy is administered with the construct. In other aspects, the chemotherapeutic agent may be administered in an amount that is anywhere between 2 to 10,000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. For example, the chemotherapeutic agent may be administered in an amount that is about 20 fold less, about 500 fold less or even about 5000 fold less than the chemotherapeutic effective dose of the chemotherapeutic agent. The chemotherapeutics of the disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct, as well as for determination of effective dosages. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, etc. In vitro testing may also be used to determine suitable combinations and dosages, as described in the examples.D. Hormone therapy

[0449] In some aspects, a cancer therapy of the present disclosure is a hormone therapy. In particular aspects, a prostate cancer therapy comprises hormone therapy. Various hormone therapies are known in the art and contemplated herein. Examples of hormone therapies include, but are not limited to, luteinizing hormone-releasing hormone (LHRH) analogs, LHRH antagonists, androgen receptor antagonists, and androgen synthesis inhibitors.E. Surgery

[0450] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and / or destroyed and may be used in conjunction with other therapies, such as the treatment of the present aspects, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to physical removal of atleast part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).

[0451] Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.F. Additional cancer therapies

[0452] Therapeutic methods disclosed herein may comprise one or more additional cancer therapies. A cancer therapy of the disclosure may comprise, for example, cryoablative therapy, high-intensity ultrasound (also “high-intensity focused ultrasound”), photodynamic therapy, laser ablation, and / or irreversible electroporation. A cancer therapy of the disclosure may comprise 1, 2, 3, 4, 5, or more distinct therapeutic methods.

[0453] It is contemplated that a cancer treatment may exclude any of the cancer treatments described herein. Furthermore, aspects of the present disclosure include patients that have been previously treated for a therapy described herein, are currently being treated for a therapy described herein, or have not been treated for a therapy described herein. In some aspects, the patient is one that has been determined to be resistant to a therapy described herein. In some aspects, the patient is one that has been determined to be sensitive to a therapy described herein.IV. Cellular Therapies

[0454] Aspects of the present disclosure include cellular therapies, including engineering and formulating cellular therapies as well as using such therapies for treatment of various conditions such as cancer.A. Cell Culture

[0455] In some aspects, cells may be cultured for at least between about 10 days and about 40 days, for at least between about 15 days and about 35 days, for at least between about 15 days and 21 days, such as for at least about 15, 16, 17, 18, 19 or 21 days. In some aspects, the cells of the disclosure may be cultured for no longer than 60 days, or no longer than 50 days, or no longer than 45 days. The cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days. The cells may be cultured in the presence of a liquid culture medium. Typically, the medium may comprise a basal medium formulation as known in the art. Many basal mediaformulations can be used to culture cells herein, including but not limited to Eagle’s Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle’s Medium (DMEM), alpha modified Minimum Essential Medium (alpha-MEM), Basal Medium Essential (BME), Iscove’s Modified Dulbecco’s Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle’s Medium (EMEM), RPMI-1640, and modifications and / or combinations thereof. Compositions of the above basal media are generally known in the art, and it is within the skill of one in the art to modify or modulate concentrations of media and / or media supplements as necessary for the cells cultured. In some aspects, a culture medium formulation may be explants medium (CEM) which is composed of IMDM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin G, 100 pg / ml streptomycin and 2 mmol / L L-glutamine. Other aspects may employ further basal media formulations, such as chosen from the ones above.

[0456] Any medium capable of supporting cells in vitro may be used to culture the cells. Media formulations that can support the growth of cells include, but are not limited to, Dulbecco’s Modified Eagle’s Medium (DMEM), alpha modified Minimal Essential Medium (aMEM), and Roswell Park Memorial Institute Media 1640 (RPMI Media 1640) and the like. Typically, up to 20% fetal bovine serum (FBS), up to 20% human AB serum, up to 20% human platelet lysate, or 1-20% horse serum is added to the above medium in order to support the growth of cells. A defined medium, however, also can be used if the growth factors, cytokines, and hormones necessary for culturing cells are provided at appropriate concentrations in the medium. Media useful in the methods of the disclosure may comprise one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culturing of cells. The cells may be grown at temperatures between 27° C to 40° C, such as 31° C to 37° C, and may be in a humidified incubator. The carbon dioxide content may be maintained between 2% to 10% and the oxygen content may be maintained between 1% and 22%. The disclosure, however, should in no way be construed to be limited to any one method of isolating and culturing cells. Rather, any method of isolating and culturing cells should be construed to be included in the present disclosure.

[0457] For use in the cell culture, media can be supplied with one or more further components. For example, additional supplements can be used to supply the cells with the necessary trace elements and substances for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components can be included in a salt solution such as, but not limited to, Hanks’ Balanced Salt Solution (HBSS), Earle’s Salt Solution. Further antioxidant supplements may be added, e.g., P-mercaptoethanol.While many media already contain amino acids, some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable when in solution. A medium may be further supplied with antibiotic and / or antimycotic compounds, such as, typically, mixtures of penicillin and streptomycin, and / or other compounds, exemplified but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. Also contemplated is supplementation of cell culture medium with mammalian plasma or sera. Plasma or sera often contain cellular factors and components that are necessary for viability and expansion. The use of suitable serum replacements is also contemplated.

[0458] Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed. In particular aspects, cells are cultured in a cell culture system comprising a cell culture medium, preferably in a culture vessel, in particular a cell culture medium supplemented with a substance suitable and determined for protecting the cells from in vitro aging and / or inducing in an unspecific or specific reprogramming.B. Cell Generation

[0459] Certain methods of the disclosure concern culturing the cells obtained from human tissue samples. In particular aspects of the present disclosure, cells are plated onto a substrate that allows for adherence of cells thereto. This may be carried out, for example, by plating the cells in a culture plate that displays one or more substrate surfaces compatible with cell adhesion. When the one or more substrate surfaces contact the suspension of cells (e.g., suspension in a medium) introduced into the culture system, cell adhesion between the cells and the substrate surfaces may ensue. Accordingly, in certain aspects cells are introduced into a culture system that features at least one substrate surface that is generally compatible with adherence of cells thereto, such that the plated cells can contact the said substrate surface, such aspects encompass plating onto a substrate, which allows adherence of cells thereto.

[0460] Cells of the present disclosure may be identified and characterized by their expression of specific marker proteins, such as cell-surface markers. Detection and isolation ofthese cells can be achieved, for example, through flow cytometry, ELISA, and / or magnetic beads. Reverse-transcription polymerase chain reaction (RT-PCR) may be used to quantify cell-specific genes and / or to monitor changes in gene expression in response to differentiation. In certain aspects, the marker proteins used to identify and characterize the cells are selected from the list consisting of c-Kit, Nanog, Sox2, Heyl, SMA, Vimentin, Cyclin D2, Snail, E- cadherin, Nkx2.5, GATA4, CD105, CD90, CD29, CD73, Wtl, CD34, CD45, CD3, CD8, CD4, and / or a combination thereof.C. Cells

[0461] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), human embryonic kidney (HEK) 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1...

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide encoding a dual-stimulatory receptor (DSR), wherein the DSR comprises: a) an extracellular binding domain comprising an extracellular costimulatory ligand domain, an antibody, or an antibody fragment; b) a transmembrane domain; and c) an intracellular cytokine signaling domain.

2. The polynucleotide of claim 1, wherein the extracellular binding domain binds a costimulatory receptor.

3. The polynucleotide of claim 1, wherein the extracellular binding domain binds a costimulatory receptor of the same cell in which it is expressed.

4. The polynucleotide of claim 1, wherein the extracellular binding domain aggregates two or more co-stimulatory receptors of the same cell in which it is expressed.

5. The polynucleotide of claim 1, wherein the extracellular binding domain can aggregate two or more co-stimulatory receptors of the same cell in which it is expressed to induce costimulatory signaling in the cell.

6. The polynucleotide of claim 1, wherein the extracellular binding domain does not bind a target antigen on a diseased cell and / or a target on a different cell than the cell in which it is expressed.

7. The polynucleotide of claim 1, wherein the extracellular binding domain does not induce co-stimulatory signaling in a different cell than the cell in which it is expressed.

8. The polynucleotide of claim 1, wherein the intracellular cytokine signaling domain does not comprise a co-stimulatory domain.

9. The polynucleotide of claim 1, wherein the intracellular cytokine signaling domain does not comprise a co-stimulatory domain derived from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and / or HVEM.

10. The polynucleotide of claim 1, wherein the intracellular cytokine signaling domain does not comprise a CD3 domain.

11. The polynucleotide of claim 1, wherein the intracellular cytokine signaling domain does not comprise a CD3zeta domain.

12. The polynucleotide of claim 1, wherein the intracellular cytokine signaling domain does not comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 immunoreceptor tyrosine-based activation motifs (IT AM).

13. The polynucleotide of claim 1, wherein the intracellular cytokine signaling domain can initiate JAK-STAT signaling.

14. The polynucleotide of claim 1, wherein the intracellular cytokine signaling domain can initiate JAK-STAT signaling and RAS / RAF signaling.

15. The polynucleotide of claim 1, wherein the extracellular binding domain comprises an extracellular costimulatory ligand domain.

16. The polynucleotide of claim 1, wherein the extracellular costimulatory ligand domain is derived from tumor necrosis factor ligand superfamily, member 9 (Tnfsf9, also known as 4- 1BBL), cluster of differentiation 80 (CD80, also known as B7-1), cluster of differentiation 86 (CD86, also known as B7-2), inducible T cell costimulator ligand (ICOS-L, also known as B7- H2), cluster of differentiation 70 (CD70, also known as CD27L), TNF superfamily member 14 (TNFSF14, also known as LIGHT), CD40 ligand (CD40L), TNF superfamily member 4 (TNFSF4, also known as OX40L), TNF superfamily member 15 (TNFSF15, also known as TL1A), TNF superfamily member 18 (TNFSF18, also known as GITRL), CD30 ligand (CD30L), T cell immunoglobulin and mucin domain containing 4 (TIM4), cluster of differentiation 150 (CD 150, also known as SLAM), cluster of differentiation 48 (CD48, also known as SLAMF2), cluster of differentiation 58 (CD58, also known as LFA-4), cluster of differentiation 155 (CD155, also known as PVR), and / or cluster of differentiation 112 (CD112, also known as Nectin-2).

17. The polynucleotide of claim 1, wherein the extracellular costimulatory ligand domain is derived from 4-1BBL.

18. The polynucleotide of claim 1, wherein the extracellular costimulatory ligand domain is derived from OX40L.

19. The polynucleotide of claim 1, wherein the encoded extracellular costimulatory ligand domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 8, 96, or 98.

20. The polynucleotide of claim 1, wherein the encoded extracellular costimulatory ligand domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 8.

21. The polynucleotide of claim 1, wherein the extracellular costimulatory ligand domain is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 7, 95, or 97.

22. The polynucleotide of claim 1, wherein the extracellular costimulatory ligand domain is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 7.

23. The polynucleotide of claim 1, wherein the transmembrane domain is derived from cluster of differentiation 28 (CD28), cluster of differentiation 8 (CD8), cluster of differentiation (CD25), cluster of differentiation 4 (CD4), cluster of differentiation 3 zeta (CD3Q, cluster of differentiation 134 (CD 134), or cluster of differentiation 7 (CD7).

24. The polynucleotide of claim 1, wherein the transmembrane domain is derived from CD28.

25. The polynucleotide of claim 1, wherein the encoded transmembrane domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 6, or 14.

26. The polynucleotide of claim 1, wherein the encoded transmembrane domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 14.

27. The polynucleotide of claim 1, wherein the transmembrane domain is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 5, or 13.

28. The polynucleotide of claim 1, wherein the transmembrane domain is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 13.

29. The polynucleotide of claim 1, wherein the intracellular cytokine signaling domain is derived from thrombopoietin receptor (cMPL, also known as TPO-R), interleukin 7 receptor (IL7R, also known as IL-7Ra, or CD127), interleukin 2 receptor subunit beta (IL-2RP, also known as CD122, or IL-15RP), myeloid differentiation primary response protein 88 (MyD88), interleukin 2 receptor subunit gamma (IL-2Ry, also known as CD 132, or yc), interleukin 1 receptor type 1 (IL-1R), interleukin 12 receptor subunit beta 1 (IL-12RP1), interleukin 12 receptor subunit beta 2 (IL-12RP2), interleukin 6 cytokine family signal transducer (IL6ST, also known as gpl30, or CD130), interleukin 4 receptor (IL-4R), interleukin 9 receptor (IL- 9R), interleukin 10 receptor (IL-10R), interleukin 13 receptor (IL-13R), interleukin 15 receptor (IL-15R), interleukin 21 receptor (IL-21R), interleukin 23 receptor (IL-23R), interferon alpha and beta receptor subunit 2 (IFNAR2, also known as IFNa / pR, or IFN-R), interleukin 10 receptor (IL-10R), interleukin 6 receptor (IL-6R), interleukin 11 receptor (IL-11R), ciliary neurotrophic factor receptor (CNTF-R), oncostatin M receptor (OSM-R), LIF receptor subunit alpha (LIF-R), calcitonin receptor (CT-R), interleukin 3 receptor (IL-3R), interleukin 5 receptor (IL-5R), granulocyte-macrophage colony-stimulating factor receptor (GM-CSF-R), growth hormone receptor (GH-R), erythropoietin receptor (EPO-R), and / or prolactin receptor (PRL-R).

30. The polynucleotide of claim 1, wherein the intracellular cytokine signaling domain is derived from cMPL (TPO-R), IL-7Ra (CD127), IL-2RP (CD122 / IL-15RP), and / or MyD88.

31. The polynucleotide of claim 1, wherein the encoded intracellular cytokine signaling domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 20, 24, 28, or 32.

32. The polynucleotide of claim 1, wherein the encoded intracellular cytokine signaling domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 32.

33. The polynucleotide of claim 1, wherein the intracellular cytokine signaling domain is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 19, 23, 27, or 31.

34. The polynucleotide of claim 1, wherein the intracellular cytokine signaling domain is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 31.

35. The polynucleotide of claim 1, wherein the extracellular costimulatory ligand domain, the transmembrane domain, and the intracellular cytokine signaling domain are operably linked in a 5' to 3' order.

36. The polynucleotide of claim 1, wherein the DSR comprises the extracellular costimulatory ligand domain derived from 4-1BBL, the transmembrane domain derived from CD28, and the intracellular cytokine signaling domain derived from IL-7Ra.

37. The polynucleotide of claim 1, wherein the DSR comprises the extracellular costimulatory ligand domain derived from 4-1BBL, the transmembrane domain derived from CD28, and the intracellular cytokine signaling domain derived from IL-2Rp.

38. The polynucleotide of claim 1, wherein the DSR comprises the extracellular costimulatory ligand domain derived from 4-1BBL, the transmembrane domain derived from CD28, and the intracellular cytokine signaling domain derived from MyD88.

39. The polynucleotide of claim 1, wherein the DSR comprises the extracellular costimulatory ligand domain derived from 4-1BBL, the transmembrane domain derived from CD28, and the intracellular cytokine signaling domain derived from cMPL.

40. The polynucleotide of claim 1, wherein the encoded DSR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 18, 22, 26, or 30.

41. The polynucleotide of claim 1, wherein the DSR is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 17, 21, 25, or 29.

42. The polynucleotide of claim 1, wherein the polynucleotide comprises a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 82-85.

43. The polynucleotide of claim 1, wherein the polynucleotide comprises a sequence at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, or 100% identical to the open reading frame of any one of SEQ ID NOs: 89-92.

44. The polynucleotide of claim 1, wherein the polynucleotide consists essentially of the DSR encoding sequence.

45. The polynucleotide of claim 1, wherein the polynucleotide does not comprise a sequence encoding an intracellular co-stimulatory domain and / or co-stimulatory signaling domain.

46. The polynucleotide of claim 1, wherein the polynucleotide does not comprise a sequence encoding an intracellular CD3z, CD28, or 4-1BBL derived domain.

47. A polypeptide encoded by the polynucleotide any one of claims 1-46.

48. A composition comprising a cell engineered to comprise the polynucleotide of any one of claims 1-46.

49. The composition of claim 48, wherein the engineered cell naturally or artificially expresses a full or partial TCR complex.

50. The composition of claim 48, wherein the engineered cell naturally or artificially expresses a full or partial CD3 complex.

51. The composition of claim 48, wherein the engineered cell naturally or artificially expresses a full or partial CD3^ chain.

52. The composition of claim 48, wherein the engineered cell naturally or artificially comprises antigen stimulation functionality.

53. The composition of claim 48, wherein the cell comprises an immune cell.

54. The composition of claim 53, wherein the immune cell is autologous or allogeneic.

55. The composition of claim 53, wherein the immune cell comprises a T cell, NK cell, dendritic cell (DC), B cell, macrophage, or iNKT cell.

56. The composition of claim 53, wherein the immune cell comprises aP T cells, virusspecific T cell, tumor-specific T cell, and / or y5 T cells.

57. The composition of claim 53, wherein the immune cell comprises Ml macrophages and / or M2 macrophages.

58. The composition of claim 53, wherein the immune cell comprises a conventional DC and / or a plasmacytoid DC.

59. The composition of claim 48, wherein the cell is derived from a pluripotent stem cell, a precursor immune cell, a cell line, cord blood, and / or a mature immune cell.

60. The composition of claim 48, wherein the cell is an embryonic stem cell or an induced pluripotent stem cell.

61. The composition of claim 48, wherein the cell is further engineered to comprise a chimeric T cell receptor (cTCR), a transgenic T cell receptor, and / or a T cell engager (TCE).

62. The composition of claim 61, wherein the cTCR targets disial oganglioside GD2 (GD2), survivin, or terminal deoxynucleotidyl transferase (TdT).

63. The composition of claim 61, wherein the cTCR comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 34, or 58.

64. The composition of claim 61, wherein the cTCR is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 33, or 57.

65. The composition of claim 61, wherein the TCE targets CD123 and CD3.

66. The composition of claim 61, wherein the TCE comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 64.

67. The composition of claim 61, wherein the TCE is encoded by a polynucleotide sequence comprising at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 63.

68. The composition of claim 48, further comprising an exogenously provided antibody.

69. A composition comprising, the polynucleotide, polypeptide, or composition of any one of claims 1-68, and a pharmaceutically acceptable carrier.

70. The composition of claim 69, wherein the composition is comprised in a delivery device.

71. A method of treating a disease in an individual, the method comprising the step of administering to the individual in need thereof a therapeutically effective amount of the composition of claim 48.

72. The method of claim 71, wherein the individual has been diagnosed, is suspected of having, or has one or more symptoms of an autoimmune disease, infection, and / or cancer.

73. The method of claim 72, wherein the cancer is of the lung, brain, breast, blood, skin, pancreas, liver, colon, head and neck, kidney, thyroid, stomach, spleen, gallbladder, bone, ovary, testes, endometrium, prostate, rectum, anus, cervix, or is hematological.

74. The method of claim 72, wherein the cancer is chronic myeloid leukemia (CML) or acute myeloid leukemia (AML).

75. The method of claim 71, wherein the individual is administered an immune cell.

76. The method of claim 75, wherein the immune cell displays an increase in in vivo anticancer functionality relative to a non-engineered immune cell and / or an immune cell engineered to comprise a transgenic TCR, cTCR, or TCE without a DSR.

77. The method of claim 76, wherein the increase in in vivo anti-cancer functionality comprises long-term cancer eradication, significant prolongation of the individual’s survival, and / or reduced toxicity or weight loss.

78. A method of providing an individual with an immune memory response against cancer, the method comprising administering to an individual in need thereof a therapeutically effective amount of the polynucleotide, polypeptide, or composition of any one of claims 1-70.

79. A kit comprising the polynucleotide, polypeptide, or composition of any one of claims 1-70.

80. A kit for the means of performing the methods of any one of claims 71-78.

81. Use of the polynucleotide, polypeptide, composition, kit, or method of any one of the preceding claims in the manufacture of a medicament.

82. A DSR polypeptide comprising an extracellular binding domain derived from 4-1BBL, a transmembrane domain derived from CD28, and an intracellular cytokine signaling domain derived from cMPL.

83. The DSR polypeptide of claim 82, wherein the extracellular binding domain binds a co-stimulatory receptor.

84. The DSR polypeptide of claim 82, wherein the extracellular binding domain binds a co-stimulatory receptor of the same cell in which it is expressed.

85. The DSR polypeptide of claim 82, wherein the extracellular binding domain aggregates two or more co-stimulatory receptors of the same cell in which it is expressed.

86. The DSR polypeptide of claim 82, wherein the extracellular binding domain can aggregate two or more co-stimulatory receptors of the same cell in which it is expressed to induce co-stimulatory signaling in the cell.

87. The DSR polypeptide of claim 82, wherein the extracellular binding domain does not bind a target antigen on a diseased cell and / or a target on a different cell than the cell in which it is expressed.

88. The DSR polypeptide of claim 82, wherein the extracellular binding domain does not induce co-stimulatory signaling in a different cell than the cell in which it is expressed.

89. The DSR polypeptide of claim 82, wherein the intracellular cytokine signaling domain does not comprise a co-stimulatory domain.

90. The DSR polypeptide of claim 82, wherein the intracellular cytokine signaling domain does not comprise a co-stimulatory domain derived from 4-1BB (CD137), CD28, ICOS, OX- 40, BTLA, CD27, CD30, GITR, and / or HVEM.

91. The DSR polypeptide of claim 82, wherein the intracellular cytokine signaling domain does not comprise a CD3 domain.

92. The DSR polypeptide of claim 82, wherein the intracellular cytokine signaling domain does not comprise a CD3zeta domain.

93. The DSR polypeptide of claim 82, wherein the intracellular cytokine signaling domain does not comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 immunoreceptor tyrosine-based activation motifs (IT AM).

94. The DSR polypeptide of claim 82, wherein the intracellular cytokine signaling domain can initiate JAK-STAT signaling.

95. The DSR polypeptide of claim 82, wherein the intracellular cytokine signaling domain can initiate JAK-STAT signaling and RAS / RAF signaling.

96. A human T cell comprising:(a) a DSR polypeptide comprising an extracellular binding domain derived from 4- 1BBL, a transmembrane domain derived from CD28, and an intracellular cytokine signaling domain derived from cMPL; and(b) a cTCR targeting TdT and / or survivin.

97. A human T cell comprising:(a) a DSR polypeptide comprising an extracellular binding domain derived from 4- 1BBL, a transmembrane domain derived from CD28, and an intracellular cytokine signaling domain derived from cMPL; and(b) a TCE that targets CD 123 and CD3.

98. A method of treating leukemia in an individual in need thereof comprising the step of administering to the individual a therapeutically effective amount of a human T cell comprising:(a) a DSR polypeptide comprising an extracellular binding domain derived from 4- 1BBL, a transmembrane domain derived from CD28, and an intracellular cytokine signaling domain derived from cMPL; and(b) a cTCR targeting TdT and / or survivin.

99. A method of treating leukemia in an individual in need thereof comprising the step of administering to the individual a therapeutically effective amount of a human T cell comprising:(a) a DSR polypeptide comprising an extracellular binding domain derived from 4- 1BBL, a transmembrane domain derived from CD28, and an intracellular cytokine signaling domain derived from cMPL; and(b) a TCE that targets CD 123 and CD3.

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